U.S. patent application number 10/278087 was filed with the patent office on 2003-07-24 for g protein coupled receptor protein, production, and use thereof.
This patent application is currently assigned to Takeda Chemical Industries, Ltd.. Invention is credited to Fujii, Ryo, Hinuma, Shuji, Ito, Yasuaki.
Application Number | 20030138817 10/278087 |
Document ID | / |
Family ID | 27571599 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138817 |
Kind Code |
A1 |
Hinuma, Shuji ; et
al. |
July 24, 2003 |
G protein coupled receptor protein, production, and use thereof
Abstract
DNA primers effective in screening G protein coupled receptor
protein-encoding DNA fragments are provided. The primers which are
complementary to nucleotide sequences that are in community with
(homologous to) the nucleotide sequences encoding amino acid
sequences corresponding to or near the first membrane-spanning
domain or the sixth membrane-spanning domain each of known various
G protein coupled receptor proteins were designed and synthesized.
Methods of amplifying G protein coupled receptor protein-encoding
DNAs using the above DNA primers, and novel target G protein
coupled receptor protein-encoding DNAs are also provided. Screening
of DNA libraries can be efficiently carried out. Human pituitary
gland or amygdala-derived and mouse pancreas-derived G protein
coupled receptor proteins, etc. or salts thereof, partial peptides
thereof, DNAs coding for the above G protein coupled receptor
proteins, processes for producing the above G protein coupled
receptor proteins, methods of determining ligands for the above G
protein coupled receptor proteins, methods of screening compounds
that inhibit the binding between the ligand and the G protein
coupled receptor proteins or screening kits therefor, compounds or
salts thereof obtained by the above screening method or the
screening kit, pharmaceutical compositions containing the above
compounds or salts thereof, and antibodies against the above
protein coupled receptor proteins or partial peptides thereof are
provided.
Inventors: |
Hinuma, Shuji; (Ibaraki,
JP) ; Ito, Yasuaki; (Ibaraki, JP) ; Fujii,
Ryo; (Ibaraki, JP) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 9169
BOSTON
MA
02209
US
|
Assignee: |
Takeda Chemical Industries,
Ltd.
Osaka
JP
|
Family ID: |
27571599 |
Appl. No.: |
10/278087 |
Filed: |
October 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10278087 |
Oct 22, 2002 |
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09461436 |
Dec 14, 1999 |
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6538107 |
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09461436 |
Dec 14, 1999 |
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09038572 |
Mar 11, 1998 |
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09038572 |
Mar 11, 1998 |
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08513974 |
Sep 14, 1995 |
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6114139 |
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08513974 |
Sep 14, 1995 |
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PCT/JP95/01599 |
Aug 10, 1995 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C12Q 1/6876 20130101;
C12Q 1/6883 20130101; C07K 14/705 20130101; C07K 14/70571 20130101;
C07K 14/723 20130101; C12Q 1/6809 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 1994 |
JP |
6-189272 |
Aug 11, 1994 |
JP |
6-189273 |
Aug 11, 1994 |
JP |
6-189274 |
Sep 30, 1994 |
JP |
6-236356 |
Sep 30, 1994 |
JP |
6-236357 |
Nov 2, 1994 |
JP |
6-270017 |
Dec 28, 1994 |
JP |
6-326611 |
Jan 20, 1995 |
JP |
7-007177 |
Mar 16, 1995 |
JP |
7-057186 |
Apr 19, 1995 |
JP |
7-093989 |
Mar 31, 1995 |
JP |
7-074314 |
Claims
1. A DNA which comprises a nucleotide sequence represented by a SEQ
ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 19.
2. A method for amplifying a DNA coding for a G protein coupled
receptor protein by polymerase chain reaction techniques, which
comprises: (i) carrying out a polymerase chain reaction in the
presence of a mixture of a DNA coding for a G protein coupled
receptor protein, said DNA being capable of acting as a template,
at least one DNA primer selected from the group consisting of DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
1, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 6, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 7, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 10, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
16 and DNA primers comprising a nucleotide sequence represented by
SEQ ID NO: 18, and at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 2, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 4, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 8, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
11, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 15, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 19; or (ii) carrying out a polymerase
chain reaction in the presence of a mixture of a DNA coding for G
protein coupled receptor protein, said DNA being capable of acting
as a template, at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 12, and at least one DNA primer
selected from the group consisting of DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 13.
3. A method for screening a DNA library for a DNA coding for a G
protein coupled receptor protein, which comprises: (i) carrying out
a polymerase chain reaction in the presence of a mixture of said
DNA library, at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 1, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 3, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 5, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
7, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16 and DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 18, and at least one
DNA primer selected from the group consisting of DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
4, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 8, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 11, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 15, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 17 and DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 19,
under conditions to amplify selectively a template DNA coding for
the G protein coupled receptor protein, contained in the DNA
library and selecting said DNA; or (ii) carrying out a polymerase
chain reaction in the presence of a mixture of said DNA library at
least one DNA primer selected from the group consisting of DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
1 and DNA primers comprising a nucleotide sequence represented by
SEQ ID NO: 12, and at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 13, under conditions to amplify
selectively a DNA coding for the G protein coupled receptor
protein, contained in the DNA library and selecting said DNA.
4. A DNA coding for a G protein coupled receptor protein or a
fragment thereof, which is obtained by the method according to
claim 2 to 3.
5. A G protein coupled receptor protein encoded by the DNA
according to claim 4, a peptide segment or fragment thereof or a
salt thereof.
6. A G protein coupled receptor protein comprising an amino acid
sequence selected from the group consisting of an amino acid
sequence represented by SEQ ID NO: 24, an amino acid sequence
represented by SEQ ID NO: 25, an amino acid sequence represented by
SEQ I D NO: 26, an amino acid sequence represented by SEQ ID NO:
27, an amino acid sequence represented by SEQ ID NO: 28, an amino
acid sequence represented by SEQ ID NO: 34, an amino acid sequence
represented by SEQ ID NO: 35, an amino acid sequence represented by
SEQ ID NO: 38, an amino acid sequence represented by SEQ ID NO: 39,
an amino acid sequence represented by SEQ ID NO: 56, and
substantial equivalents to the amino acid sequence represented by
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 39,
or SEQ ID NO: 56; a peptide segment (or fragment) thereof, a
modified peptide derivative thereof or a salt thereof.
7. The G protein coupled receptor protein according to claim 6,
comprising an amino acid sequence selected from the group
consisting of an amino acid sequence represented by SEQ ID NO: 38,
an amino acid sequence represented by SEQ ID NO: 39, an amino acid
sequence represented by SEQ ID NO: 56 and substantial equivalents
to the amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO:
39, or SEQ ID NO: 56.
8. The G protein coupled receptor protein according to claims 6 or
7, wherein said receptor is a purinoceptor.
9. The G protein coupled receptor protein according to any of
claims 6 to 8, wherein an agonist to said receptor is useful as an
immunomodulator or an antitumor agent, in addition it is useful in
therapeutically or prophylactically treating hypertension, diabetes
or cystic fibrosis, and an antagonist to said receptor is useful as
a hypotensive agent, an analgesic, or an agent for therapeutically
or prophylactically treating incontinence of urine.
10. A DNA which comprises a nucleotide sequence coding for a G
protein coupled receptor protein of claim 6.
11. The DNA according to claim 10 comprising a nucleotide sequence
coding for the G protein coupled receptor protein according to
claim 7.
12. The DNA according to claim 11 comprising a nucleotide sequence
represented by SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 57.
13. A transformant containing a vector comprising the DNA according
to claim 4 or 10; or an expression system comprising an open
reading frame (ORF) of DNA derived from a G protein coupled
receptor protein DNA according to claim 4 or 10, wherein the ORF is
operably linked to a control sequence compatible with a desired
host cell.
14. A method for determining a ligand to the G protein coupled
receptor protein according to any of claims 5 to 8, which comprises
contacting (i) at least one component selected from the group
consisting of G protein coupled receptor proteins or salts thereof
according to any of claims 5 to 8, peptide segments or salts
thereof, and mixtures thereof, with (ii) at least one compound to
be tested and determining whether said compound to be tested bound
to the component of (i).
15. A screening method for a compound capable of inhibiting the
binding of a G protein coupled receptor protein according to any of
claims 5 to 8 with a ligand, which comprises carrying out a
comparison between: (i) at least one case where said ligand is
contacted with at least one component selected from the group
consisting of G protein coupled receptor proteins or salts thereof
according to any of claims 5 to 8, peptide segments or salts
thereof, and mixtures thereof, and (ii) at least one case where
said ligand together with a compound to be tested is contacted with
at least one component selected from the group consisting of G
protein coupled receptor proteins or salts thereof according to any
of claims 5 to 8, peptide segments or salts thereof, and mixtures
thereof.
16. A compound which is determined through the method according to
claim 15 or a salt thereof.
17. The compound according to claim 16, which is an agonist or
antagonist to a G protein coupled receptor protein according to any
of claims 5 to 8.
18. A ligand to a G protein coupled receptor protein according to
any of claims 5 to 8, which is determined through the method
according to claim 14.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel DNAs which are useful
as DNA primers for a polymerase chain reaction (PCR); methods for
amplifying DNAs each coding for a G protein coupled receptor
protein via PCR techniques using said DNA; screening methods for
DNAs each encoding a G protein coupled receptor protein via PCR
techniques using said DNA; G protein coupled receptor
protein-encoding DNAs obtained by said screening method; G protein
coupled receptor proteins which are encoded by the DNA obtained via
said screening method, peptide fragments or segments thereof, and
modified peptide derivatives thereof; etc.
[0002] The present invention also relates to novel G protein
coupled receptor proteins; novel G protein coupled receptor
protein-encoding DNAs; processes for producing said G protein
coupled receptor protein; use of said receptor protein and said
protein-encoding DNA; etc.
[0003] The present invention also relates to novel human amygdaloid
nucleus-derived G protein coupled receptor proteins; novel DNAs
each coding for said G protein coupled receptor protein; processes
for producing said G protein coupled receptor protein; use of said
receptor protein and said protein-encoding DNA; etc.
[0004] The present invention also relates to novel mouse pancreatic
.beta. cell line MIN6-derived G protein coupled receptor proteins;
novel DNAs each coding for said G protein coupled receptor protein;
processes for producing said G protein coupled receptor protein;
use of said receptor protein and said protein-encoding DNA; etc.
Further, the present invention relates to novel human-derived G
protein coupled receptor proteins (human prinoceptors); novel DNAs
each coding for said G protein coupled receptor protein; processes
for producing said G protein coupled receptor protein; use of said
receptor protein and said protein-encoding DNA; etc.
BACKGROUND OF THE INVENTION
[0005] A variety of hormones, neurotransmitters and the like
control, regulate or adjust the functions of living bodies via
specific receptors located in cell membranes. Many of these
receptors mediate the transmission of intracellular signals via
activation of guanine nucleotide-binding proteins (hereinafter,
sometimes referred to as G proteins) with which the receptor is
coupled and possess the common (homologous) structure, i.e. seven
transmembranes (membrane-spanning regions (domains)). Therefore,
such receptors are generically referred to as G protein coupled
receptors or seven transmembrane (membrane-spanning) receptors.
[0006] G protein coupled receptor proteins have a very important
role as targets for molecules such as hormones, neurotransmitters
and physiologically active substances, which molecules control,
regulate or adjust the functions of living bodies. Each molecule
has its own receptor protein which is specific thereto, whereby the
specificities of individual physiologically active substances,
including specific target cells and organs, specific
pharmacological actions, specific action strength, action time,
etc., are decided. Accordingly, it has been believed that, if G
protein coupled receptor genes or cDNA can be cloned, those will be
helpful not only for the clarification of structure, function,
physiological action, etc. of the G protein coupled receptor but
also for the development of pharmaceuticals by investigating the
substances which act on the receptor. Until now, only several G
protein coupled receptor genes or cDNAs have been cloned but it is
believed that there are many unknown G protein coupled receptor
genes which have not been recognized yet.
[0007] The characteristic feature of the G protein coupled receptor
proteins which have been known up to now is that seven clusters of
hydrophobic amino acid residues are located in the primary
structure and pass through (span) the cell membrane at each region
thereof. It has been known that such a structure is common among
all of the known G protein coupled receptor proteins and further
that the amino acid sequences corresponding to the area where the
protein passes through the membrane (membrane-spanning region or
transmembrane region) and the amino acid sequences near the
membrane-spanning region are often highly conserved among the
receptors. When an unknown protein has such a structure, it is
strongly suggested that said protein is within a category of the G
protein coupled receptor proteins. In addition, some amino acid
residue alinements are common (homologous) and, by taking it as a
characteristic feature, it is further strongly suggested that said
protein is a G protein coupled receptor protein.
[0008] Libert, F, et al. (Science, 244:569-571; 1989) reported a
method for cloning novel receptor genes by means of a polymerase
chain reaction (hereinafter, sometimes referred to as PCR or a PCR
technique) for a synthetic DNA primer which was synthesized based
upon the information of common amino acid sequences obtained from a
comparison among known G protein coupled receptor proteins. Libert,
F. et al. used a pair of synthetic DNA primers corresponding to the
portions of the third and the sixth membrane-spanning regions.
However, in general, the design of primers used for the PCR
regulates the molecular species of DNAs which are to be amplified.
In addition, when a similarity (homology) in the amino acid
sequence level is used as a basis, the use of different codons
affects on the binding (hybridization) of the primer thereby
resulting in a decrease in the amplifying efficiency. Accordingly,
although various novel receptor protein DNAs have been obtained
using said DNA primers, it is not possible to succeed in amplifying
DNAs for all receptor proteins in the prior art.
[0009] Further, the amino acid sequence which is common to from the
first to the seventh membrane-spanning regions among 74 G protein
coupled receptor proteins was reported by William C. Probst, et al.
(DNA and Cell Biology, Vol. 11, No. 1, 1992, pp. 1-20). In this
report, however, there is no suggestion for a method in which DNA
coding for a novel G protein coupled receptor protein is screened
by means of PCR using DNA primers which are complementary to the
DNA coding for those amino acid sequences.
[0010] It would be desirable to develop DNA primers for PCR
techniques which allow selective and efficient screenings of DNAs
coding for the areas (regions) more nearer the full length of novel
G protein coupled receptor proteins by utilizing the common
(homologous) sequence(s) of the G protein coupled receptor protein
or the DNA coding therefor.
[0011] It would also be desirable to develop synthetic DNA primers
corresponding to the portions of the third and the sixth
membrane-spanning regions, said primer being useful in screening
for DNA coding for G protein coupled receptor proteins in more
selective and efficient manner as compared with a series of the
synthetic DNA primers corresponding to the sequences of the third
to the sixth membrane-spanning regions as reported by Libert, F. et
al.
[0012] G protein coupled receptor proteins are important for
investigating substances which control the function of living
organisms and proceeding developments thereof as pharmaceuticals.
Finding and development of candidate compounds for new
pharmaceuticals can be efficiently proceeded by using G protein
coupled receptor proteins and by conducting receptor binding
experiments and evaluating experiments on agonists/antagonists
using intracellular information transmittance systems as indexes.
Especially when the presence of a novel G protein coupled receptor
protein can be clarified, the presence of a substance having a
specific action thereon can be suggested.
[0013] If a novel DNA which codes for a novel G protein coupled
receptor protein can be efficiently screened and isolated, it will
now be possible to proceed with the isolation of DNA having an
entire coding region, the construction of an expression system
therefor and the screening of an acting ligand.
[0014] A hypothalamo-hypophysial system is one of the passages for
controlling, regulating or adjusting the functions of organisms
relying upon interactions of hormones and neurotransmitters with G
protein coupled receptors. In the hypothalamo-hypophysial system,
the secretion of pituitary hormones from the pituitary body
(hypophysis) is regulated by hypothalamic hormones
(hypophysiotropic releasing factors), and the functions of target
cells and organs are controlled by pituitary hormones released into
the blood. Functions which are important for the living body are
regulated through this system, such as maintenance of homeostasis
and control of development and growth of a genital system and an
individual organism. Representative examples of the hypothalamic
hormones include TRH, LH-RH, CRF, GRF, somatostatin, galanin, etc.
Representative examples of the pituitary hormones include TSH,
ACTH, FSH, LH, prolactin, growth hormone, oxytocin, vasopressin,
etc. In particular, the secretion of pituitary hormones is
regulated according to a positive feedback mechanism or a negative
feedback mechanism relied on the hypothalamic hormones and
peripheral hormones secreted from the target endocrine glands. A
variety of receptor proteins present in the pituitary gland play a
major role for regulating the hypothalamo-hypophysial system.
[0015] It has been widely known that these hormones, factors and
receptors are widely distributed in the brain instead of existing
only locally in the hypothalamo-hypophysial system. This fact
suggests that the substances which are called "hypothalamic
hormones" are working as neurotransmitters or neuroregulators in
the central nervous system. It is further considered that these
substances are similarly distributed even in the peripheral tissues
to play the role of important functions. The pancreas plays an
important role of carrying out the carbohydrate metabolism by
secreting not only a digestive fluid but also glucagon and insulin.
Insulin is secreted from the .beta. cells and its secretion is
promoted chiefly by glucose. It has, however, been known that a
variety of receptors exist in the .beta. cells, and the secretion
of insulin is controlled by various factors such as peptide
hormones (galanin, somatostatin, gastric inhibitory polypeptide,
glucagon, amylin, etc.), sugars (mannose, etc.), amino acids, and
neurotransmitters in addition to glucose.
[0016] It has thus been known that in the pituitary gland and in
the pancreas are present receptor proteins for many hormones and
neurotransmitters, said receptor proteins playing important roles
for regulating the functions. As for the galanin and amylin,
however, there has not yet been reported any discovery concerning
the structure of their receptor protein cDNAs. It is not known
whether there exist any unknown receptor proteins or receptor
protein subtypes.
[0017] For substances regulating the functions of the pituitary
gland and pancreas, there exist receptor proteins specific to said
substance on the surfaces of various functional cells of the
pituitary gland and pancreas. The pituitary gland and the pancreas
are associations of a plurality of functional cells, and the
actions of the individual substances are defined by the
distributions of their target receptor proteins among the
functional cells. Accordingly, a substance, in many cases, exhibits
an extensive variety of actions. To comprehend such complex
systems, it is necessary to clarify the relations between the
acting substances and the specific receptor proteins. It is further
necessary to efficiently screen for receptor protein agonists and
antagonists capable of regulating the pituitary gland and pancreas,
to clarify the structures of genes of receptor proteins from the
standpoint of investigating and developing pharmaceuticals, and
further to express them in a suitable expression system.
[0018] By utilizing the fact that a G protein coupled receptor
protein exhibits homology in part of the structure thereof at the
amino acid sequence level, an experiment of looking at DNAs coding
for novel receptor proteins relying upon a polymerase chain
reaction (hereinafter simply referred to as "PCR") has recently
been made.
[0019] In the central nervous system, many receptor proteins such
as dopamine receptor protein, LH-RH receptor protein, neurotensin
receptor protein, opioid receptor protein, CRF receptor protein,
CRF receptor protein, somatostatin receptor protein, galanin
receptor protein, TRH receptor protein, etc. are G protein coupled
receptor proteins, and it has been clarified that ligands to these
receptors exert a variety of effects in the central nervous
system.
[0020] In the immune system, an .alpha.- or a .beta.-chemokine
receptor protein, an MIPI.alpha. receptor protein, an IL-8 receptor
protein, a C5a receptor protein, etc. have been known as such G
protein coupled receptor proteins, and are working as receptor
proteins responsive to immunoregulating substances to play
important roles for regulating the functions of the living body.
There is, for example, an IL-6 receptor protein that acts both in
the above-mentioned central nervous system and in the immune
system. IL-6 is both a ..beta.-cell differentiating factor and a
biologically active factor related to the proliferation and
differentiation of nerve cells.
[0021] It has been widely known that these hormones, factors and
receptor proteins are usually widely distributed up to the
peripheral tissues instead of existing only locally in the central
nervous system and in the immune system and are producing important
functions, respectively. Agonists and antagonists for these
receptor proteins are now being developed as various useful
pharmaceuticals.
[0022] For substances regulating the functions of the central
nervous system and the immune system, there exist receptor proteins
specific to said substance on the surfaces of various functional
cells of the central nervous system and the immune system. The
central nervous system and the immune system are associations of a
plurality of functional cells, and the actions of the individual
substances are defined by the distributions of their target
receptor proteins among the functional cells. Accordingly, a
substance, in many cases, exhibits an extensive variety of actions.
Moreover, there is an example wherein many factors play a part in a
physiological phenomenon. To comprehend such complex systems, it is
necessary to clarify relations between the acting substances and
the specific receptor proteins.
[0023] As discussed herein above, the G protein coupled receptor
protein is present on the cell surface of living body cells and
organs and has a very important role as a target for molecules such
as hormones, neurotransmitters and physiologically active
substances, which molecules control, regulate or adjust the
functions of living body cells and organs.
SUMMARY OF THE INVENTION
[0024] One object of the present invention is to provide novel DNAs
which are useful as DNA primers for a polymerase chain reaction;
methods for amplifying a DNA coding for a G protein coupled
receptor protein using said DNA; screening methods for the DNA
coding for a G protein coupled receptor protein using said DNA;
DNAs obtained by said screening method; and G protein coupled
receptor proteins encoded by the DNA obtained by said screening
method, peptide fragments or segments thereof, modified peptide
derivatives thereof or salts thereof.
[0025] Another object of the present invention is to provide
processes for producing said receptor protein; transformants
capable of expressing said receptor protein; cell membrane
fractions obtained from said transformant; methods for determining
a ligand to the receptor protein; screening methods for a compound
or a salt thereof capable of inhibiting the binding of the ligand
with the receptor protein; kits for said screening method,
pharmaceutical compositions comprising an effective amount of the
inhibitory compound; antibodies against said receptor protein;
immunoassays using said receptor protein or said antibody and use
of said receptor protein and encoding DNA.
[0026] Yet another object of the present invention is to provide
novel G protein coupled receptor proteins which are expressed in
pituitary glands or pancreatic .beta. cells; DNAs comprising a DNA
coding for said G protein coupled receptor protein; processes for
producing said receptor protein; transformants capable of
expressing said receptor protein; cell membrane fractions obtained
from said transformant; methods for determining a ligand to the
receptor protein; screening methods for a compound or a salt
thereof capable of inhibiting the binding of the ligand with the
receptor protein; kits for said screening method, pharmaceutical
compositions comprising the inhibitory compound; antibodies against
said receptor protein; immunoassays using said receptor protein or
said antibody and use of said receptor protein and encoding
DNA.
[0027] Still another object of the present invention is to provide
novel human amygdaloid nucleus-derived G protein coupled receptor
proteins; DNAs comprising a DNA coding for said G protein coupled
receptor protein; processes for producing said receptor protein;
transformants capable of expressing said receptor protein; cell
membrane fractions obtained from said transformant; methods for
determining a ligand to the receptor protein; screening methods for
a compound or a salt thereof capable of inhibiting the binding of
the ligand with the receptor protein; kits for said screening
method, pharmaceutical compositions comprising the inhibitory
compound; antibodies against said receptor protein; immunoassays
using said receptor protein or said antibody and use of said
receptor protein and encoding DNA.
[0028] Yet another object of the present invention is to provide
novel mouse pancreatic .beta. cell line MIN6-derived G protein
coupled receptor proteins; DNAs comprising a DNA coding for said G
protein coupled receptor protein; processes for producing said
receptor protein; transformants capable of expressing said receptor
protein; cell membrane fractions obtained from said transformant;
methods for determining a ligand to the receptor protein; screening
methods for a compound or a salt thereof capable of inhibiting the
binding of the ligand with the receptor protein; kits for said
screening method, pharmaceutical compositions comprising the
inhibitory compound; antibodies against said receptor protein;
immunoassays using said receptor protein or said antibody and use
of said receptor protein and encoding DNA.
[0029] The present inventors have succeeded in synthesizing novel
DNA primers based upon the similarity (homology) with the base
sequences coding for the first membrane-spanning region or the
sixth membrane-spanning region each of known G protein coupled
receptor proteins. It is to be particularly noted that there has
been no report of a DNA primer pair which has been synthesized
paying attention to the similarity with the base sequence coding
for the first and the sixth membrane-spanning region of the known G
protein coupled receptor protein.
[0030] Next the present inventors have succeeded in synthesizing
other novel DNA primers based upon the similarity (homology) with
the base sequences coding for the third or the sixth
membrane-spanning region each of known G protein coupled receptor
proteins. They have also unexpectedly succeeded in efficiently
amplifying DNAs (DNA fragments) coding for G protein coupled
receptor proteins by means of PCR using those DNA primers.
[0031] They have further succeeded in synthesizing novel DNA
primers based upon the similarity (homology) with the base
sequences coding for the second or the seventh membrane-spanning
region each of known G protein coupled receptor proteins; upon the
similarity (homology) with the base sequences coding for first or
the third membrane-spanning region each of known G protein coupled
receptor proteins; and upon the similarity (homology) with the base
sequences coding for the second or the sixth membrane-spanning
region each of known G protein coupled receptor proteins. They have
furthermore and unexpectedly succeeded in efficiently amplifying
DNAs (DNA fragments) coding for G protein coupled receptor proteins
by conducting PCR using those DNA primers.
[0032] Moreover, the present inventors have succeeded in
efficiently cloning full-length DNA coding for said G protein
coupled receptor protein via using amplified DNAs (DNA fragments)
coding for said G protein coupled receptor protein. Thus, they have
found that novel DNA coding for novel G protein coupled receptor
proteins can be isolated, characterized or prepared via conducting
amplifications and analyses of various DNA using said DNA
primers.
[0033] To be more specific, the present inventors have selected
amino acid sequences which are each common to the portion
corresponding to or near the first and the sixth membrane-spanning
region of the known individual G protein coupled receptor proteins
and have designed the DNA primer (SEQ ID NO: 1) coding for the
amino acid sequence common (homologous) to the first
membrane-spanning region and the DNA primer (SEQ ID NO: 2) which is
complementary to the nucleotide sequence coding for the amino acid
sequence common (homologous) to the area near the sixth
membrane-spanning region. Those DNA primers have a different
nucleotide sequence as compared with reported DNA primers (e.g. a
set of synthetic DNA primers corresponding to the third and the
sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO: 61)
as reported by Libert, F. et al.) and such instant primers are
novel and unique.
[0034] Especially for an object of conducting an efficient
elongation reaction in the PCR, the 3'-terminal region of the
instant primers contains the nucleotide sequence which is common
(homologous) among many receptor proteins. Even in other areas, the
similarity (homology) at the nucleotide sequence level (base
sequence level) is utilized for setting the mixed base (nucleotide)
parts wherein their nucleotide sequences (base sequences) are
matched for as many nucleotides (bases) as possible among many DNA
for the receptor proteins. Then the present inventors have
amplified cDNA derived from human brain amygdala, human pituitary
gland and rat brain, found the amplified products as shown in FIG.
17 and, from those products, obtained the G protein coupled
receptor protein cDNAs having the sequence as shown in FIG. 18,
FIG. 19, FIG. 20, FIG. 21, FIG. 22, FIG. 23, FIG. 27, FIG. 29, FIG.
34, FIG. 37, FIG. 40, FIG. 43 or FIG. 46. Among them, the G protein
coupled receptor protein cDNAs having the sequence as shown in FIG.
22, FIG. 23, FIG. 27, FIG. 29, FIG. 34, FIG. 37, FIG. 40, FIG. 43
or FIG. 46 are novel.
[0035] Further, the present inventors have selected the amino acid
sequences common (homologous) to the third and the sixth
membrane-spanning region each of the known G protein coupled
receptor proteins and designed the DNA primers coding for the amino
acid sequence common (homologous) to the third membrane-spanning
region (SEQ ID NO: 3; SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7)
and the DNA primers which are complementary to the nucleotide
sequence coding for the amino acid sequence common (homologous) to
the portion near the sixth membrane-spanning region (SEQ ID NO: 4,
SEQ ID NO: 8 and SEQ ID NO: 9). Again, those DNA primers have
different base sequences from those of the DNA primers previously
reported (e.g., a set of synthetic DNA primers corresponding to the
sequence of the third and the sixth membrane-spanning regions (SEQ
ID NO: 60 and SEQ ID NO: 61) as reported by Libert, F. et al.) and
such instant primers are novel and unique. The present inventors
amplified cDNA derived from the smooth muscles of gastric pylorus
of rabbits using said DNA primer and obtained G protein coupled
receptor protein cDNA having the sequence of FIG. 49 or FIG. 52.
Those cDNAs are novel.
[0036] Still further, the present inventors have selected the amino
acid sequences common (homologous) to the second and the seventh
membrane-spanning region each of the known G protein coupled
receptor proteins and designed the DNA primer coding for the amino
acid sequence common (homologous) to the second membrane-spanning
region (SEQ ID NO: 10) and the DNA primer which is complementary to
the base sequence coding for the amino acid sequence common
(homologous) to the portions near the seventh membrane-spanning
region (SEQ ID NO: 11). Those DNA primers have different base
sequences from those of DNA primers previously reported (e.g., a
set of synthetic DNA primers corresponding to the part of the third
and the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID
NO: 61) as reported by Libert, F. et al) and such instant primers
are novel and unique. The present inventors amplified cDNA derived
from the smooth muscles of gastric pylorus of rabbits using said
DNA primer and obtained G protein coupled receptor protein cDNAs
having each the sequence of FIG. 55, FIG. 56, FIG. 72, or FIG. 73.
Those cDNAs are novel.
[0037] Furthermore, the present inventors have selected the amino
acid sequences common (homologous) to the first and the third
membrane-spanning region each of the known G protein coupled
receptor proteins and designed the DNA primer coding for the amino
acid sequence common (homologous) to the first membrane-spanning
region (SEQ ID NO: 12) and the DNA primer which is complementary to
the base sequence coding for the amino acid sequence common
(homologous) to the portions near the third membrane-spanning
region (SEQ ID NO: 13). Still further, the present inventors have
selected the amino acid sequences common (homologous) to the third
and the sixth membrane-spanning region each of the known G protein
coupled receptor proteins and designed the DNA primers coding for
the amino acid sequence common (homologous) to the third
membrane-spanning region (SEQ ID NO: 10 and SEQ ID NO: 18) and the
DNA primers which are complementary to the base sequence coding for
the amino acid sequence common (homologous) to the parts near the
sixth membrane-spanning region (SEQ ID NO: 15 and SEQ ID NO: 19).
Further, the present inventors have selected the amino acid
sequences common (homologous) to the second and the sixth
membrane-spanning region each of the known G protein coupled
receptor proteins and designed the DNA primer coding for the amino
acid sequence common (homologous) to the second membrane-spanning
region (SEQ ID NO: 16) and the DNA primer which is complementary to
the base sequence coding for the amino acid sequence common
(homologous) to the parts near the sixth membrane-spanning region
(SEQ ID NO: 17). Those DNA primers have different base sequences
from those of DNA primers previously reported (e.g., a set of
synthetic DNA primers corresponding to the part of the third and
the sixth membrane-spanning regions (SEQ ID NO: 60 and SEQ ID NO:
61) as reported by Libert, F. et al) and such instant primers are
novel and unique.
[0038] Still another object of the present invention is to provide
a G protein coupled receptor protein expressed in the pituitary
gland and pancreatic a cells, a DNA comprising a DNA coding for
said protein, a process for producing said protein, and use of said
protein and DNA.
[0039] In order to achieve the above-mentioned aims, the present
inventors have made extensive investigations. As a result, the
present inventors have succeeded in amplifying cDNA derived from
the human pituitary gland and the mouse pancreatic .beta.-cell
strain, MIN 6, with a synthetic DNA primer for efficiently
isolating G protein coupled receptor protein-encoding DNA, and have
forwarded the analysis. Thus, the present inventors have succeeded
in isolating novel human and mouse-derived G protein coupled
receptor protein-encoding cDNAs, in determining the partial
structure thereof, and have considered that these cDNA sequences
are preserved very well in the human and in the mouse, and are
coding for novel receptor proteins for the same ligand. Based upon
the above knowledge, the present inventors have discovered that
these DNAs make it possible to obtain a cDNA having a full length
open reading frame (ORF) of the receptor protein, hence, to produce
the receptor protein. The inventors have further discovered that
the above-mentioned receptor protein obtained when the G protein
coupled receptor protein-encoding cDNA is expressed by a suitable
means permits screening for a ligand to the receptor protein from
the living body or from natural or non-natural compounds under
guidance of data obtainable in receptor coupling tests or
measurements of intracellular second messengers, etc. and further
allows screening for a compound that inhibits the binding of the
ligand and the receptor protein.
[0040] In one embodiment, the present inventors have carried out
PCR amplification of novel human pituitary gland-derived cDNA
fragments as shown in FIGS. 22 and 23, and have subcloned them to
obtain a plasmid vector (p19P2). From analysis of the partial
sequence, it has been clarified that the cDNA has been encoded a
novel receptor protein. The synthetic DNA primers used for
amplifying the cDNA are corresponding to seven hydrophobic clusters
that exist in the known G protein coupled receptor proteins in
common, i.e., corresponding to the first and sixth
membrane-spanning regions among the membrane-spanning domains. The
nucleotide sequence (SEQ ID NO: 29) has been determined from the
primer region at the 5' side (first membrane-spanning domain side)
and has been translated into an amino acid sequence (SEQ ID NO: 24)
[FIG. 221. As a result, the second and third membrane-spanning
domains have been confirmed on the hydrophobicity plotting (FIG.
58). Similarly, the nucleotide sequence (SEQ ID NO: 30) has been
determined from the primer region at the 3' side (sixth
membrane-spanning domain side) and has been translated into an
amino acid sequence (SEQ ID NO: 25) [FIG. 231. As a result, the
presence of the sixth and fifth membrane-spanning domains has been
confirmed on the hydrophobicity plots [FIG. 59]. The size of the
amplified cDNA is about 700 bp which is nearly comparable with the
number of bases between the first membrane-spanning domain and the
sixth membrane-spanning domain of the known G protein coupled
receptor protein.
[0041] G protein coupled receptor proteins exert common property to
some extent at an amino acid sequence level, and are forming one
protein family. Therefore, data base retrieval has been carried out
based upon the amino acid sequence of the subject novel receptor
protein (protein encoded by cDNA included in p19P2). As a result, a
high homology has been exhibited as compared with the known G
protein coupled receptor protein (rat neuropeptide Y receptor
protein encoded by S12863) that is shown in FIG. 60. This fact
tells that the novel receptor protein of the present invention
belongs to the G protein coupled receptor protein family. Moreover,
the data base has been retrieved using, as a template, the amino
acid sequence encoded by the DNA of the invention. It exhibits high
homology to the amino acid sequences of the known G protein coupled
receptor proteins, mouse-derived ligand unknown RP-23 (B40470),
human-derived ligand unknown K-opioid receptor protein (P30098) and
human-derived NK-2 receptor protein (JQ 1059). However, none of
them are in perfect agreement, from which it is learned that a
novel receptor protein had been encoded. The aforementioned
abbreviations in parentheses are reference numbers that are
assigned when they are registered as data to NBRF-PIR/Swiss-PROT
and are, usually, each called "Accession Number".
[0042] Next, by using the novel G protein coupled receptor
protein-encoding cDNA fragment (p19P2) of the present invention, a
cDNA having a full-length open reading frame of the receptor
protein of the present invention has been obtained from human
pituitary gland cDNA libraries. The nucleotide sequence analysis of
a plasmid (phGR3) carrying the cDNA having a full length open
reading frame of the receptor protein shows that the nucleotide
sequence of a coding region of this receptor protein is represented
by SEQ ID NO: 31, and the amino acid sequence deduced therefrom is
represented by SEQ ID NO: 26 [FIG. 34]. Based upon the amino acid
sequence, hydrophobicity plotting has been carried out. The results
are shown in FIG. 36. From the hydrophobicity plotting, it has been
clarified that the receptor protein of the present invention
possessed seven hydrophobic domains. That is, it has been confirmed
that the receptor protein encoded by the cDNA obtained according to
the present invention is a seven transmembrane (membrane-spanning)
G protein coupled receptor protein. An expression of mRNA for
receptor genes encoded by the cDNA of the present invention has
been checked by northern blotting techniques at a mRNA level, and
it has been confirmed that the receptor gene has been expressed in
the human pituitary gland [FIG. 35].
[0043] Thee present inventors have further succeeded in PCR
amplification of a mouse pancreatic .beta. cell strain, MIN6
derived cDNA fragment, and cloning of pG3-2 and pG1-10. Then, based
on the nucleotide sequence of cDNA included in these two plasmid
vectors, the nucleotide sequence shown in FIG. 27 has been derived.
It was learned from the nucleotide sequence that the cDNA encodes a
novel receptor protein. Upon translating the nucleotide sequence
into an amino acid sequence, the presence of the third, fourth,
fifth and sixth membrane-spanning domains has been confirmed on the
hydrophobicity plots [FIG. 28). The size of the amplified cDNA is
about 400 bp which is nearly comparable with the number of bases
between the third membrane-spanning domain and the sixth
membrane-spanning domain of the known G protein coupled receptor
protein. The amino acid sequence has been compared with amino acid
sequences [FIGS. 22 and 23] encoded by the G protein coupled
receptor protein cDNA included in p19P2 cloned from the human
pituitary gland. As a result, homology is more than 95% [FIG. 61].
From this fact, it was estimated that the protein encoded by the
cDNA included in pG3-2 is a mouse type G protein coupled receptor
protein relative to the human-derived one encoded by the cDNA
included in p19P2.
[0044] The present inventors have further amplified a mouse
pancreatic .beta.-cell strain, MIN6-derived cDNA fragment by the
PCR followed by subcloning into a plasmid vector to obtain a clone
(p5S38) having a nucleotide sequence as shown in FIG. 62. From the
nucleotide sequence (SEQ ID NO: 33), it has been clarified that the
cDNA encodes a novel receptor protein. Upon translating the
nucleotide sequence into an amino acid sequence (SEQ ID NO: 28),
the presence of the third, fourth, fifth and sixth
membrane-spanning domains has been confirmed on the hydrophobicity
plots [FIG. 641. The size of the amplified DNA is about 400 bp that
is nearly comparable with the known G protein coupled receptor
protein. The amino acid sequence has been compared with amino acid
sequences [FIGS. 22 and 231 encoded by the G protein coupled
receptor protein cDNA included in p19P2 cloned from the human
pituitary gland and with amino acid sequences of proteins encoded
by pG3-2 and pG1-10 derived from the mouse pancreatic .beta.-cell
strain. As a result, homology is more than 95% to them [FIG. 63].
This fact suggests that the protein encoded by the human-derived
pituitary gland-derived p 19P2, the proteins encoded by the mouse
pancreatic .beta.-cell strain-derived pG3-2 and pG1-10, and the
protein encoded by the mouse pancreatic .beta.-cell strain-derived
p5S38, pertain to a receptor family that recognizes the same
ligand.
[0045] Another object of the present invention is to provide a
novel human amygdaloid nucleus-derived protein coupled receptor
protein, a DNA containing a DNA coding for said G protein coupled
receptor protein, a process for producing said G protein coupled
receptor protein, and use of said protein and DNA.
[0046] The present inventors have synthesized DNA primers for
efficiently isolating a DNA coding for G protein coupled receptor
proteins, amplified an amygdaloid nucleus-derived cDNA with the
above primer, and have analyzed it.
[0047] As a result, the present inventors have succeeded in
isolating, from the human amygdaloid nucleus, a cDNA coding for a
novel G protein coupled receptor protein and have determined its
partial structure. The nucleotide sequence of the isolated cDNA is
preserved very well as compared with that of the mouse
glucocorticoid-induced receptor (hereinafter sometimes referred to
as "GIR") and is considered to be encoding a receptor protein to
the same ligand (Molecular Endocrinology 5:1331-1338, 1991). It is
reputed that, in the mouse, the GIR is a receptor which is induced
by glucocorticoid and expressed in T-cells and is working as a
receptor to immunoregulating factors in the immune system on the
T-cells. The present inventors have succeeded in the isolation of
this human type GIR from the human amygdaloid nucleus. Accordingly,
it is suggested that the isolated GIR is expressed even in the
human central nervous system to carry out some function. From these
facts, it is considered that the receptor protein is strongly
expressed in the human brain and in the immune system and is also
functioning therein. These characterized DNAs allow one to obtain a
cDNA having a full length open reading frame of the receptor and
production of the receptor proteins. The receptor proteins
expressed by a suitable means, furthermore, permit screening for a
ligand to the receptor proteins from the living body or from
natural and non-natural compounds depending on indications
obtainable in receptor protein-binding experiments, measurements of
intracellular second messengers, etc. It further allows one to
screen for compounds capable of inhibiting the binding between the
ligand and the receptor protein.
[0048] To be more specific, the present inventors have amplified,
as a novel human amygdaloid nucleus-derived cDNA, one species, as
shown in FIGS. 29 and 30, by PCR, cloned it, and clarified from the
analysis of a partial sequence thereof that a novel receptor
protein is encoded. The synthetic DNA primers used for amplifying
the cDNA are corresponding to seven hydrophobic clusters that exist
in the G protein coupled receptor proteins in common, i.e.,
corresponding to the first and sixth membrane-spanning regions
among the membrane-spanning domains. The nucleotide sequence has
been determined from the primer region at the 5' side (first
membrane-spanning domain side) and has been translated into an
amino acid sequence. As a result, the second and third
membrane-spanning domains have been confirmed on the hydrophobicity
plotting [FIG. 31). Similarly, the nucleotide sequence has been
determined from the primer region at the 3' side (sixth
membrane-spanning domain side) and has been translated into an
amino acid sequence. As a result, the presence of the fifth and
fourth membrane-spanning domains has been confirmed on the
hydrophobicity plots [FIG. 32). The size of the amplified cDNA is
about 700 bp which is nearly comparable with the number of bases of
the known G protein coupled receptor protein.
[0049] The inventors have further retrieved the data base based on,
as a template, the nucleotide sequence of the isolated DNA and
observed high homology to the DNA that codes for mouse-derived
glucocorticoid-induced receptor protein which is a widely known G
protein coupled receptor protein [FIG. 33]. This result strongly
suggests that the DNA of the present invention is encoding a
human-type receptor protein of GIR.
[0050] Yet another object of the present invention is to provide a
novel mouse pancreatic .beta.-cell strain, MIN6-derived protein
coupled receptor protein, a DNA containing a DNA coding for said G
protein coupled receptor protein, a process for producing said G
protein coupled receptor protein, and use of said protein and DNA.
The present inventors have synthesized DNA primers for efficiently
isolating a DNA coding for G protein coupled receptor proteins,
amplified a mouse pancreatic .beta.-cell strain, MIN6-derived cDNA
with the above primer, and have analyzed it.
[0051] As a result, the present inventors have succeeded in
isolating a mouse-derived cDNA coding for a novel G protein coupled
receptor protein and have determined its partial structure. The
isolated cDNA is homologous to known G protein coupled receptors at
the nucleotide sequence level and at the amino acid sequence level
and is considered to be encoding a novel receptor protein which is
expressed in the mouse pancreas and is also functioning therein.
These characterized DNAs allow one to obtain a cDNA having a full
length open reading frame of the receptor and production of the
receptor proteins. Human-derived cDNAs may be cloned by using, as a
probe, said mouse-derived cDNA. The receptor proteins expressed by
a suitable means, furthermore, permit screening for a ligand to the
receptor protein from the living body or from natural and
non-natural compounds relying on indications obtainable in receptor
protein-binding experiments, measurements of intracellular second
messengers, etc. It further allows one to screen for compounds
capable of inhibiting the binding of the ligand with the receptor
protein.
[0052] To be more specific, the present inventors have amplified,
as a novel mouse pancreatic .beta.-cell strain, MIN6-derived cDNA,
p3H2-17, as shown in FIG. 37, by PCR, cloned it, and clarified from
the analysis of a partial sequence thereof that a novel receptor
protein is encoded. The nucleotide sequence has been translated
into an amino acid sequence. As a result, the presence of the
third, fourth, fifth and sixth membrane-spanning domains has been
confirmed on the hydrophobicity plots [FIG. 38]. The size of the
amplified cDNA is about 400 by which is nearly comparable with that
of the known G protein coupled receptor protein.
[0053] The inventors have retrieved the data base based on, as a
template, the nucleotide sequence of the isolated DNA and observed
30% homology to chicken ATP receptor (P34996), 25% homology to
human somatostatin receptor subtype 3 (A46226), 27% homology to
human somatostatin receptor subtype 4 (JN0605), and 28% homology to
bovine neuropeptide Y receptor (S28787), respectively (FIG. 39),
which are known G protein coupled receptor proteins. The
aforementioned abbreviations in parentheses are reference numbers
that are assigned when they are registered as data to
NBRF-PIR/Swiss-PROT and are, usually, each called "Accession
Number".
[0054] An expression of receptor genes encoded by the cDNA fragment
included in p3H2-17 of the present invention has been checked by
northern blotting techniques at a mRNA level, and it has been
confirmed that the receptor gene has been intensely expressed in
the mouse thymus and spleen. It has been also confirmed that the
receptor gene has been expressed in the mouse brain and pancreas
(FIG. 65).
[0055] Next, by utilizing the information on the nucleotide
sequence of the fragment included in p3H2-17, cDNA encoding a
full-length open reading frame of the mouse pancreatic B -cell
strain, MINE-derived G protein coupled receptor protein of the
present invention has been obtained from mouse thymic and spleenic
poly(A).sup.+RNA by 5RACE (5' rapid amplification of cDNA ends)
techniques (Frohman M. A. et al., Proc. Natl. Acad. Sci. USA,
85:8998-9002 (1988); Belyavsky A. et al., Nucleic Acids Res.,
17:2919-2932 (1989); Edwards J. B. D. M. et al., Nucleic Acids
Res., 19:5227-5232 (1991)) and 3RACE (3' rapid amplification of
cDNA ends) techniques (Frohman M. A. et al., Proc. Natl. Acad. Sci.
USA, 85:8998-9002 (1988); Belyavsky A. et al., Nucleic Acids Res.,
17:2919-2932 (1989)).
[0056] The plasmid (pMAH2-17) carrying cDNA encoding a full-length
open reading frame of the receptor protein of the present invention
has been subjected to sequencing analysis. As a result, the
nucleotide sequence of the region coding for the receptor protein
is represented by SEQ ID NO: 41 and the amino acid sequence deduced
therefrom is represented by SEQ ID NO: 39 (FIG. 69). Based on the
amino acid sequence, hydrophobicity plotting has been carried out.
The results are shown in FIG. 70.
[0057] It has been clarified from the hydrophobicity plotting that
the mouse pancreatic .beta.-cell strain, MIN6-derived receptor
protein of the present invention has seven hydrophobic domains.
Thus, it has been confirmed that the receptor protein encoded by
the cDNA included in pMAH2-17 according to the present invention is
a seven transmembrane G protein coupled receptor protein.
[0058] Data base retrieval has been carried out based on the
full-length amino acid sequence encoded by the cDNA included in
pMAH2-17, and it has been observed that the amino acid sequence has
44.0% homology to mouse P.sub.2Upurinoceptor (P35383) and 38.1%
homology to chicken P.sub.2Ypurinoceptor (P34996), respectively
(FIG. 71), which are known G protein coupled receptor proteins. The
aforementioned abbreviations in parentheses are reference numbers
that are assigned when they are registered as data to
NBRF-PIR/Swiss-PROT and are, usually, each called "Accession
Number". Since the receptor protein encoded by pMAH2-17 is highly
homologous to prinoceptors, it is considered that there are strong
possibility of a subtype within prinoceptor families. Therefore,
the present inventors have carried out an electrophysiological
analysis of the receptor gene in Xenopus oocytes and found
significant inward currents elicited by Xenopus oocytes carrying
the subject receptor gene in response to ATP stimulation (FIG. 75).
As a result, it has been determined that the receptor encoded by
pMAH2-17 is one of the subtypes within prinoceptor families. It has
been discussed and expected that there are a variety of subtypes
among purinoceptors (Pharmac. Ther., Vol. 64, pp. 445-475
(1994).
[0059] All data are supporting that the mouse pancreatic
.beta.-cell strain, MIN6-derived receptor protein of the present
invention (e.g., SEQ ID NO: 38 and SEQ ID NO: 39, or proteins
encoded by pMAH2-17) is a novel purinoceptor subtype which is
clearly distinct from chicken P.sub.2y1 purinoceptor (FEBS LETTERS,
Vol. 324(2), 219-225 (1993)); mouse P.sub.2y2 or P.sub.2u
purinoceptor (Proc. Natl. Acad. Sci. USA, Vol. 90, pp.5113-5117
(1993)); rat P.sub.2u or P.sub.2y2 purinoceptor (Am. J. Respir.
Cell Mol. Biol., Vol. 12, pp. 27-32 (1995)); human P.sub.2u or
P.sub.2y2 purinoceptor (Proc. Natl. Acad. Sci. USA, Vol. 91, pp.
3275-3279 (1994)); and rat P.sub.2x purinoceptor (Nature, Vol.
371.6, pp. 516-519 (1994).
[0060] It is also strongly suggested that agonists and/or
antagonists related to the receptor encoded by pMAH2-17 would be
useful in therapeutic or prophylactic treatment of diseases or
syndromes in connection with purine ligand compounds. It is
expected that the agonists of the receptor encoded by pMAH2-17 are
useful as an immunomodulator or an antitumor agent, in addition
they are useful in therapeutically or prophylactically treating
hypertension, diabetes, cystic fibrosis, etc. It is still expected
that the antagonists of the receptor encoded by pMAH2-17 are useful
as hypotensive agents, analgesics, agents for therapeutically or
prophylactically treating incontinence of urine, etc.
[0061] Another object of the present invention is to provide a
novel human-derived protein coupled receptor protein of prinoceptor
type, a DNA containing a DNA coding for said G protein coupled
receptor protein, a process for producing said G protein coupled
receptor protein, and use of said protein and DNA. The present
inventors have synthesized DNA primers for efficiently isolating a
DNA coding for prinoceptor type G protein coupled receptor proteins
on the basis of the nucleotide sequence of mouse purinoceptor,
amplified a human-derived cDNA with the above primer, and have
analyzed it.
[0062] As a result, the present inventors have succeeded in
isolating a human-derived cDNA coding for a novel G protein coupled
receptor protein and have determined its full-length structure
[FIG. 77]. The isolated cDNA is homologous to mouse G protein
coupled receptor (purinoceptor) at the nucleotide sequence level
and at the amino acid sequence level (87% homology; FIG. 79) and is
considered to be encoding a novel purinoceptor protein. The
receptor proteins expressed by a suitable means, furthermore,
permit screening for a ligand to the receptor protein from the
living body or from natural and non-natural compounds relying on
indications obtainable in receptor protein-binding experiments,
etc. It further allows one to screen for compounds capable of
inhibiting the binding of the ligand with the receptor protein.
[0063] It is also strongly suggested that agonists and/or
antagonists related to the human receptor encoded by phAH2-17 would
be useful in therapeutic or prophylactic treatment of diseases or
syndromes in connection with purine ligand compounds. It is
expected that the agonists of the human receptor are useful as an
immunomodulator or an antitumor agent, in addition they are useful
in therapeutically or prophylactically treating hypertension,
diabetes, cystic fibrosis, etc. It is still expected that the
antagonists of the human receptor are useful as hypotensive agents,
analgesics, agents for therapeutically or prophylactically treating
incontinence of urine, etc.
[0064] Accordingly, one aspect of the present invention is
[0065] (1) DNAs comprising a nucleotide sequence represented by a
SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ
ID NO: 19;
[0066] (2) DNAs according to the above (1) comprising a nucleotide
sequence represented by a SEQ ID NO selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 9;
[0067] (3) DNAs according to the above (1) comprising a nucleotide
sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
[0068] (4) DNAs according to the above (1) wherein the DNA is a
primer for polymerase chain reaction in order to amplify a DNA
coding for a G protein coupled receptor protein;
[0069] (5) a method for amplifying a DNA coding for a G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises:
[0070] (i) carrying out a polymerase chain reaction in the presence
of a mixture of
[0071] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0072] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 5, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO. 6, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 7, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
14, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 16 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 18, and
[0073] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 11, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
17 and DNA primers comprising a nucleotide sequence represented by
SEQ ID NO: 19; or
[0074] (ii) carrying out a polymerase chain reaction in the
presence of a mixture of
[0075] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0076] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0077] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13;
[0078] (6) a method for screening a DNA library for a DNA coding
for a G protein coupled receptor protein, which comprises:
[0079] (i) carrying out a polymerase chain reaction in the presence
of a mixture of
[0080] said DNA library,
[0081] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 5, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 6, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 7, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
14, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 16 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 18, and
[0082] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 11, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
17 and DNA primers comprising a nucleotide sequence represented by
SEQ ID NO: 19,
[0083] to amplify selectively a template DNA coding for G protein
coupled receptor protein, contained in the DNA library; or
[0084] (ii) carrying out a polymerase chain reaction in the
presence of a mixture of
[0085] said DNA library
[0086] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0087] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13,
[0088] to amplify selectively a DNA coding for G protein coupled
receptor protein, contained in the DNA library;
[0089] (7) a DNA coding for a G protein coupled receptor protein,
which is obtained by a method according to the above (5) or (6);
and
[0090] (8) G protein coupled receptor proteins encoded by a DNA
according to the above (7), their peptide segments or fragments and
salts thereof.
[0091] Another specific aspect of the invention is:
[0092] (9) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
first to sixth membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0093] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0094] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0095] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19;
[0096] (10) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
first to seventh membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0097] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0098] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0099] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11;
[0100] (11) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
third to sixth membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof) by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0101] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0102] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 6, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 7, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 14 and DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 18,
and
[0103] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19;
[0104] (12) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
third to seventh membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0105] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0106] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 6, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 7, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 14 and DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 18,
and
[0107] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11;
[0108] (13) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
second to sixth membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0109] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0110] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16, and
[0111] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19;
[0112] (14) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
second to seventh membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof) by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0113] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0114] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16, and
[0115] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11;
[0116] (15) a method for amplifying a DNA coding for G protein
coupled receptor protein (e.g. a region corresponding to from the
first to third membrane-spanning domains of G protein coupled
receptor proteins or other domains thereof) by polymerase chain
reaction techniques, which comprises carrying out a polymerase
chain reaction in the presence of a mixture of
[0117] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0118] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0119] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13;
[0120] (16) a method for amplifying a DNA coding for G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0121] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0122] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1, and
[0123] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2;
[0124] (17) a method for amplifying a DNA coding for G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0125] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0126] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, and
[0127] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 4;
[0128] (18) a method for amplifying a DNA coding for G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0129] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0130] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 6, and
[0131] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 8;
[0132] (19) a method for amplifying a DNA coding for G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0133] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0134] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10, and
[0135] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11;
[0136] (20) a method for amplifying DNA coding for a G protein
coupled receptor protein which comprises
[0137] (i) carrying out a polymerase chain reaction in the presence
of a mixture of
[0138] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0139] at least one DNA primer which is capable of binding with the
3'-side nucleotide sequence of the 31 chain (minus chain) of the
template DNA coding for G protein coupled receptor protein to allow
the extension of the + chain (plus chain) in the 5' .quadrature.3'
direction, said DNA primer being selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 5, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 6, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 7, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
12, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 14, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 18, and
[0140] at least one DNA primer which is capable of binding with the
3'-side nucleotide sequence of the +chain (plus chain) of the
template DNA coding for G protein coupled receptor protein to allow
the extension of the - chain (minus chain) in the 5' 3' direction,
said DNA primer being selected from the group consisting of DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
2, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 9, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 11, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19, or
[0141] (ii) carrying out a polymerase chain reaction in the
presence of a mixture of
[0142] a DNA coding for G protein coupled receptor protein, said
DNA being capable of acting as a template,
[0143] at least one DNA primer which is capable of binding with the
3'-side nucleotide sequence of the - chain (minus chain) of the
template DNA coding for G protein coupled receptor protein to allow
the extension of the + chain (plus chain) in the 5' .quadrature.3'
direction, said DNA primer being selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0144] at least one DNA primer which is capable of binding with the
3'-side nucleotide sequence of the +chain (plus chain) of the
template DNA coding for G protein coupled receptor protein to allow
the extension of the - chain (minus chain) in the 5' .quadrature.3'
direction, said DNA primer being selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13;
[0145] (21) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the first to
sixth membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0146] said DNA library,
[0147] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0148] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19,
[0149] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the first to sixth
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0150] (22) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the first to
seventh membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0151] said DNA library,
[0152] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0153] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11,
[0154] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the first to seventh
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0155] (23) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the third to
sixth membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0156] said DNA library,
[0157] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 6, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 7, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 14 and DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 18,
and
[0158] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19,
[0159] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the third to sixth
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0160] (24) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the third to
seventh membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0161] said DNA library,
[0162] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 6, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 7, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 14 and DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 18,
and
[0163] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11,
[0164] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the third to seventh
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0165] (25) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the second to
sixth membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0166] said DNA library,
[0167] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16, and
[0168] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 15, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 17 and
DNA primers comprising a nucleotide sequence represented by SEQ ID
NO: 19,
[0169] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the second to sixth
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0170] (26) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the second to
seventh membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0171] said DNA library,
[0172] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16, and
[0173] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11,
[0174] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the second to seventh
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0175] (27) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein (e.g. from the first to
third membrane-spanning domains or other domains of G protein
coupled receptor protein), which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0176] said DNA library,
[0177] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1 and DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 12, and
[0178] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13,
[0179] to amplify selectively a template DNA coding for G protein
coupled receptor protein (e.g. from the first to third
membrane-spanning domains or other domains of G protein coupled
receptor protein), contained in the DNA library;
[0180] (28) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0181] said DNA library,
[0182] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 1, and
[0183] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2,
[0184] to amplify selectively the template DNA coding for G protein
coupled receptor protein, contained in the DNA library;
[0185] (29) a method for screening DNA libraries to detect a DNA
coding for G protein coupled receptor protein, which comprises
carrying out a polymerase chain reaction in the presence of a
mixture of
[0186] said DNA library,
[0187] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 3, and
[0188] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 4,
[0189] to amplify selectively a template DNA coding for G protein
coupled receptor protein, contained in the DNA library;
[0190] (30) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0191] said DNA library,
[0192] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 6, and
[0193] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 8,
[0194] to amplify selectively a template DNA coding for G protein
coupled receptor protein, contained in the DNA library;
[0195] (31) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0196] said DNA library,
[0197] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10, and
[0198] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 11,
[0199] to amplify selectively a template DNA coding for G protein
coupled receptor protein, contained in the DNA library; and
[0200] (32) a method for screening DNA libraries according to any
of the above (6), and (21) to (31) wherein said DNA library is
derived from an origin selected from the group consisting of human
tissues and human cells. Examples of such human tissues include
adrenal, umbilical cord, brain, tongue, liver, lymph gland, lung,
thymus, placenta, peritoneum, retina, spleen, heart, smooth muscle,
intestine, vessel, bone, kidney, skin, fetus, mammary gland, ovary,
testis, pituitary gland, pancreas, submandibular gland, spine,
prostate gland, stomach, thyroid gland, trachea (windpipe),
skeletal muscle, uterus, adipose tissue, urinary bladder, cornea,
olfactory bulb, bone marrow, amnion, etc. Examples of such human
cells include nerve cells, epithelial cells, endothelial cells,
leukocytes, lymphocytes, gliacytes, fibroblasts, keratinized cells,
osteoblasts, osteoclasts, astrocytes, melanocytes, various
carcinomas, various sarcomas, various cells derived from the
above-mentioned human tissues.
[0201] Yet another aspect of the present invention is a degenerate
deoxynucleotide which has an oligonucleotide sequence to which a
SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ
ID NO. 19 is assigned.
[0202] Another aspect of the present invention is (33) a G protein
coupled receptor protein comprising an amino acid sequence selected
from the group consisting of amino acid sequences represented by
SEQ ID NO: 24 and/or SEQ ID NO: 25 and substantial equivalents to
the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO:
25; or a salt thereof;
[0203] (34) a G protein coupled receptor protein according to the
above (33) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 26 and substantial equivalents to the amino acid sequence
represented by SEQ ID NO: 26; or a salt thereof;
[0204] (35) a G protein coupled receptor protein comprising an
amino acid sequence selected from the group consisting of an amino
acid sequence represented by SEQ ID NO. 27 and substantial
equivalents to the amino acid sequence represented by SEQ ID NO:
27; or a salt thereof;
[0205] (36) a G protein coupled receptor protein comprising an
amino acid sequence selected from the group consisting of an amino
acid sequence represented by SEQ ID NO: 28 and substantial
equivalents to the amino acid sequence represented by SEQ ID NO:
28; or a salt thereof;
[0206] (37) a G protein coupled receptor protein comprising an
amino acid sequence selected from the group consisting of amino
acid sequences represented by SEQ ID NO: 34 and/or SEQ ID NO: 35
and substantial equivalents to the amino acid sequence represented
by SEQ ID NO: 34 or SEQ ID NO: 35; or a salt thereof;
[0207] (38) a G protein coupled receptor protein comprising an
amino acid sequence selected from the group consisting of an amino
acid sequence represented by SEQ ID NO: 38 and substantial
equivalents to the amino acid sequence represented by SEQ ID NO:
38; or a salt thereof;
[0208] (39) a G protein coupled receptor protein according to the
above (38) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 39 and substantial equivalents to the amino acid sequence
represented by SEQ ID NO: 39; or a salt thereof;
[0209] (40) a G protein coupled receptor protein comprising an
amino acid sequence represented by SEQ ID NO: 56 and substantial
equivalents to the amino acid sequence represented by SEQ ID NO:
56; or a salt thereof;
[0210] (41) a peptide segment or fragment of a G protein coupled
receptor protein according to any of the above (33) to (40), a
modified derivative thereof or a salt thereof;
[0211] (42) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (33);
[0212] (43) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (34);
[0213] (44) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (35);
[0214] (45) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (36);
[0215] (46) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (37);
[0216] (47) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (38);
[0217] (48) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (39);
[0218] (49) a DNA which comprises a nucleotide sequence coding for
a G protein coupled receptor protein of the above (40);
[0219] (50) a DNA of the above (42) comprising a nucleotide
sequence represented by SEQ ID NO: 29 and/or SEQ ID NO: 30;
[0220] (51) a DNA of the above (43) comprising a nucleotide
sequence represented by SEQ ID NO: 31;
[0221] (52) a DNA of the above (44) comprising a nucleotide
sequence represented by SEQ ID NO: 32;
[0222] (53) a DNA of the above (45) comprising a nucleotide
sequence represented by SEQ ID NO: 33;
[0223] (54) a DNA of the above (46) comprising a nucleotide
sequence represented by SEQ ID NO: 36 and/or SEQ ID NO: 37;
[0224] (55) a DNA of the above (47) comprising a nucleotide
sequence represented by SEQ ID NO: 40;
[0225] (56) a DNA of the above (48) comprising a nucleotide
sequence represented by SEQ ID NO: 41;
[0226] (57) a DNA of the above (49) comprising a nucleotide
sequence represented by SEQ ID NO: 57;
[0227] (58) a vector comprising a DNA according to any of the above
(42) to (57);
[0228] (59) a transformant (including a transfectant) carrying a
vector of the above (58);
[0229] (60) a process for producing a G protein coupled receptor
protein or a salt thereof according to any of the above (33) to
(40), which comprises culturing a transformant of the above (59) to
express said G protein coupled receptor protein on the membrane of
the transformant;
[0230] (61) a method for determining a ligand to a G protein
coupled receptor protein according to any of the above (33) to
(40), which comprises contacting
[0231] (i) at least one component selected from the group
consisting of G protein coupled receptor proteins or salts thereof
according to any of the above (33) to (40), peptide fragments or
segments or salts thereof according to the above (41), and mixtures
thereof,
[0232] with
[0233] (ii) at least one compound to be tested;
[0234] (62) a screening method for a compound capable of inhibiting
the binding of a G protein coupled receptor protein according to
any of the above (33) to (40) with a ligand, which comprises
carrying out a comparison between:
[0235] (i) at least one case where said ligand is contacted with at
least one component selected from the group consisting of G protein
coupled receptor proteins or salts thereof according to any of the
above (33) to (40), peptide fragments or segments or salts thereof
according to the above (41), and mixtures thereof,
[0236] and
[0237] (ii) at least one case where said ligand together with a
compound to be tested is contacted with at least one component
selected from the group consisting of G protein coupled receptor
proteins or salts thereof according to any of the above (33) to
(40), peptide fragments or segments or salts thereof according to
the above (41), and mixtures thereof;
[0238] (63) a kit for the screening of one or more compounds
capable of inhibiting the binding of a G protein coupled receptor
protein according to any of the above (33) to (40), with a ligand,
which comprises at least one component selected from the group
consisting of G protein coupled receptor proteins or salts thereof
according to any of the above (33) to (40), peptide fragments or
segments or salts thereof according to the above (41), and mixtures
thereof; and
[0239] (64) an antibody against at least one component selected
from the group consisting of G protein coupled receptor proteins or
salts thereof according to any of the above (33) to (40), peptide
fragments or segments or salts thereof according to the above (41),
and mixtures thereof.
[0240] Yet another aspect of the present invention is
[0241] (65) a G protein coupled receptor protein according to the
above (33) comprising
[0242] (i) an amino acid sequence selected from the group
consisting of an amino acid sequence represented by SEQ ID NO: 24,
amino acid sequences wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) are deleted from the amino acid
sequence of SEQ ID NO: 24, amino acid sequences wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are added to the
amino acid sequence of SEQ ID NO: 24, and amino acid sequences
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 24 are
substituted with one or more other amino acid residues, or/and
[0243] (ii) an amino acid sequence selected from the group
consisting of an amino acid sequence represented by SEQ ID NO: 25,
amino acid sequences wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) are deleted from the amino acid
sequence of SEQ ID NO: 25, amino acid sequences wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are added to the
amino acid sequence of SEQ ID NO: 25, and amino acid sequences
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 25 are
substituted with one or more other amino acid residues,
[0244] or a salt thereof;
[0245] (66) a G protein coupled receptor protein according to the
above (34) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 26, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 26, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 26, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 26 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0246] (67) a G protein coupled receptor protein according to the
above (35) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 27, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 27, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 27, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 27 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0247] (68) a G protein coupled receptor protein according to the
above (36) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 28, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 28, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 28, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 28 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0248] (69) a G protein coupled receptor protein according to the
above (37) comprising
[0249] (i) an amino acid sequence selected from the group
consisting of an amino acid sequence represented by SEQ ID NO: 34,
amino acid sequences wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) are deleted from the amino acid
sequence of SEQ ID NO: 34, amino acid sequences wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are added to the
amino acid sequence of SEQ ID NO: 34, and amino acid sequences
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 34 are
substituted with one or more other amino acid residues, or/and
[0250] (ii) an amino acid sequence selected from the group
consisting of an amino acid sequence represented by SEQ ID NO: 35,
amino acid sequences wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) are deleted from the amino acid
sequence of SEQ ID NO: 35, amino acid sequences wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are added to the
amino acid sequence of SEQ ID NO: 35, and amino acid sequences
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 35 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0251] (70) a G protein coupled receptor protein according to the
above (38) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 38, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO. 38, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 38, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 38 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0252] (71) a G protein coupled receptor protein according to the
above (39) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 39, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 39, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 39, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 39 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0253] (72) a G protein coupled receptor protein according to the
above (40) comprising an amino acid sequence selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO: 56, amino acid sequences wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 56, amino acid sequences wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 56, and amino acid
sequences wherein one or more amino acid residues (preferably from
2 to 30 amino acid residues, more preferably from 2 to 10 amino
acid residues) in the amino acid sequence of SEQ ID NO: 56 are
substituted with one or more other amino acid residues, or a salt
thereof;
[0254] (73) a method for determining a ligand according to the
above (61) wherein said ligand is selected from the group
consisting of angiotensin, bombesin, canavinoid, cholecystokinin,
glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine,
vasopressin, oxytocin, VIP (vasoactive intestinal and related
peptides), somatostatin, dopamine, motilin, amylin, bradykinin,
CGRP (calcitonin gene related peptides), adrenomedullin,
leukotriene, pancreastatin, prostaglandin, thromboxanes, adenosine,
adrenaline, .alpha.- and .beta.-chemokine (IL-8, GRO.alpha.,
GRO.beta., GRO.gamma., NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1,
HC14, MCP-3, 1-309, MIP1.alpha., MIP-1.beta., RANTES, etc.),
endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic
polypeptides and galanin;
[0255] (74) a method for the screening of a compound or a salt
thereof capable of inhibiting the binding of a ligand with a G
protein coupled receptor protein according to any of the above (33)
to (40), which comprises measuring amounts of a labeled ligand
bound to the said G protein coupled receptor protein in at least
two cases:
[0256] (i) where the labeled ligand is contacted with at least one
component selected from the group consisting of G protein coupled
receptor proteins or salts thereof according to any of the above
(33) to (40), peptide fragments or segments or salts thereof
according to the above (41), and mixtures thereof, and
[0257] (ii) where the labeled ligand together with a compound to be
tested is contacted with at least one component elected from the
group consisting of G protein coupled receptor proteins or salts
thereof according to any of the above (33) to (40), peptide
fragments or segments or salts thereof according to the above (41),
and mixtures thereof,
[0258] and comparing the measured amounts of the labeled ligand;
(75) a method for the screening of a compound or a salt thereof
capable of inhibiting the binding of a ligand with a G protein
coupled receptor protein according to any of the above (33) to
(40), which comprises measuring amounts of a labeled ligand bound
to a cell comprising the said G protein coupled receptor protein in
at least two cases:
[0259] (i) where the labeled ligand is contacted with the said
cell, and
[0260] (ii) where the labeled ligand together with a compound to be
tested is contacted with the said cell,
[0261] and comparing the measured amounts of the labeled
ligand;
[0262] (76) a method for the screening of a compound or a salt
thereof capable of inhibiting the binding of a ligand with a G
protein coupled receptor protein according to any of the above (33)
to (40), which comprises measuring amounts of a labeled ligand
bound to a membrane fraction of a cell comprising the said G
protein coupled receptor protein in at least two cases:
[0263] (i) where the labeled ligand is contacted with the said
membrane fraction, and
[0264] (ii) where the labeled ligand together with a compound to be
tested is contacted with the membrane fraction,
[0265] and comparing the measured amounts of the labeled
ligand;
[0266] (77) a method for the screening of a compound or a salt
thereof capable of inhibiting the binding of a ligand with a G
protein coupled receptor protein according to any of the above (33)
to (40), which comprises measuring amounts of a labeled ligand
bound to said G protein coupled receptor protein in at least two
cases:
[0267] (i) where the labeled ligand is contacted with a G protein
coupled receptor protein according to any of the above (33) to (40)
which is expressed on the membrane of a transformant according to
the above (59) during incubation of the transformant, and
[0268] (ii) where the labeled ligand together with a compound to be
tested is contacted with the G protein coupled receptor protein
according to any of the above (33) to (40) which is expressed on
the membrane of a transformant according to the above (59) during
incubation of the transformant,
[0269] and comparing the measured amounts of the labeled
ligand;
[0270] (78) a method for the screening of a compound or a salt
thereof capable of inhibiting the binding of a ligand with a G
protein coupled receptor protein according to any of the above (33)
to (40), which comprises measuring G protein coupled receptor
protein-mediated cell-stimulating activities in at least two
cases:
[0271] (i) where a compound capable of activating the G protein
coupled receptor protein according to any of the above (33) to (40)
is contacted with a cell comprising the said G protein coupled
receptor protein, and
[0272] (ii) where the compound capable of activating the G protein
together with a compound to be tested is contacted with the cell
comprising the said G protein coupled receptor protein,
[0273] and comparing the measured cell-stimulating activities;
[0274] (79) a method for the screening of a compound or a salt
thereof capable of inhibiting the binding of a ligand with a G
protein coupled receptor protein according to any of the above (33)
to (40), which comprises measuring G protein coupled receptor
protein-mediated cell-stimulating activities in at least two
cases:
[0275] (i) where a compound capable of activating the G protein
coupled receptor protein according to any of the above (33) to (40)
is contacted with a G protein coupled receptor protein according to
any of the above (33) to (40) which is expressed on the membrane of
a transformant according to the above (59) during incubation of the
transformant, and
[0276] (ii) where the compound capable of activating the G protein
together with a compound to be tested is contacted with the G
protein coupled receptor protein according to any of the above (33)
to (40) which is expressed on the membrane of a transformant
according to the above (59) during incubation of the
transformant,
[0277] and comparing the measured cell-stimulating activities;
[0278] (80) a method according to the above (78) or (79) wherein
said compound capable of activating the G protein coupled receptor
protein according to any of the above (33) to (40) is selected from
the group consisting of angiotensin, bombesin, canavinoid,
cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y,
opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal
and related peptides), somatostatin, dopamine, motilin, amylin,
bradykinin, CGRP (calcitonin gene related peptides),
adrenomedullin, leukotriene, pancreastatin, prostaglandin,
thromboxane, adenosine, adrenaline, .alpha.- and .beta.-chemokine
(IL-8, GRO.alpha., GRO.beta., GRO.gamma., NAP-2, ENA-78, PF4, IP10,
GCP-2, MCP-1, HC14, MCP-3, 1-309, MIP1.alpha., MIP-1.beta., RANTES,
etc.), endothelin, enterogastrin, histamine, neurotensin, TRH,
pancreatic polypeptides and galanin;
[0279] (81) a compound which is determined through a method
according to any of the above (62) and (74) to (80) or a salt
thereof;
[0280] (82) a pharmaceutical composition comprising an effective
amount of a compound according to the above (81) or a salt
thereof;
[0281] (83) a screening kit according to the above (63), comprising
a cell comprising a G protein coupled receptor protein according to
any of the above (33) to (40);
[0282] (84) a screening kit according to the above (63), comprising
a membrane fraction derived from a cell comprising a G protein
coupled receptor protein according to any of the above (33) to
(40);
[0283] (85) a screening kit according to the above (63), comprising
a cell of the (59) or (109) mentioned herein below;
[0284] (86) a screening kit according to the above (63), comprising
a membrane fraction derived from a cell of the (59) or (109);
[0285] (87) a compound which is determined by means of a screening
kit according to any of the above (63) and (83) to (86) or a salt
thereof;
[0286] (88) a pharmaceutical composition comprising an effective
amount of a compound according to the above (87) or a salt thereof;
and
[0287] (89) a method for measuring at least one component selected
from the group consisting of G protein coupled receptor proteins or
salts thereof according to any of the above (33) to (40), peptide
fragments or segments or salts thereof according to the above (41),
and mixtures thereof, which comprises contacting an antibody
according to the above (64) with the component selected from the
group consisting of G protein coupled receptor proteins or salts
thereof according to any of the above (33) to (40), peptide
segments or salts thereof according to the above (41), and mixtures
thereof.
[0288] Still another aspect of the present invention is
[0289] (90) a ligand to a G protein coupled receptor protein
according to any of the above (33) to (40), which is determined
through the following step of:
[0290] contacting (i) at least one component selected from the
group consisting of G protein coupled receptor proteins or salts
thereof according to any of the above (33) to (40), peptide
fragments or segments or salts thereof according to the above (41),
and mixtures thereof,
[0291] with (ii) at least one compound to be examined; and
[0292] (91) a compound capable of inhibiting the binding of a G
protein coupled receptor protein according to any of the above (33)
to (40) with a ligand, which is determined through carrying out a
comparison between:
[0293] (i) at least one case where said ligand is contacted with at
least one component selected from the group consisting of G protein
coupled receptor proteins or salts thereof according to any of the
above (33) to (40), peptide fragments or segments or salts thereof
according to the above (41), and mixtures thereof, and
[0294] (ii) at least one case where said ligand together with a
compound to be tested is contacted with at least one component
selected from the group consisting of G protein coupled receptor
proteins or salts thereof according to any of the above (33) to
(40), peptide fragments or segments or salts thereof according to
the above (41), and mixtures thereof.
[0295] Another aspect of the present invention is
[0296] (92) a recombinant G protein coupled receptor protein and a
salt thereof which is obtained by the expression of a DNA according
to any of the above (42) to (57), or a modified or fragmented
derivative thereof;
[0297] (93) a method for amplifying a DNA coding for G protein
coupled receptor protein by polymerase chain reaction techniques,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0298] (1) a DNA coding for G protein coupled receptor protein,
said DNA being capable of acting as a template, and
[0299] (2) at least one DNA primer selected from the group
consisting of DNA primers comprising either SEQ ID NO: 1 or SEQ ID
NO: 2; and
[0300] (94) a method for screening DNA libraries for a DNA coding
for G protein coupled receptor protein, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0301] (1) said DNA library, and
[0302] (2) at least one DNA primer selected from the group
consisting of DNA primers comprising either SEQ ID NO: 1 or SEQ ID
NO: 2,
[0303] to amplify selectively the DNA coding for G protein coupled
receptor protein, contained in the DNA library.
[0304] Yet another aspect of the present invention is
[0305] (95) a monoclonal antibody against at least one component
selected from the group consisting of G protein coupled receptor
proteins or salts thereof according to any of the above (33) to
(40), peptide fragments or segments or salts thereof according to
the above (41), and mixtures thereof;
[0306] (96) a preparation of purified polyclonal antibodies against
at least one component selected from the group consisting of G
protein coupled receptor proteins or salts thereof according to any
of the above (33) to (40), peptide fragments or segments or salts
thereof according to the above (41), and mixtures thereof;
[0307] (97) an immunoassay for detecting a G protein coupled
receptor protein which comprising
[0308] (i) incubating a sample to be tested with an antibody
according to the above (64) to allow formation of an
antigen-antibody complex; and
[0309] (ii) detecting an antigen-antibody complex formed in step
(i); and
[0310] (98) an immunoassay for detecting antibodies against a G
protein coupled receptor protein which comprising
[0311] (i) incubating a sample to be tested with at least one
component selected from the group consisting of G protein coupled
receptor proteins or salts thereof according to any of the above
(33) to (40), peptide fragments or segments or salts thereof
according to the above (41), and mixtures thereof to allow
formation of an antigen-antibody complex; and
[0312] (ii) detecting an antigen-antibody complex formed in step
(a).
[0313] Still another aspect of the present invention is
[0314] (99) an antisense DNA or RNA which comprises a nucleotide
sequence complementary to at least a portion of a DNA according to
any of the above (42) to (57), said antisense DNA or RNA being
hybridizable to said DNA according to any of the above (42) to
(57);
[0315] (100) an antisense DNA or RNA according to the above (99)
wherein said antisense DNA or RNA comprises the 5' end hairpin
loop, 5' end 6-base-pair repeat, 5' end untranslated region,
protein translation initiation site or codon, ORF translation
initiation site or codon, 3'-untranslated region, 3' end palindrome
region, or 3' end hairpin loop of a G protein coupled receptor
protein DNA according to any of the above (42) to (57);
[0316] (101) an antisense DNA or RNA according to the above (99) in
a pharmaceutically acceptable carrier;
[0317] (102) an antisense DNA or RNA according to the above (99)
comprising from 2 to 50 nucleotides;
[0318] (103) a method for modulating the activity of a G protein
coupled receptor protein comprising contacting cells expressing the
G protein coupled receptor protein with an antisense DNA or RNA
according to the above (99);
[0319] (104) a method for producing an antibody against a G protein
coupled receptor protein according to any of the above (33) to
(40), which comprises administering to an individual at least one
component selected from the group consisting of G protein coupled
receptor proteins or salts thereof according to any of the above
(33) to (40), peptide fragments or segments or salts thereof
according to the above (41), and mixtures thereof; and
[0320] (105) a method for producing a hybridoma which produces a
monoclonal antibody against a G protein coupled receptor protein
according to any of the above (33) to (40), which comprises
[0321] (i) immunizing an individual with at least one component
selected from the group consisting of G protein coupled receptor
proteins or salts thereof according to any of the above (33) to
(40), peptide fragments or segments or salts thereof according to
the above (41), and mixtures thereof;
[0322] (ii) immortalizing antibody producing cells from the
immunized individual;
[0323] (iii) selecting an immortal cell which produces antibodies
reactive with the G protein coupled receptor protein; and
[0324] (iv) growing said immortal cell.
[0325] Yet another aspect of the present invention is
[0326] (106) a PCR screening kit for a DNA (or nucleotide sequence)
coding for G protein coupled receptor protein in a DNA library
which comprises
[0327] (i) at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 1, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 3, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 5, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
7, DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 10, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 16 and DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 18, and
[0328] at least-one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 2, DNA primers comprising a nucleotide sequence represented
by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 8, DNA primers comprising a nucleotide
sequence represented by SEQ ID NO; 9, DNA primers comprising a
nucleotide sequence represented by SEQ ID NO: 11, DNA primers
comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA
primers comprising a nucleotide sequence represented by SEQ ID NO:
17 and DNA primers comprising a nucleotide sequence represented by
SEQ ID NO: 19; or
[0329] (ii) at least one DNA primer selected from the group
consisting of DNA primers comprising a nucleotide sequence
represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide
sequence represented by SEQ ID NO: 12, and
[0330] at least one DNA primer selected from the group consisting
of DNA primers comprising a nucleotide sequence represented by SEQ
ID NO: 13;
[0331] (107) a vector comprising the DNA according to the above
(7);
[0332] (108) an expression system comprising an open reading frame
(ORF) of DNA derived from a G protein coupled receptor protein DNA
according to any of the above (7) and (42) to (57), wherein the ORF
is operably linked to a control sequence compatible with a desired
host cell;
[0333] (109) a transformant (including a transfectant) carrying a
vector of the above (107) or an expression system of the above
(108);
[0334] (110) a process for producing a G protein coupled receptor
protein or a salt thereof, which comprises culturing the
transformant of the above (109) to express said G protein coupled
receptor protein on the membrane of the transformant;
[0335] (111) a method for expressing a polypeptide of G protein
coupled receptor protein, comprising:
[0336] (a) providing a transformant of the above (59) or (109);
and
[0337] (b) incubating the transformant under conditions which allow
expression of the polypeptide of G protein coupled receptor
protein;
[0338] (112) a method for preparing a transformant according to the
above (59) or (109), comprising;
[0339] (a) providing a host cell capable of transformation;
[0340] (b) providing a vector according to the above (58) or (107)
or an expression system according to the above (108); and
[0341] (c) incubating (a) with (b) under conditions which allow
transformation of the host cell with the vector or the expression
system;
[0342] (113) a pharmaceutical composition according to the above
(82) or (88), comprising an effective amount of a compound
according to the above (81) or (87) or a pharmaceutically
acceptable salt thereof in admixture with a pharmaceutically
acceptable carrier, excipient or diluent;
[0343] (114) the pharmaceutical composition according to the above
(82) or (88), for inhibiting the binding of a G protein coupled
receptor protein according to the present invention with a
ligand;
[0344] (115) a method for inhibiting the binding of a G protein
coupled receptor protein according to the present invention with a
ligand in a medium which comprises contacting an effective amount
of a compound according to the above (81) or (87) or a salt thereof
with said medium;
[0345] (116) a method for modulating the activity of a G protein
coupled receptor protein comprising contacting cells expressing the
G protein coupled receptor protein with a an effective amount of a
compound according to the above (81) or (87) or a salt thereof;
[0346] (117) the ligand according to the above (90) being labeled
with a detectable reporter;
[0347] (118) the antibody according to the above (64) wherein the
antibody is labeled with a detectable reporter;
[0348] (119) a pharmaceutical composition for controlling an
expression of G protein coupled receptor protein, which comprises
an effective amount of the antisense DNA according to the above
(99), and
[0349] (120) a culture product produced by a transformant according
to the above (59) or (109).
[0350] Yet another aspect of the present invention is
[0351] (121) a DNA according to the above (1) wherein the DNA is an
oligonucleotide having from 8 to 60 base residues;
[0352] (122) a DNA according to the above (1) wherein the DNA is
synthetic;
[0353] (123) a DNA (or nucleotide sequence) coding for a G protein
coupled receptor protein or a fragment thereof, which is obtained
through the method according to any of the above (5) to (32);
[0354] (124) a DNA (or nucleotide sequence) according to the above
(123), wherein said G protein coupled receptor protein is selected
from the group consisting of angiotensin receptor, bombesin
receptor, canavinoid receptor, cholecystokinin receptor, glutamine
receptor, serotonin receptor, melatonin receptor, neuropeptide Y
receptor, opioid receptor, purine receptor, vasopressin receptor,
oxytocin receptor, VIP receptor (vasoactive intestinal and related
peptide receptor), somatostatin receptor, dopamine receptor,
motilin receptor, amylin receptor, bradykinin receptor, CGRP
receptor (calcitonin gene related peptide receptor), adrenomedullin
receptor, leukotriene receptor, pancreastatin receptor,
prostaglandin receptor, thromboxane receptor, adenosine receptor,
adrenaline receptor, .alpha.- and .beta.-chemokine receptor
including IL-8, GRO.alpha., GRO.beta., GRO.gamma., NAP-2, ENA-78,
PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, 1-309, MIP1.alpha.,
MIP-1.beta., and RANTES receptors, endothelin receptor,
enterogastrin receptor, histamine receptor, neurotensin receptor,
TRH receptor, pancreatic polypeptide receptor, and galanin
receptor; and
[0355] (125) a culture product produced by a transformant according
to the above (59) or (109).
[0356] As used herein the term "substantial equivalent(s)" means
that the activity of the protein, e.g., nature of the ligand
binding activity, and physical characteristics are substantially
the same. Substitutions, deletions or insertions of amino acids
often do not produce radical changes in the physical and chemical
characteristics of a polypeptide, in which case polypeptides
containing the substitution, deletion, or insertion would be
considered to be substantially equivalent to polypeptides lacking
the substitution, deletion, or insertion. Substantially equivalent
substitutes for an amino acid within the sequence may be selected
from other members of the class to which the amino acid belongs.
The non-polar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine, The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0357] FIG. 1 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (HS-1) having a nucleotide sequence
represented by SEQ ID NO: 1 with the nucleotide sequence each of
other G protein coupled receptor protein-encoding cDNAs and
genes,
[0358] FIG. 2 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (HS-2)
having a nucleotide sequence represented by SEQ ID NO: 2 with the
nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0359] FIG. 3 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (3A) having a nucleotide sequence
represented by SEQ ID NO: 5 or 5' side synthetic DNA primers (3B)
having a nucleotide sequence represented by SEQ ID NO: 6 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0360] FIG. 4 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (3C) having a nucleotide sequence
represented by SEQ ID NO: 7 or 5' side synthetic DNA primers (3D)
having a nucleotide sequence represented by SEQ ID NO: 3 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0361] FIG. 5 depicts the community (homology) of the sequence (6A)
which is complementary to 3' side synthetic DNA primers having a
nucleotide sequence represented by SEQ ID NO: 8 or the nucleotide
sequence (6B) which is complementary to 3' side synthetic DNA
primers having a nucleotide sequence represented by SEQ ID NO: 9
relative to the nucleotide sequence each of other G protein coupled
receptor protein-encoding cDNAs and genes.
[0362] FIG. 6 depicts the community (homology) of the sequence
(6C).which is complementary to 3' side synthetic DNA primers having
a nucleotide sequence represented by SEQ ID NO: 4 relative to the
nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0363] FIG. 7 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (T2A) having a nucleotide sequence
represented by SEQ ID NO: 10 with the nucleotide sequence each of
other G protein coupled receptor protein-encoding cDNAs and
genes.
[0364] FIG. 8 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (T7A)
having a nucleotide sequence represented by SEQ ID NO: 11 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0365] FIG. 9 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (TM1-A2) having a nucleotide sequence
represented by SEQ ID NO: 12 relative to the nucleotide sequence
each of other G protein coupled receptor protein-encoding cDNAs and
genes.
[0366] FIG. 10 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (TM3-B2)
having a nucleotide sequence represented by SEQ ID NO: 13 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0367] FIG. 11 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (TM3-C2) having a nucleotide sequence
represented by SEQ ID NO: 14 relative to the nucleotide sequence
each of other G protein coupled receptor protein-encoding cDNAs and
genes.
[0368] FIG. 12 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (TM6-E2)
having a nucleotide sequence represented by SEQ ID NO: 15 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0369] FIG. 13 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (TM2F18) having a nucleotide sequence
represented by SEQ ID NO: 16 relative to the nucleotide sequence
each of other G protein coupled receptor protein-encoding cDNAs and
genes.
[0370] FIG. 14 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (TM6R21)
having a nucleotide sequence represented by SEQ ID NO: 17 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0371] FIG. 15 depicts the community (homology) of the sequence of
5' side synthetic DNA primers (S3A) having a nucleotide sequence
represented by SEQ ID NO: 18 relative to the nucleotide sequence
each of other G protein coupled receptor protein-encoding cDNAs and
genes.
[0372] FIG. 16 depicts the community (homology) of the sequence
which is complementary to 3' side synthetic DNA primers (S6A)
having a nucleotide sequence represented by SEQ ID NO: 19 relative
to the nucleotide sequence each of other G protein coupled receptor
protein-encoding cDNAs and genes.
[0373] FIG. 17 is the 1.2% agarose gel electrophoresis profile of
cDNA products each obtained from human brain amygdala (1, 2, 7),
human pituitary body (3, 4, 8) and rat brain (5, 6, 9) by PCR
amplification using the synthetic DNA primers having a nucleotide
sequence represented by SEQ ID NO: 1 and the synthetic DNA primers
having a nucleotide sequence represented by SEQ ID NO: 2, wherein
lanes 1 to 6 show the results of when PCR is carried out under
severe conditions as disclosed in Examples, lanes 7 to 9 show the
results of when PCR is carried out under mild conditions, and M
denotes a size marker which is obtained by cutting .lambda.-phage
DNA with restriction enzyme, EcoT14I.
[0374] FIG. 18 shows the nucleotide sequence determined by
sequencing of clone A58, with a T7 primer wherein the clone A58 is
obtained by amplifying human brain amygdala-derived cDNA by PCR
under mild conditions and subcloning it to pCR.TM. II.
[0375] FIG. 19 shows the nucleotide sequence determined by
sequencing of clone A58 with an SP6 primer.
[0376] FIG. 20 shows the nucleotide sequence determined by
sequencing of clone 57-A-2 by using a -21M 13 primer wherein the
clone 57-A-2 is obtained by amplifying human brain amygdala-derived
cDNA by PCR under severe conditions and subcloning it to pCR.TM.
II.
[0377] FIG. 21 shows the nucleotide sequence determined by
sequencing of clone B54 with a T7 primer wherein the clone B54 is
obtained by amplifying rat whole brain-derived cDNA by PCR under
mild conditions and subcloning it to pCR TM II.
[0378] FIG. 22 illustrates the nucleotide sequence of the human
pituitary gland-derived G protein coupled receptor protein cDNA
fragment included in the cDNA clone p19P2 isolated by PCR using a
human pituitary gland-derived cDNA and the amino acid sequence
encoded thereby, wherein the primer used for sequencing is -21M 13,
and the underlined part corresponds to the synthetic primer.
[0379] FIG. 23 illustrates the nucleotide sequence of the human
pituitary gland-derived G protein coupled receptor protein cDNA
fragment included in the cDNA clone p19P2 isolated by PCR using a
human pituitary gland-derived cDNA and the amino acid sequence
encoded thereby, wherein the primer used for sequencing is M 13RV-N
(Takara, Japan), and the underlined part corresponds to the
synthetic primer.
[0380] FIG. 24 is the partial hydrophobicity plotting profile of
the protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p19P2, prepared
based upon the amino acid sequence shown in FIG. 22.
[0381] FIG. 25 is the partial hydrophobicity plotting profile of
the protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p19P2, prepared
based upon the amino acid sequence shown in FIG. 23.
[0382] FIG. 26 shows the partial amino acid sequence (p19P2) of the
protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p19P2, as shown
in FIGS. 22 and 23, relative to the known G protein coupled
receptor protein, S12863, wherein reverse amino acid residues are
in agreement, the 1st to 99th amino acid residues of the p19P2
sequence correspond to the 1st to 99th amino acid residues in FIG.
22, and the 156th to 230th amino acid residues thereof correspond
to the 1st to 68th amino acid residues in FIG. 23.
[0383] FIG. 27 is the nucleotide sequence of the MIN6-derived G
protein coupled receptor protein cDNA fragment derived based upon
the nucleotide sequences of the MIN6-derived G protein coupled
receptor protein cDNA fragments each included in the cDNA clones,
pG3-2 and pG1-10, isolated by PCR using a MIN6-derived cDNA and the
amino acid sequence encoded thereby, wherein the underlined parts
corresponds to the synthetic primers.
[0384] FIG. 28 is the partial hydrophobicity plotting profile of
the MIN6-derived G protein coupled receptor protein, prepared based
upon the partial amino acid sequence shown in FIG. 27.
[0385] FIG. 29 is the partial nucleotide sequence of the novel
receptor protein cDNA clone, p63A2, obtained from the human
amygdaloid nucleus by PCR amplification and the amino acid
sequence, encoded thereby, wherein the underlined part corresponds
to the synthetic primer.
[0386] FIG. 30 is the partial nucleotide sequence of the novel
receptor protein cDNA clone, p63A2, obtained from the human
amygdaloid nucleus by PCR amplification and the amino acid sequence
encoded thereby, wherein the underlined part corresponds to the
synthetic primer.
[0387] FIG. 31 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 29, suggesting the
presence of hydrophobic domains as designated by 1 to 3.
[0388] FIG. 32 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 30, suggesting the
presence of hydrophobic domains as designated by 4 to 6.
[0389] FIG. 33 is the partial amino acid sequence (p63A2) of the
protein encoded by the novel receptor protein cDNA fragment
included in p63A2, relative to the partial amino acid sequence of
the G protein coupled receptor protein (P30731) expressed and
induced by a mouse T cell-derived glucocorticoid, wherein reverse
amino acid residues are in agreement.
[0390] FIG. 34 is the whole nucleotide sequence of the human
pituitary gland-derived G protein coupled receptor protein cDNA,
included in the cDNA clone, phGR3, isolated from the human-derived
cDNA library by plaque hybridization using an DNA insert in the
p19P2 as a probe, and the amino acid sequence encoded thereby.
[0391] FIG. 35 is the northern blotting profile of the human
pituitary gland mRNA of the receptor gene encoded by the human
pituitary gland-derived cDNA clone, phGR3.
[0392] FIG. 36 is the hydrophobicity plotting profile of the
protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA included in phGR3, prepared based
upon the amino acid sequence shown in FIG. 34.
[0393] FIG. 37 is the partial nucleotide sequence of the novel
receptor protein cDNA clone, p3H2-17, obtained from mouse
pancreatic .beta.-cell strain, MIN6, by PCR amplification and the
amino acid sequence encoded thereby, wherein the underlined part
corresponds to the synthetic primer used for the PCR
amplification.
[0394] FIG. 38 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 37, suggesting the
presence of hydrophobic domains as designated by 3 to 6.
[0395] FIG. 39 is the partial amino acid sequence encoded by the
novel receptor protein cDNA included in p3H2-17, relative to the
partial amino acid sequence each of chicken ATP receptor protein
(P34996), human somatostatin receptor subtype 3 protein (A46226),
human somatostatin receptor subtype 4 protein (JN0605) and bovine
neuropeptide Y receptor protein (S28787), wherein reverse amino
acid residues are in agreement.
[0396] FIG. 40 is the partial nucleotide sequence of the novel
receptor protein cDNA clone, p3H2-34, obtained from mouse
pancreatic .beta.-cell strain, MIN6, by PCR amplification and the
amino acid sequence encoded thereby, wherein the underlined parts
correspond to the synthetic primers used for the PCR
amplification.
[0397] FIG. 41 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 40, wherein the
axis of ordinate represents an index of hydrophobicity, the axis of
abscissa represents the number of amino acids and numerals 3 to 6
represent the presence of hydrophobic domains.
[0398] FIG. 42 is the partial amino acid sequence encoded by the
novel receptor protein cDNA included in p3H2-34, relative to the
partial amino acid sequence each of human somatostatin receptor
subtype 4 protein (JN0605), human somatostatin receptor subtype 2
protein (B41795) and rat-derived ligand unknown receptor protein
(A39297), wherein reverse amino acid residues are in agreement.
[0399] FIG. 43 is the nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in the novel receptor protein cDNA
clone, pMD4, obtained from rabbit gastropyrolic part smooth muscles
by PCR amplification, and the amino acid sequence encoded thereby,
wherein the underlined parts correspond to the synthetic primers
used for the PCR amplification.
[0400] FIG. 44 is the hydrophobicity plotting profile of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMD4, prepared based upon the amino acid sequence shown
in FIG. 35, wherein numerals 1 to 3 suggest the presence of
hydrophobic domains.
[0401] FIG. 45 is the partial amino acid sequence (pMD4) of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMD4 as shown in FIG. 43, relative to the known G
protein coupled receptor protein, rat ligand unknown receptor
protein (A35639), wherein reverse amino acid residues are in
agreement, the 1st to 88th amino acid residues of the pMD4 sequence
correspond to the 1st to 88th amino acid residues in FIG. 43.
[0402] FIG. 46 shows the nucleotide sequence of the mouse-derived
galanin receptor protein cDNA clone, pMGR20, which has been cloned
with, as a probe, the cDNA insert in p3H2-34 and the amino acid
sequence encoded thereby.
[0403] FIG. 47 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 46, wherein the
axis of ordinate represents an index of hydrophobic property, the
axis of abscissa represents the number of amino acids, and numerals
1 to 7 represent the presence of hydrophobic domains.
[0404] FIG. 48 is the amino acid sequence (MOUSEGALRECE) of the
mouse-derived galanin receptor protein encoded by pMGR20, relative
to the amino acid sequence (HUMAGALAMI) of the human-derived
galanin receptor protein, wherein reverse amino acid residues are
in agreement.
[0405] FIG. 49 is the nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in the novel receptor protein cDNA
clone, pMJ10, obtained from rabbit gastropyrolic part smooth
muscles by PCR amplification and the amino acid sequence encoded
thereby, wherein the underlined parts corresponds to the synthetic
primers used for the PCR amplification.
[0406] FIG. 50 is the hydrophobicity plotting profile of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDBA fragment
included in pMJ10, prepared based upon the amino acid sequence
shown in FIG. 49, wherein numerals 4 to 6 suggest the presence of
hydrophobic domains.
[0407] FIG. 51 is the partial amino acid sequence (pMJ10) of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMJ10 shown in FIG. 49, relative to human ligand
unknown receptor protein (B42009), human N-formylpeptide receptor
protein (JC2014), rabbit N-formylpeptide receptor protein (A46520),
mouse C5a anaphylatoxin receptor protein (A46525) and bovine
neuropeptide Y receptor protein (S28787) which are known G protein
coupled receptor proteins, wherein reverse amino acid residues are
in agreement, and the 1st to 125th amino acid residues of pMJ10
correspond to the 1st to 125th amino acid residues in FIG. 49.
[0408] FIG. 52 is the nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in the novel receptor protein cDNA
clone, pMH28, obtained from rabbit gastropyrolic part smooth
muscles by PCR amplification and the amino acid sequence encoded
thereby, wherein the underlined parts correspond to the synthetic
primers used for the PCR amplification.
[0409] FIG. 53 is the hydrophobicity plotting profile of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDBA fragment
included in pMH28, prepared based upon the amino acid sequence
shown in FIG. 52, wherein numerals 4 to 6 suggest the presence of
hydrophobic domains.
[0410] FIG. 54 is the partial amino acid sequence (pMH28) of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMH28 shown in FIG. 52, relative to mouse IL-8 receptor
protein (P35343), human somatostatin receptor protein 1 (A41795)
and human somatostatin receptor protein 4 (A47457) which are known
G protein coupled receptor proteins, wherein reverse amino acid
residues are in agreement, and the 1st to 119th amino acid residues
of pMH28 correspond to the 1st to 119th amino acid residues in FIG.
52.
[0411] FIG. 55 is the nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in the novel receptor protein cDNA
clone, pMN7, obtained from rabbit gastropyrolic part smooth muscles
by PCR amplification and the amino acid sequence encoded thereby,
wherein the underlined 5'-end nucleotide sequence part corresponds
to the synthetic primer used for the PCR amplification..
[0412] FIG. 56 is the nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in the novel receptor protein cDNA
clone, pMN7, obtained from rabbit gastropyrolic part smooth muscles
by PCR amplification and the amino acid sequence encoded thereby,
wherein the underlined 3'-end nucleotide sequence part corresponds
to the synthetic primer used for the PCR amplification.
[0413] FIG. 57 is the hydrophobicity plotting profile of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMN7, prepared based upon the amino acid sequences
shown in FIGS. 55 and 56, wherein numerals TM2 to TM6 suggest the
presence of hydrophobic domains.
[0414] FIG. 58 is the partial hydrophobicity plotting profile of
the protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p19P2, prepared
based upon the amino acid sequence shown in FIG. 22.
[0415] FIG. 59 is the partial hydrophobicity plotting profile of
the protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p 19P2, prepared
based upon the amino acid sequence shown in FIG. 23.
[0416] FIG. 60 shows the partial amino acid sequence (p 19P2).,of
the protein encoded by the human pituitary gland-derived G protein
coupled receptor protein cDNA fragment included in p19P2, as shown
in FIGS. 22 and 23, relative to the known G protein coupled
receptor protein, S 12863, wherein reverse amino acid residues are
in agreement, the 1st to 99th amino acid residues of the p19P2
sequence correspond to the 1st to 99th amino acid residues in FIG.
22, and the 156th to 230th amino acid residues thereof correspond
to the 1st to 68th amino acid residues in FIG. 23.
[0417] FIG. 61 is the partial amino acid sequence (pG3-2/pG1-10) of
the MIN6-derived G protein coupled receptor protein, as shown in
FIG. 27, relative to the partial amino acid sequence (p19P2) of the
protein encoded by p19P2, as shown in FIGS. 22 and 23, wherein
reverse amino acid residues are in agreement, the 1st to 99th amino
acid residues of the p 19P2 sequence correspond to the 1st to 99th
amino acid residues in FIG. 22, the 156th to 223rd amino acid
residues thereof correspond to the 1st to 68th amino acid residues
in FIG. 23, and the 1st to 223rd amino acid residues of the
pG3-2/pG1-10 sequence correspond to the 1st to 223rd amino acid
residues in FIG. 27.
[0418] FIG. 62 is the nucleotide sequence of the MIN6-derived G
protein coupled receptor protein cDNA fragment included in the cDNA
clone, p5S38, isolated by PCR using a MIN6-derived cDNA and the
amino acid sequence encoded thereby, wherein the underlined parts
corresponds to the synthetic primers.
[0419] FIG. 63 is the partial amino acid sequence (p5S38) of the
MIN6-derived G protein coupled receptor protein, as shown in-Figure
62, relative to the partial amino acid sequence (p19P2) of the G
protein coupled receptor protein encoded by p19P2, as shown in
FIGS. 22 and 23, as well as the partial amino acid sequence of the
G protein coupled receptor protein encoded by the nucleotide
sequence derived from the nucleotide sequence of the cDNA fragment
included in pG3-2 and pG1-10, as shown in FIG. 27, wherein reverse
amino acid residues are in agreement, the 1st to 144th amino acid
residues of the p5S38 sequence correspond to the 1st to 144th amino
acid residues in FIG. 62, the 1st to 99th amino acid residues of
the p19P2 sequence correspond to the 1st to 99th amino acid
residues in FIG. 22, the 156th to 223rd amino acid residues thereof
correspond to the 1st to 68th amino acid residues in FIG. 23, and
the 1st to 223rd amino acid residues of the pG3-2/pG1-10 sequence
correspond to the 1st to 223rd amino acid residues in FIG. 27.
[0420] FIG. 64 is the partial hydrophobicity plotting profile of
the protein encoded by the MIN6-derived G protein coupled receptor
protein cDNA fragment included in p5S38, prepared based upon the
amino acid sequence shown in FIG. 62.
[0421] FIG. 65 shows the northern blot analysis profile of the
receptor gene encoded by the cDNA included in the mouse pancreatic
.beta.-cell strain MIN6-derived novel receptor protein cDNA clone,
p3H2-17, for mouse cell line, MIN6, Neuro-2a cell and mouse brain,
thymus, spleen and pancreas poly(A)+RNA, wherein each arrow and
number indicates the size marker position (unit of number: kb).
[0422] FIG. 66 shows the agarose gel electrophoresis analysis
profile of the PCR products obtained by 5' RACE PCR of the receptor
gene included in p3H2-17 using mouse thymus and spleen
poly(A)+RNA.
[0423] Lane 1 indicates the size marker 6 (Wako Pure Chemical,
Japan).
[0424] Lane 2 indicates the internal control which is the
thymus-derived PCR product obtained by PCR amplification using the
primer having SEQ ID NO: 20 and the primer having SEQ ID NO: 22
with Taq polymerase.
[0425] Lane 3 indicates the negative control which is the PCR
product obtained by Ex Taq polymerase PCR amplification of thymus
cDNA prior to addition of anchors.
[0426] Lane 4 indicates the negative control which is the PCR
product obtained by Taq polymerase PCR amplification of thymus cDNA
prior to addition of anchors.
[0427] Lane 5 indicates the PCR product obtained by 5'RACE of
thymus poly(A).sup.+ RNA with Pfu polymerase.
[0428] Lane 6 indicates the PCR product obtained by 5'RACE of
thymus poly(A).sup.+ RNA with Vent polymerase.
[0429] Lane 7 indicates the PCR product obtained by 5RACE of thymus
poly(A)+RNA with Ex Tag polymerase.
[0430] Lane 8 indicates the PCR product obtained by 5RACE of thymus
poly(A)+RNA with Taq polymerase.
[0431] Lane 9 indicates the size marker 5 (Wako Pure Chemical,
Japan).
[0432] Lane 10 indicates the internal control which is the
spleen-derived PCR product obtained by PCR amplification using the
primer having SEQ ID NO: 20 and the primer having SEQ ID NO: 22
with Taq polymerase.
[0433] Lane 11 indicates the negative control which is the PCR
product obtained by Ex Taq polymerase PCR amplification of spleen
cDNA prior to addition of anchors.
[0434] Lane 12 indicates the negative control which is the PCR
product obtained by Taq polymerase PCR amplification of spleen cDNA
prior to addition of anchors.
[0435] Lane 13 indicates the PCR product obtained by 5RACE of
poly(A) RNA.sup.+ with Pfu polymerase.
[0436] Lane 14 indicates the PCR product obtained by 5RACE of
spleen poly(A).sup.+ RNA with Vent polymerase.
[0437] Lane 15 indicates the PCR product obtained by 5RACE of
spleen poly(A).sup.+ RNA with Ex Taq polymerase.
[0438] Lane 16 indicates the PCR product obtained by 5RACE of
spleen poly(A).sup.+ RNA with Taq polymerase.
[0439] Lane 17 indicates the size marker 5 (Wako Pure Chemical,
Japan).
[0440] Each blacked triangle indicates the band recovered.
[0441] FIG. 67 shows the agarose gel electrophoresis analysis
profile of the PCR products obtained by 3RACE PCR of the receptor
gene included in p3H2-17 using mouse thymus and spleen
poly(A).sup.+ RNA.
[0442] Lane 1 indicates the size marker 5 (Wako Pure Chemical,
Japan).
[0443] Lane 2 indicates the PCR product obtained by 3RACE of spleen
poly(A).sup.+ RNA with Taq polymerase.
[0444] Lane 3 indicates the PCR product obtained by 3'RACE of
spleen poly(A).sup.+ RNA with Ex Taq polymerise.
[0445] Lane 4 indicates the PCR product obtained by 3'RACE of
spleen poly(A) RNA with Vent polymerase.
[0446] Lane 5 indicates the PCR product obtained by 3'RACE of
spleen poly(A).sup.+ RNA with Pfu polymerase.
[0447] Lane 6 indicates the PCR product obtained by 3'RACE of
thymus poly(A).sup.+ RNA with Taq polymerase.
[0448] Lane 7 indicates the PCR product obtained by 3'RACE of
thymus poly(A).sup.+ RNA with Ex Taq polymerase.
[0449] Lane 8 indicates the PCR product obtained by 3'RACE of
thymus poly(A).sup.+ RNA with Vent polymerase.
[0450] Lane 9 indicates the PCR product obtained by 3RACE of thymus
poly(A).sup.+ RNA with Pfu polymerase.
[0451] Lane 10 indicates the size marker 6 (Wako Pure Chemical,
Japan).
[0452] Each blacked triangle indicates the band recovered.
[0453] FIG. 68 depicts the model of the RACE products of the
receptor protein cDNA fragment included in p3H2-17 obtained by
5'RACE and 3'RACE. Open squares represent regions which have
already been isolated and included in p3H2-17. Small arrows, (D,
{circle over (1)}, {circle over (2)}, {circle over (3)} and {circle
over (4)}, indicate the positions and directions of the primers
designed in Working Example 19. The big arrow shows a predicted
full-length open reading frame of the receptor protein held by
p3H2-17. Numbers at both ends, N26, N64, N75, C2, C13 and C15,
indicate clone numbers of the RACE products obtained. Among these
RACE products, N26, N64 and N75 are inserted into pCR.TM. II vector
and C2, C13 and C15 are inserted into the SmaI site of pUC18. The
solid triangle indicates the PCR error position which has been
clarified through sequencing.
[0454] FIG. 69 is the nucleotide sequence of the open reading frame
and neighboring regions thereof of mouse G protein coupled receptor
protein cDNA included in the cDNA clone pMAH2-17 obtained from
mouse spleen and thymus poly(A) RNA by RACE techniques based on the
nucleotide sequence of the cDNA fragment included in p3H2-17 and
the amino acid sequence encoded thereby.
[0455] FIG. 70 is the hydrophobicity plotting profile of the
protein encoded by the receptor protein cDNA included in pMAH2-17,
prepared based upon the amino acid sequence shown in FIG. 69.
[0456] FIG. 71 is the amino acid sequence (75+13CODING) of the
protein encoded by the mouse-derived G protein coupled receptor
protein cDNA fragment included in pMAH2-17, as shown in FIG. 69,
relative to the known G protein coupled receptor proteins, mouse
P.sub.2Upurinoceptor (P2UR MOUSE) and chicken P.sub.2Y purinoceptor
(P2YR CHICK), wherein reverse amino acid residues are in
agreement.
[0457] FIG. 72 is the nucleotide sequence (from 1st to 540th
nucleotides) of the rabbit gastropyrolic part smooth muscle-derived
G protein coupled receptor protein cDNA fragment included in the
novel receptor protein cDNA clone, pMN128, obtained from rabbit
gastropyrolic part smooth muscles by PCR amplification, and the
amino acid sequence encoded thereby, wherein the underlined 5' part
corresponds to the synthetic primer used for the PCR
amplification.
[0458] FIG. 73 is the nucleotide sequence (from 541st to 843rd
nucleotides) of the rabbit gastropyrolic part smooth muscle-derived
G protein coupled receptor protein cDNA fragment included in the
novel receptor protein cDNA clone, pMN128, obtained from rabbit
gastropyrolic part smooth muscles by PCR amplification, and the
amino acid sequence encoded thereby, wherein the underlined 3' part
corresponds to the synthetic primer used for the PCR
amplification.
[0459] FIG. 74 is the hydrophobicity plotting profile of the
protein encoded by the rabbit gastropyrolic part smooth
muscle-derived G protein coupled receptor protein cDNA fragment
included in pMN128, prepared based upon the amino acid sequences
shown in FIGS. 72 and 73, suggesting the presence of hydrophobic
domains.
[0460] FIG. 75 shows inward currents evoked by ATP in Xenopus
oocytes injected with cDNA of pMAH2-17-encoded receptor.
[0461] FIG. 76 is the nucleotide sequence of the human-derived G
protein coupled receptor protein cDNA fragment included in
ph3H2-17, relative to the nucleotide sequence of the mouse-derived
G protein coupled receptor protein cDNA fragment included in
p3H2-17, wherein reverse base residues are in agreement.
[0462] FIG. 77 is the nucleotide sequence of the open reading frame
and neighboring regions thereof of human-derived G protein coupled
receptor protein cDNA included in phAH2-17 and the amino acid
sequence encoded thereby.
[0463] FIG. 78 is the hydrophobicity plotting profile of the
protein encoded by the human-derived G protein coupled receptor
protein cDNA included in phAH2-17.
[0464] FIG. 79 is the amino acid sequence of human type
purinoceptor encoded by phAH2-17, relative to the mouse
purinoceptor encoded by p3H2-17, wherein reverse amino acid
residues are in agreement.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0465] According to the present invention, DNA sequences comprising
each a nucleotide sequence indicated by a SEQ ID NO selected from
the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19 have been
synthesized and characterized. The DNA is a potent primer for
polymerase chain reaction in order to amplify DNA sequences
encoding part or all of the polypeptide sequence of G protein
coupled receptor protein. PCR amplification methods of the DNA
coding for part or all of the polypeptide sequence of G protein
coupled receptor protein can be advantageously carried out with the
said primer DNA. Screening of DNA libraries for the DNA encoding
part or all of the polypeptide sequence of G protein coupled
receptor protein can be successfully carried out through polymerase
chain reaction techniques with the said primer DNA. As a result,
template DNAs coding for part or all of the polypeptide sequence of
G protein coupled receptor protein, contained in the DNA library,
can be selectively amplified and various DNA sequences encoding
part or all of the polypeptide sequence of G protein coupled
receptor protein may be isolated and characterized. Further, G
protein coupled receptor proteins, peptide segments or fragments
derived from the G protein coupled receptor protein, modified
derivatives or analogues thereof, and salts thereof may be
recognized, predicted, deduced, produced, expressed, isolated and
characterized.
[0466] The primer DNA useful in PCR amplification of the DNA
sequence encoding part or all of the polypeptide sequence of G
protein coupled receptor protein is a degenerate deoxynucleotide
which has an oligonucleotide sequence to which a SEQ ID NO selected
from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19 is
assigned.
[0467] The nucleotide sequence represented by SEQ ID NO: 1 is a
base sequence having the following formula:
[0468] 5'-CGTGGSCMTSSTGGGCAACN.sub.1YCCTG-3'
[0469] wherein S is G or C, M is A or C, N.sub.1=A, G, C, or T,
and
[0470] Y is T or C (FIG. 1: HS-1).
[0471] The nucleotide sequence represented by SEQ ID NO: 2 (HS-2)
is a base sequence having the following formula:
[0472] 5'-GTN.sub.1GWRRGGCAN.sub.1CCAGCAGAKGGCAAA-3'
[0473] wherein N.sub.1=A, G, C, or T, W is A or T, R is A or G,
and
[0474] K is G or T, which is complementary to a nucleotide sequence
having the following formula:
[0475] 5'-TTTGCCMTCTGCTGGNTGCCYYWCNAC-3'
[0476] wherein N =A, C, G, or T, M is A or C, Y is T or C, and W is
A or T (FIG. 2).
[0477] The nucleotide sequence represented by SEQ ID NO: 3 is a
base sequence having the following formula:
[0478] 5'-CTCGCSGCYMTN.sub.2RGYATGGAYCGN.sub.2TAT-3'
[0479] wherein S is G or C, Y is C or T, M is A or C, R is A or G,
and N.sub.2=I (FIG. 4: 3D).
[0480] The nucleotide sequence represented by SEQ ID NO: 4 is a
base sequence having the following formula:
[0481] 5'-CATGTRGWAGGGAAN.sub.2CCAGSAMAN.sub.2RARRAA-3'
[0482] wherein R is A or G, W is T or A, S is G or C, M is A or C,
and N.sub.2=I, which is complementary to a nucleotide sequence
having the following formula:
[0483] 5' -TTYYTYN.sub.1TKTSCTGGN.sub.1TTCCCTWCYACATG-3'
[0484] wherein Y is C or T, N.sub.1=A, G, C, or T, K is G or T, K
is G or T, S is G or C, W is A or T (FIG. 6: 6C).
[0485] The nucleotide sequence represented by SEQ ID NO: 5 is a
base sequence having the following formula:
[0486] 5'-CTGACYGYTCTN.sub.2RSN.sub.2RYTGACMGVTAC-3'
[0487] wherein Y is C or T, R is A or G, S is G or C, M is A or C,
and V is A, C or G, and N.sub.2 is I (FIG. 3: 3A).
[0488] The nucleotide sequence represented by SEQ ID NO: 6 is a
base sequence having the following formula:
[0489] 5'-CTGACYGYTCTN.sub.2RSN.sub.2RYTGACMGVTAT-3'
[0490] wherein Y is C or T, R is A or G, S is G or C, M is A or C,
and V is A, C or G, and N.sub.2 is I (FIG. 3: 3B).
[0491] The nucleotide sequence represented by SEQ ID NO: 7 is a
base sequence having the following formula:
[0492] 5'-CTCGCSGCYMTN.sub.2RGYATGGAYCGN.sub.2TAC-3'
[0493] wherein S is G or C, Y is C or T, M is A or C, R is A or G,
and N.sub.2 is I (FIG. 4: 3C).
[0494] The nucleotide sequence represented by SEQ ID NO: 8 is a
base sequence having the following formula:
[0495] 5'-GATGTGRTARGGSRN.sub.2CCAACAGAN.sub.2GRYAAA-3'
[0496] wherein R is A or G, S is G or C, Y is C or T, and N.sub.2
is I, which is complementary to a nucleotide sequence having the
following formula:
[0497] 5'-TTTRYCN.sub.1TCTGTTGGN.sub.1YSCCYTAYCACATC-3'
[0498] wherein R is A or G, Y is C or T, S is G or C, and N.sub.1
is A, T, G, or C (FIG. 5: 6A).
[0499] The nucleotide sequence represented by SEQ ID NO: 9 is a
base sequence having the following formula:
[0500] 5'-GATGTGRTARGGSRN.sub.2CCAACAGAN.sub.2GRYGAA-3'
[0501] wherein R is A or G, S is G or C, Y is C or T, and N.sub.2
is I, which is complementary to a nucleotide sequence having the
following formula:
[0502] 5'-TTCRYCN.sub.1TCTGTTGGN.sub.1YSCCYTAYCACATC-3'
[0503] wherein R is A or G, Y is C or T, S is G or C, and N.sub.1
is A, T, G, or C (FIG. 5: 6B).
[0504] The nucleotide sequence represented by SEQ ID NO: 10 is a
base sequence having the following formula:
[0505] 5'-GYCACCAACN.sub.2WSTTCATCCTSWN2HCTG-3'
[0506] wherein S is G or C, Y is C or T, W is A or T, H is A, C or
T, and N.sub.2 is I (FIG. 7: T2A).
[0507] The nucleotide sequence represented by SEQ ID NO: 11 (FIG.
8: T7A) is a base sequence having the following formula:
[0508]
5'-ASN.sub.2SAN.sub.2RAAGSARTAGAN.sub.2GAN.sub.2RGGRTT-3'
[0509] wherein R is A or G, S is G or C, and N.sub.2 is I, which is
complementary to a nucleotide sequence having the following
formula:
[0510]
5'-AAYCCYN.sub.2TCN.sub.2TCTAYTSCTTYN.sub.2TSN.sub.2ST-3'
[0511] wherein Y is C or T, N.sub.2 is I, and S is G or C (FIG.
8).
[0512] The nucleotide sequence represented by SEQ ID NO: 12 is a
base sequence having the following formula:
[0513]
5'-TGN.sub.2TSSTKMTN.sub.2GSN.sub.2GTKGTN.sub.2GGN.sub.2AA-3'
[0514] wherein S is G or C, K is G or T, M is A or C, and N.sub.2
is I (FIG. 9: TM 1-A2).
[0515] The nucleotide sequence represented by SEQ ID NO: 13 (FIG.
10: TM3-B2) is a base sequence having the following formula:
[0516] 5'-AYCKGTAYCKGTCCAN.sub.2KGWN.sub.2ATKGC-3'
[0517] wherein Y is C or T, K is G or T, W is A or T, and N.sub.2
is I, which is complementary to a nucleotide sequence having the
following formula:
[0518] 5'-GCMATN.sub.2WCMN.sub.2TGGACMGRTACMGRT-3'
[0519] wherein M is A or C, W is A or T, R is A or G, and N.sub.2
is I (FIG. 10).
[0520] The nucleotide sequence represented by SEQ ID NO: 14 is a
base sequence having the following formula:
[0521] 5'-CATKKCCSTGGASAGN.sub.2TAYN.sub.2TRGC-3'
[0522] wherein K is G or T, S is G or C, Y is C or T, R is A or G,
and N.sub.2 is I (FIG. 11: TM3-C2).
[0523] The nucleotide sequence represented by SEQ ID NO: 15 (FIG.
12: TM6-E2) is a base sequence having the following formula:
[0524] 5'-GWWGGGSAKCCAGCASAN.sub.2000RAA-3'
[0525] wherein W is A or T, S is G or C, K is G or T, R is A or G,
and N.sub.2 is I, which is complementary to a nucleotide sequence
having the following formula:
[0526] 5'-TTYGCCN.sub.2TSTGCTGGMTSCCCWWC-3'
[0527] wherein Y is C or T, S is G or C, M is A or C, W is A or T,
and N.sub.2 is I (FIG. 12).
[0528] The nucleotide sequence represented by SEQ ID NO: 16 is a
base sequence having the following formula:
[0529] 5'-ARYYTN.sub.2GCN.sub.2 N.sub.2 TN.sub.2GCN.sub.1GAY-3'
[0530] wherein R is A or G, Y is C or T, N.sub.1 is A, T, G, or C,
and N.sub.2 is I (FIG. 13: TM2F18).
[0531] The nucleotide sequence represented by SEQ ID NO: 17 (FIG.
14: TM6R21) is a base sequence having the following formula:
[0532]
5'-N.sub.2GGN.sub.2AN.sub.2CCARCAN.sub.1AN.sub.1N.sub.1RN.sub.1RAA--
3'
[0533] wherein R is A or G, N.sub.1 is A, T, G, or C, and N.sub.2
is I which is complementary to a nucleotide sequence having the
following formula:
[0534]
5'-TTYN.sub.1YN.sub.1N.sub.1TN.sub.1TGYTGGN.sub.2TN.sub.2CCN23'
[0535] wherein Y is C or T, N.sub.1 is A, T, G, or C, and N.sub.2
is I (FIG. 14).
[0536] The nucleotide sequence represented by SEQ ID NO: 18 is a
base sequence having the following formula:
[0537]
5'-GCCTSN.sub.2TN.sub.2RN.sub.2SATGWSTGTGGAN.sub.2MGN.sub.2T-3'
[0538] wherein S is G or C, R is A or G, W is A or T, M is A or C,
and N.sub.2 is I (FIG. 15: S3A).
[0539] The nucleotide sequence represented by SEQ ID NO: 19 (FIG.
16: S6A) is a base sequence having the following formula:
[0540]
5'-GAWSN.sub.2TGMYN.sub.2AN.sub.2RTGGWAGGGN.sub.2AN.sub.2CCA-3'
[0541] wherein W is A or T, S is G or C, M is A or C, Y is C or T,
R is A or G, and N.sub.2 is I, which is complementary to a
nucleotide sequence having the following formula:
[0542]
5'-TGGN.sub.2TN.sub.2CCCTWCCAYN.sub.2TN.sub.2RKCAN.sub.2SWTC-3'
[0543] wherein W is A or T, Y is C or T, R is A or G, K is G or T,
and S is G or C (FIG. 16).
[0544] In a specific embodiment, symbols in the aforementioned SEQ
ID NOs (R, Y, M, K, S, W, H, V and N) indicate the incorporation of
plural bases, leading to multiple oligonucleotides in the primer
preparation. In other words, SEQ ID NO: 1 to SEQ ID NO: 19 are
degenerate nucleotide primers.
[0545] The nucleotide sequence represented by SEQ ID NO: 1 (FIG. 1:
HS-1) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence corresponding to or
near the first membrane-spanning (transmembrane) domain each of
known G protein coupled receptor-proteins such as human-derived TRH
receptor protein (HTRHR), human-derived RANTES receptor protein
(L10918, HUMRANTES), human Burkitt's lymphoma-derived receptor
protein with an unknown ligand (X68149, HSBLRIA), human-derived
somatostatin receptor protein (L14856, HUMSOMATO), rat-derived
.mu.-opioid receptor protein (U02083, RNU02083), rat-derived
.kappa.-opioid receptor protein (U00442, U00442), human-derived
neuromedin B receptor protein (M73482, HUMNMBR), human-derived
muscarinic acetylcholine receptor protein (X15266, HSHM4),
rat-derived adrenaline .alpha..sub.1B receptor protein (L08609,
RATAADRE01), human-derived somatostatin 3 receptor protein (M96738,
HUMSSTR3X), human-derived C.sub.5a receptor protein (HUMC5AAR),
human-derived receptor protein with an unknown ligand (HUMRDC1A),
human-derived receptor protein with an unknown ligand (M84605,
HUMOPIODRE), rat-derived adrenaline a 2 B receptor protein (M91466,
RATA2BAR) and the like [FIG. 1).
[0546] The nucleotide sequence represented by SEQ ID NO: 2 (HS-2)
is a nucleotide sequence which is complementary to the nucleotide
sequence (FIG. 2) highly homologous to the DNA sequence coding for
the amino acid sequence corresponding to or near the sixth
membrane-spanning domain of known G protein coupled receptor
proteins such as mouse-derived receptor protein with an unknown
ligand (M80481, MUSGIR), human-derived bombesin receptor protein
(L08893, HUMBOMB3S), human-derived adenosine A2 receptor protein
(S46950, S46950), mouse-derived receptor protein with an unknown
ligand (D21061, MUSGPCR), mouse-derived TRH receptor protein
(S43387, S43387), rat-derived neuromedin K receptor protein
(J05189, RATNEURA), rat-derived adenosine A1 receptor protein
(M69045, RATA1ARA), human-derived neurokinin A receptor protein
(M57414, HUMNEKAR), rat-derived adenosine A3 receptor protein
(M94152, RATADENREC), human-derived somatostatin 1 receptor protein
(M81829, HUMSTRI1A), human-derived neurokinin 3 receptor protein
(S86390, S86371S4), rat-derived receptor protein with an unknown
ligand (X61496, RNCGPCR), human-derived somatostatin 4 receptor
protein (L07061, HUMSSTR4Z), rat-derived GnRH receptor protein
(M31670, RATGNRHA) and the like (FIG. 2).
[0547] The nucleotide sequence represented by SEQ ID NO: 5 (FIG. 3:
3A) or the nucleotide sequence represented by SEQ ID NO: 6 (FIG. 3:
3B) is a nucleotide sequence highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the
third membrane-spanning domain each of known G protein coupled
receptors such as mouse-derived .kappa.-opioid receptor protein
(L11064), mouse-derived .delta.-opioid receptor protein (L11065),
rat-derived .mu.-opioid receptor protein (D16349), mouse-derived
bradykinin B2 receptor protein (X69676), rat-derived bradykinin B2
receptor protein (M59967), mouse-derived bombesin receptor protein
(M35328), human-derived neuromedin B receptor protein (M73482),
human-derived gastrin releasing peptide receptor protein (M73481),
human-derived bombesin receptor protein subtype 3 (L08893),
mouse-derived substance K receptor protein (X62933), mouse-derived
substance P receptor protein (X62934), rat-derived neurokinin 3
receptor protein (J05189), rat-derived endothelin receptor protein
(M60786), rat-derived receptor protein with an unknown ligand
(L04672), rat-derived receptor protein with an unknown ligand
(X61496), rat-derived receptor protein with an unknown ligand
(X59249), rat-derived receptor protein with an unknown ligand
(L09249), mouse-derived receptor protein with an unknown ligand
(P30731), human-derived receptor protein with an unknown ligand
(M31210), human-derived receptor protein with an unknown ligand
(U03642) and the like [FIG. 3].
[0548] The nucleotide sequence represented by SEQ ID NO: 7 (FIG. 4:
3C) or the nucleotide sequence represented by SEQ ID NO: 3 (FIG. 4:
3D) is a nucleotide sequence highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the
third membrane-spanning domain each of known G protein coupled
receptors such as mouse-derived angiotensin II receptor protein
(L32840), rat-derived angiotensin Ib receptor protein (X64052),
rat-derived angiotensin receptor protein subtype (M90065),
human-derived angiotensin Ia receptor protein (M91464), rat-derived
cholecystokinin a receptor protein (M88096), rat-derived
cholecystokinin b receptor protein (M99418), human-derived
cholecystokinin b receptor protein (L04473), mouse-derived low
affinity interleukin 8 receptor protein (M73969), human-derived
high affinity interleukin 8 receptor protein (X65858),
mouse-derived C5a anaphylatoxin receptor protein (S46665),
human-derived N-formylpeptide receptor protein (M60626) and the
like FIG. 4).
[0549] The nucleotide sequence represented by SEQ ID NO: 10 (FIG.
7: T2A) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence corresponding to or
near the second membrane-spanning domain each of known G protein
coupled receptors such as human galanin receptor (HUMGALAREC), rat
.alpha.-1B-adrenergic receptor (RATADR1B), human
.beta.-1-adrenergic receptor (HUMADRB1), rabbit IL-8 receptor
(RABIL8RSB), human opioid receptor (HUMOPIODRE), bovine substance K
receptor (BTSKR), human somatostatin receptor-2 (HUMSRI2A), human
somatostatin receptor-3 (HUMSSTR3Y), human gastrin receptor
(HUMGARE), human cholecystokininA receptor (HUMCCKAR), human
dopamine receptor-D5 (HUMD1B), human serotonin receptor 5HT1E
(HUM5HT1E), human dopamine receptor D4 (HUMD4C), mouse serotonin
receptor-2 (MMSERO), rat .alpha.-1A-adrenergic receptor
(RATADRAIA), rat histamine H2 receptor (S57565) and the like [FIG.
7).
[0550] The nucleotide sequence represented by SEQ ID NO: 8
(complementary to 6A of FIG. 5) or the nucleotide sequence
represented by SEQ ID NO: 9 (complementary to 6B of FIG. 5) is a
nucleotide sequence which is complementary to the nucleotide
sequence (FIG. 5) highly homologous to the DNA sequence coding for
the amino acid sequence corresponding to or near the sixth
membrane-spanning domain of known G protein coupled receptors such
as mouse-derived .kappa.-opioid receptor protein (L11064),
mouse-derived .delta.-opioid receptor protein (L11065), rat-derived
.mu.-opioid receptor protein (D16349), mouse-derived bradykinin B2
receptor protein (X69676), rat-derived bradykinin B2 receptor
protein (M59967), mouse-derived bombesin receptor protein (M35328),
human-derived neuromedin B receptor protein (M73482), human-derived
gastrin releasing peptide receptor protein (M73481), human-derived
bombesin receptor protein subtype 3 (L08893), mouse-derived
substance K receptor protein (X62933), mouse-derived substance P
receptor protein (X62934), rat-derived neurokinin 3 receptor
protein (J05189), rat-derived endothelin receptor protein (M60786),
rat-derived receptor protein with an unknown ligand (L04672),
rat-derived receptor protein with an unknown ligand (X61496),
rat-derived receptor protein with an unknown ligand (X59249),
rat-derived receptor protein with an unknown ligand (L09249),
mouse-derived receptor protein with an unknown ligand (P30731),
human-derived receptor protein with an unknown ligand (M31210)
human-derived receptor protein with an unknown ligand (U03642) and
the like FIG. 5).
[0551] The nucleotide sequence represented by SEQ ID NO: 4
(complementary to 6C of FIG. 6) is a nucleotide sequence which is
complementary to the nucleotide sequence (FIG. 6) highly homologous
to the DNA sequence coding for the amino acid sequence
corresponding to or near the sixth membrane-spanning domain of
known G protein coupled receptors such as mouse-derived angiotensin
II receptor protein (L32840), rat-derived angiotensin Ib receptor
protein (X64052), rat-derived angiotensin receptor protein subtype
(M90065), human-derived angiotensin Ia receptor protein (M91464),
rat-derived cholecystokinin a receptor protein (M88096),
rat-derived cholecystokinin b receptor protein (M99418),
human-derived cholecystokinin 8 receptor protein (L04473),
mouse-derived low affinity interleukin 8 receptor protein (M73969),
human-derived high affinity interleukin 8 receptor protein
(X65858), mouse-derived C5a anaphylatoxin receptor protein
(S46665), human-derived N-formylpeptide receptor protein (M60626)
and the like [FIG. 6).
[0552] The nucleotide sequence represented by SEQ ID NO: 11 (FIG.
8: T7A) is a nucleotide sequence which is complementary to the
nucleotide sequence (FIG. 8) highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the
seventh membrane-spanning domain each of known G protein coupled
receptors such as human galanin receptor (HUMGALAREC), rat A1
adenosine receptor (RAT1DREC), porcine angiotensin receptor
(PIGA2R), rat serotonin receptor (RAT5HTRTC), human dopamine
receptor (S58541), human gastrin releasing peptide receptor
(HUMGRPR), mouse GRP/bombesin receptor (MUSGRPBOM), rat vascular
type 1 angiotensin receptor (RRVT1AIIR), human muscarinic
acetylcholine receptor (HSHM4), human .beta.-1 adrenergic receptor
(HUMDRB1), human gastrin receptor (HUMGARE), rat cholecystokinin
receptor (RATCCKAR), rat receptor with an unknown ligand (S59748),
human somatostatin receptor (HUMSST28A), rat receptor with an
unknown ligand (RNGPROCR), mouse somatostatin receptor-1
(MUSSRI1A)., human .alpha.-A1-adrenergic receptor (HUMA1AADR),
mouse delta-opioid receptor (S66181), human somatostatin receptor-3
(HUMSSTR3Y) and the like [FIG. 8).
[0553] The nucleotide sequence represented by SEQ ID NO: 12 (FIG.
9: TM 1-A2) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence within.the first
membrane-spanning (transmembrane) domain each of known G protein
coupled receptors such as mouse-derived bradykinin B.sub.2 receptor
(MUSBB2R), bovine-derived substance K receptor (BTSKR),
bovine-derived endothelin ET.sub.B receptor (BOVEETBR),
human-derived neuropeptide Y receptor (MMSUBKREC), human-derived
prostaglandin E.sub.2 receptor (HUMPGE2R), human-derived
prostacyclin receptor (HUMPIR), human-derived .kappa.-opioid
receptor (HSU11053), rat-derived melanocortin 3 receptor (RRMC3RA),
human-derived melanocortin receptor (HUMMR), mouse-derived
bombesin/GRP receptor (MUSGRPBOM), rat-derived cholecystokinin B
receptor (RATCHOLREC), rat-derived cholecystokinin A receptor
(RATCCKAR) and the like [FIG. 9).
[0554] The nucleotide sequence represented by SEQ ID NO: 13 (FIG.
10: TM3-B2) is a nucleotide sequence which is complementary to the
nucleotide sequence (FIG. 10) highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the end
of the third membrane-spanning domain of known G protein coupled
receptors such as human-derived cholecystokinin receptor (HUMCCKR),
human-derived cholecystokinin B receptor (HUMCCKBGR), mouse-derived
melanocortin 5 receptor (MMGMC5R), human-derived vasopressin
receptor (HUMV2R), rat-derived neuromedin K receptor (RATNEURA),
dog-derived gastrin receptor (DOGGSTRN), rat-derived serotonin
receptor (RAT5HT5A), mouse-derived .alpha..sub.2 -adrenaline
receptor (MUSALP2ADA), human-derived adenosine A.sub.1 receptor
(HUMADORA1X), human-derived opioid (presumed) receptor
(HUMOPIODRE), mouse-derived bombesin/GRP receptor (MUSGRPBOM),
rat-derived cholecystokinin A receptor (RATCCKAR), human-derived
TRH receptor (HSTRHREC) and the like [FIG. 10).
[0555] The nucleotide sequence represented by SEQ ID NO: 14 (FIG.
11: TM3-C2) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence corresponding to or
near the end of the third membrane-spanning domain of known G
protein coupled receptors such as human-derived neurokinin 3
receptor (HUMNK3R), human-derived oxytocin receptor (HSMRNAOXY),
guinea pig-derived cholecystokinin A receptor (S68242), dog-derived
cholecystokinin A receptor with an unknown ligand (CFGPCR4),
mouse-derived substance P receptor (MMSUBPREC), human-derived
receptor with an unknown ligand (HUMOPIODRE), human-derived galanin
receptor (HUMGALAREC), human-derived serotonin receptor (HSS31G),
human-derived .beta..sub.3-adrenaline receptor (HUMARB3A),
human-derived prostacyclin receptor (HUMHPR), rat-derived
cholecystokinin A receptor (RATCCKAR) and the like [FIG. 11).
[0556] The nucleotide sequence represented by SEQ ID NO: 15 (FIG.
12: TM6-E2) is a nucleotide sequence which is complementary to the
nucleotide sequence (FIG. 12) highly homologous to the DNA sequence
coding for the amino acid sequence within the sixth
membrane-spanning domain of known G protein coupled receptors such
as human-derived neurokinin A receptor (HUMNEKAR), human-derived
substance P receptor (HUMSUBPRA), rat-derived substance K receptor
(RATSKR), mouse-derived bombesin/ GRP receptor (MUSGRPBOM),
human-derived opioid (presumed) receptor (HUMOPIODRE),
human-derived adenosine A.sub.2 receptor (HUMA2XXX), human-derived
.beta..sub.2-adrenaline receptor (HUMADRBR), canine-derived
receptor RDC5 with an unknown ligand (CFGPCR8), human-derived
endothelin receptor (HUMETSR), mouse-derived neuropeptide Y1
receptor (MMNPY1CDS), human-derived oxytocin receptor (HSMRNAOXY),
rat-derived cholecystokinin A receptor (RATCCKAR) and the like
[FIG. 12).
[0557] The nucleotide sequence represented by SEQ ID NO: 16 (FIG.
13: TM2F18) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence corresponding to or
near the second membrane-spanning domain of known G protein coupled
receptors such as human-derived TSH receptor (HUMTSHX),
human-derived neurokinin A receptor (HUMNEKAR), human-derived FMLP
receptor (HUMFMLP), human-derived IL8 receptor B (HUMINTLEU8),
human-derived .alpha.-A1 adrenergic receptor (HUMA1AADR),
human-derived IL8 receptor A (HUMIL8RA), human-derived dopamine D2
receptor (HSDD2), human-derived angiotensin type I receptor
(HUMANTIR), human-derived somatostatin receptor (HUSOMAT),
human-derived TRH receptor (HSTRHREC), human-derived delta-opioid
receptor (HSU07882) and the like [FIG. 13).
[0558] The nucleotide sequence represented by SEQ ID NO: 17 (FIG.
14: TM6R21) is a nucleotide sequence which is complementary to the
nucleotide sequence (FIG. 14) highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the
sixth membrane-spanning domain of known G protein coupled receptors
such as human-derived .beta.-adrenergic receptor (HSBAR),
human-derived neurokinin A receptor (HUMNEKAR), human-derived
endothelin-1 receptor (HUMETN1R), human-derived histamine H.sub.2
receptor (HUMHISH2R), human-derived .alpha.-A1 adrenergic receptor
(HUMA1AADR), human-derived IL8 receptor A (HUMIL8RA), human-derived
neuromedin B receptor (HUMNMBR), human-derived neurokinin 1
receptor (HUMNKIRX), human-derived substance P receptor
(HUMSUBPRA), human-derived 5-HT1D serotonin receptor (HUM5HT1DA),
human-derived formylpeptide receptor (HUMPFPR2A), human-derived
dopamine D2 receptor (HSDD2), human-derived neuropeptide Y receptor
(HUMNEUYREC), human-derived adenosine A2 receptor (HUMA2XXX),
human-derived bradykinin receptor BK-2 (HUMBK2A), human-derived
FMLP-related receptor II (HUMFMLPX), human-derived somatostatin
receptor subtype 3 (HUMSSTR3X), human-derived cholecystokinin
receptor (HUMCCKR), human-derived neurotensin receptor (HSNEURA)
and the like [FIG. 14).
[0559] The nucleotide sequence represented by SEQ ID NO: 18 (FIG.
15: S3A) is a nucleotide sequence highly homologous to the DNA
sequence coding for the amino acid sequence corresponding to or
near the third membrane-spanning domain of known G protein coupled
receptors such as human-derived galanin receptor (HUMGALAREC),
human-derived CCK-B receptor (S70057), human-derived ETA receptor
(S67127), human-derived ET.sub.B receptor (S44866), human-derived
C5A receptor (HUMC5AAR), human-derived angiotensin II receptor
(HUMANTIR), human-derived bradykinin receptor (HUMBK2R),
human-derived neurotensin receptor (HSNEURA), human-derived GRP
receptor (HUMGRPR), human-derived somatostatin 5 receptor
(HUMFSRS), human-derived IL-8 receptor (HUMIL8RA), human-derived
neurokinin 2 (neurokinin A) receptor (HUMNEKAR) and the like [FIG.
15).
[0560] The nucleotide sequence represented by SEQ ID NO: 19 (FIG.
16: S6A) is a nucleotide sequence which is complementary to the
nucleotide sequence (FIG. 16) highly homologous to the DNA sequence
coding for the amino acid sequence corresponding to or near the
sixth membrane-spanning domain of known G protein coupled receptors
such as humanderived galanin receptor (HUMGLAREC), human-derived
CCK-B receptor (S70057), human-derived ETA receptor (S67127),
human-derived ET.sub.B receptor (S44866), human-derived C5A
receptor (HUMC5AAR), human-derived angiotensin II receptor
(HUMANTIR), human-derived bradykinin receptor (HUMBK2R),
human-derived neurotensin receptor (HSNEURA), human-derived GRP
receptor (HUMGRPR), human-derived somatostatin 5 receptor
(HUMFSRS), human-derived IL-8 receptor (HUMIL8RA), human-derived
neurokinin 2 (neurokinin A) receptor (HUMNEKAR) and the like [FIG.
16).
[0561] The above-mentioned abbreviations in the parentheses are the
identifiers (or reference numbers) which are shown when GenBank/
EMBL Data Bank is searched using a DNASIS Gene/Protein Sequence
Data Base (CDO19; Hitachi Software Engineering, Japan) and are
usually called "Accession Numbers" or "Entry Names". HTRHR is,
however, the sequence as described in Japanese Patent Application
No. Hei 5-286986 (or No.286986/1993) (EPA 638645).
[0562] The DNA (or nucleotides) of the present invention may be
manufactured by DNA synthetic methods which are known per se or by
methods similar thereto. The DNA (or nucleotides) of the present
invention may be an oligonucleotide sequence having 8 to 60 base
residues, preferably 12 to 50 base residues, more preferably 15 to
40 residues and most preferably 18 to 30 residues.
[0563] Among the DNAs of the present invention, the DNA having the
nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 12 is
a nucleotide sequence which is commonly present in the nucleotide
sequence of the DNA encoding the amino acid sequence corresponding
to or near the first membrane-spanning domain of the
above-mentioned known G protein coupled receptor protein.
Therefore, it can be complementarily bonded (i.e. is hybridizable)
with RNA or DNA (including genome DNA, cDNA) coding for the amino
acid sequence corresponding to or near the first membrane-spanning
domain of known or unknown G protein coupled receptor proteins and,
furthermore, it can be complementarily bonded (i.e. is
hybridizable) with nucleotide sequences encoding other
membrane-spanning domains as well.
[0564] The DNA having a nucleotide sequence represented by SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 or
SEQ ID NO: 18 is a nucleotide sequence which is commonly present in
the nucleotide sequence of the DNA coding for the amino acid
sequence corresponding to or near the third membrane-spanning
domain of the above-mentioned known G protein coupled receptor
protein. Therefore, it can be complementarily bonded with RNA or
DNA (including genome DNA, cDNA) coding for the part corresponding
to or near the third membrane-spanning domain of known or unknown G
protein coupled receptor proteins and, furthermore, it can be
complementarily bonded with nucleotide sequences encoding other
membrane-spanning domains as well.
[0565] The DNA having a nucleotide sequence represented by SEQ ID
NO: 10 or SEQ ID NO: 16 is a nucleotide sequence which is commonly
present in the nucleotide sequence of the DNA coding for the amino
acid sequence corresponding to or near the second membrane-spanning
domain of the above-mentioned known G protein coupled receptor
protein. Therefore, it can be complementarily bonded with RNA or
DNA (including genome DNA, cDNA) coding for the part corresponding
to or near the second membrane-spanning domain of known or unknown
G protein coupled receptor proteins and, furthermore, it can be
complementarily bonded with nucleotide sequences encoding other
membrane-spanning domains as well.
[0566] The DNA having a nucleotide sequence represented by SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 15, SEQ
ID NO: 17 or SEQ ID NO: 19 is a nucleotide sequence which is
commonly present in the nucleotide sequence of the DNA coding for
the amino acid sequence corresponding to or near the sixth
membrane-spanning domain of the above-mentioned known G protein
coupled receptor protein. Therefore, it can be complementarily
bonded with RNA or DNA (including genome DNA, cDNA) coding for the
part corresponding to or near the sixth membrane-spanning domain of
known or unknown G protein coupled receptor proteins and,
furthermore, it can be complementarily bonded with nucleotide
sequences encoding other membrane-spanning domains as well.
[0567] The DNA having a nucleotide sequence represented by SEQ ID
NO: 11 is a nucleotide sequence which is commonly present in the
nucleotide sequence of the DNA coding for the amino acid sequence
corresponding to or near the seventh membrane-spanning domain of
the above-mentioned known G protein coupled receptor protein.
Therefore, it can be complementarily bonded with RNA or DNA
(including genome DNA, cDNA) coding for the part corresponding to
or near the seventh membrane-spanning domain of known or unknown G
protein coupled receptor proteins and, further more, it can be
complementarily bonded with nucleotide sequences encoding other
transmembrane domains as well.
[0568] The DNA having a nucleotide sequence represented by SEQ ID
NO: 13 is a nucleotide sequence which is commonly present in the
nucleotide sequence of the DNA coding for the amino acid sequence
corresponding to or near the third membrane-spanning domain of the
above-mentioned known G protein coupled receptor protein.
Therefore, it can be complementarily bonded with RNA or DNA
(including genome DNA, cDNA) coding for the part corresponding to
or near the third membrane-spanning domain of known or unknown G
protein coupled receptor proteins and, furthermore, it can be
complementarily bonded with nucleotide sequences encoding other
membrane-spanning domains as well.
[0569] Accordingly, the DNAs (or nucleotides) of the present
invention can be used as DNA primers for a polymerase chain
reaction (hereinafter, sometimes referred to as PCR).
[0570] For example:
[0571] (i) a polymerase chain reaction is carried out by mixing
[0572] (1) a small amount of DNA (or DNA fragment(s)) which codes
for G protein coupled receptor protein, said DNA (or DNA
fragment(s)) acting as a template,
[0573] (2) at least one DNA primer selected from the group
consisting of DNA primers having a nucleotide sequence represented
by SEQ ID NO: 1, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 3, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 5, DNA primers having a
nucleotide sequence represented by SEQ ID NO: 6, DNA primers having
a nucleotide sequence represented by SEQ ID NO: 7, DNA primers
having a nucleotide sequence represented by SEQ ID NO: 10, DNA
primers having a nucleotide sequence represented by SEQ ID NO: 12,
DNA primers having a nucleotide sequence represented by SEQ ID NO:
14, DNA primers having a nucleotide sequence represented by SEQ ID
NO: 16 and DNA primers having a nucleotide sequence represented-by
SEQ ID NO: 18 and
[0574] (3) at least one DNA primer selected from the group
consisting of DNA primers having a nucleotide sequence represented
by SEQ ID NO: 2, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 4, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 8, DNA primers having a
nucleotide sequence represented by SEQ ID NO: 9, DNA primers having
a nucleotide sequence represented by SEQ ID NO: 11, DNA primers
having a nucleotide sequence represented by SEQ ID NO: 15, DNA
primers having a nucleotide sequence represented by SEQ ID NO: 17
and DNA primers having a nucleotide sequence represented by SEQ ID
NO: 19; or
[0575] (ii) a polymerase chain reaction is carried out by
mixing
[0576] (1) a small amount of DNA (or DNA fragment(s)) coding for G
protein coupled receptor protein, said DNA (or DNA fragment(s))
acting as a template,
[0577] (2) at least one DNA primer selected from the group
consisting of DNA primers having a nucleotide sequence represented
by SEQ ID NO: 1 and DNA primers having a nucleotide sequence
represented by SEQ ID NO: 12 and
[0578] (3) at least one DNA primer selected from the group
consisting of DNA primers having a nucleotide sequence represented
by SEQ ID NO: 13
[0579] so that it is possible to amplify the target DNA (or DNA
fragment(s)) coding for said receptor protein.
[0580] When the PCR is carried out using at least one DNA primer
selected from the group consisting of DNA primers having a
nucleotide sequence represented by SEQ ID NO: 2, DNA primers having
a nucleotide sequence represented by SEQ ID NO: 4, DNA primers
having a nucleotide sequence represented by SEQ ID NO: 8, DNA
primers having a nucleotide sequence represented by SEQ ID NO: 9,
DNA primers having a nucleotide sequence represented by SEQ ID NO:
11, DNA primers having a nucleotide sequence represented by SEQ ID
NO: 15, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 17 and DNA primers having a nucleotide sequence represented
by SEQ ID NO: 19, said DNA primer(s) is(are) bonded (hybridized)
with the nucleotide sequence at the 3'-side of the +chain (plus
chain) of template RNA or DNA (or fragment(s) thereof coding for
the sixth membrane-spanning domain or other membrane-spanning
domains of G protein coupled receptor protein whereupon an
elongation of the - chain (minus chain) proceeds in the 5'
.quadrature.3' direction.
[0581] When the PCR is carried out using at least one DNA primer
selected from the group consisting of DNA primers having a
nucleotide sequence represented by SEQ ID NO: 11, said DNA primer
is bonded with the nucleotide sequence at the 3'-side of the +chain
(plus chain) of template RNA or DNA (or fragment(s) thereof coding
for the seventh membrane-spanning domain or other membrane-spanning
domains of the G protein coupled receptor protein whereupon an
elongation of the - chain (minus chain) proceeds in the 5'
.quadrature.3' direction.
[0582] When the PCR is carried out using at least one DNA primer
selected from the group consisting of DNA primers having a
nucleotide sequence represented by SEQ ID NO: 1 and DNA primers
having a nucleotide sequence represented by SEQ ID NO: 12, said DNA
primer is bonded with the nucleotide sequence at the 3'-side of the
- chain (minus chain) of template RNA or DNA (or fragment(s)
thereof) coding for the first membrane-spanning domain or other
membrane-spanning domains of G protein coupled receptor protein
whereupon an elongation of the + chain (plus chain) proceeds in the
5' .quadrature.3' direction.
[0583] When the PCR is carried out using at least one DNA primer
selected from the group consisting of DNA primers having a
nucleotide sequence represented by SEQ ID NO: 10 and DNA primers
having a nucleotide sequence represented by SEQ ID NO 16, said DNA
primer is bonded with the nucleotide sequence at the 3'-side of the
- chain (minus chain) of template RNA or DNA (or fragment(s)
thereof coding for the second membrane-spanning domain or other
membrane-spanning domains of G protein coupled receptor protein
whereupon an elongation of the +chain (plus chain) proceeds in the
5' .quadrature.3' direction.
[0584] When the PCR is carried out using at least one DNA primer
selected from the group consisting of DNA primers having a
nucleotide sequence represented by SEQ ID NO: 3, DNA primers having
a nucleotide sequence represented by SEQ ID NO: 5, DNA primers
having a nucleotide sequence represented by SEQ ID NO: 6, DNA
primers having a nucleotide sequence represented by SEQ ID NO: 7,
DNA primers having a nucleotide sequence represented by SEQ ID NO:
14 and DNA primers having a nucleotide sequence represented by SEQ
ID NO: 18, said DNA primer is bonded with the nucleotide sequence
at the 3'-side of the - chain (minus chain) of template RNA or DNA
(or fragment(s) thereof) coding for the third membrane-spanning
domain or other membrane-spanning domains of G protein coupled
receptor protein whereupon an elongation of the + chain (plus
chain) proceeds in the 5' .quadrature.3' direction.
[0585] Accordingly, when the DNA primers having nucleotide
sequences represented by any of SEQ ID NO: 1 to SEQ ID NO: 19 of
the present invention are used in combination each other, DNA (or
DNA fragment(s)) coding for G protein coupled receptor protein can
be successfully amplified.
[0586] One embodiment of the present invention provides:
[0587] (A) a method of amplifying DNA coding for the G protein
coupled receptor protein (e.g., from the first to sixth
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0588] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0589] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0590] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19;
[0591] (B) a method of amplifying DNA coding for the G protein
coupled receptor protein (e.g., from the first to seventh
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0592] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0593] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0594] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11;
[0595] (C) a method of amplifying a DNA coding for the G protein
coupled receptor protein (e.g., from the second to sixth
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0596] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0597] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 10 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 16 and
[0598] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19;
[0599] (D) a method of amplifying a DNA coding for the G protein
coupled receptor protein (e.g., from the second to seventh
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0600] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0601] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 10 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 16 and
[0602] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11;
[0603] (E) a method of amplifying a DNA coding for the G protein
coupled receptor protein (e.g., from the third to sixth
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0604] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0605] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 3, DNA primers having a nucleotide sequence represented by SEE
ID NO: 5, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 6, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 7, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 14 and DNA primers having a nucleotide
sequence represented by SEQ ID NO: 18 and
[0606] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19;
[0607] (F) a method of amplifying a DNA coding for the G protein
coupled receptor protein (e.g., from the third to seventh
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0608] a DNA coding for the G protein coupled receptor protein,
said DNA acting as a template,
[0609] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 3, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 5, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 6, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 7, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 14 and DNA primers having a nucleotide
sequence represented by SEQ ID NO: 18 and
[0610] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11; and
[0611] (G) a method of amplifying a DNA coding for the G protein
coupled receptor protein (e.g., from the first to third
membrane-spanning (transmembrane) domains or other segments of the
G protein coupled receptor protein), characterized in that a
polymerase chain reaction is carried out by mixing
[0612] DNA coding for the G protein coupled receptor protein, said
DNA acting as a template,
[0613] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0614] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 13.
[0615] An example of more preferred combination of the DNA primers
in the amplification according to the above-mentioned (A) includes
a combination of a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 1 with a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 2 and the like.
[0616] An example of more preferred combination of the DNA primers
in the amplification according to the above-mentioned (D) includes
a combination of a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 10 with a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 11 and the like.
[0617] An example of more preferred combination of the DNA primers
in the amplification according to the above-mentioned (E)
includes:
[0618] (i) a combination of a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 5 or a DNA primer having a
nucleotide sequence represented by SEQ ID NO: 6 with a DNA primer
having a nucleotide sequence represented by SEQ ID-NO: 8 or a DNA
primer having a nucleotide sequence represented by SEQ ID NO:
9;
[0619] (ii) a combination of a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 3 or a DNA primer having a
nucleotide sequence represented by SEQ ID NO: 7 with a DNA primer
having a nucleotide sequence represented by SEQ ID NO: 4 and the
like.
[0620] An example of more preferred combination of the DNA primers
in the amplification according to the above-mentioned (G) includes
a combination of a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 12 with a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 13 and the like.
[0621] The amplification may be carried out in accordance with
known PCR techniques. For example, it may be carried out by the
method described in Saiki, R. K. et al., Science, 239:487-491
(1988). Temperature, time, buffer, number of reaction cycles,
enzyme such as DNA polymerase, addition of
2'-deoxy-7-deazaguanosine triphosphate or inosine, etc. in the PCR
amplification may be suitably selected depending upon the type of
target DNA and other factors. When RNA is used as a template, PCR
amplification may be carried out, for example, by the method
described in Saiki, R. K. et al., Science, 239:487-491(1988).
[0622] Moreover, the DNA having a nucleotide sequence represented
by SEQ ID NO: 1 or SEQ ID NO: 12 of the present invention can be
selectively and complementarily bonded (hybridized) with the
nucleotide sequence at the 3'-side of the - chain of the DNA coding
for the amino acid sequence corresponding to or near the first
membrane-spanning domain of the G protein coupled receptor protein;
the DNA having a nucleotide sequence represented by SEQ ID NO: 10
or SEQ ID NO: 16 of the present invention can be selectively and
complementarily bonded (hybridized) with the nucleotide sequence at
the 3'-side of the - chain of the DNA coding for the amino acid
sequence corresponding to or near the second membrane-spanning
domain of the G protein coupled receptor protein; the DNA having a
nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 or SEQ ID NO: 18 of the
present invention can be selectively and complementarily bonded
(hybridized) with the nucleotide sequence at the 3'-side of the -
chain of the DNA coding for the amino acid sequence corresponding
to or near the third membrane-spanning domain of the G protein
coupled receptor protein; the DNA having a nucleotide sequence
represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 of the present
invention can be selectively and complementarily bonded
(hybridized) with the nucleotide sequence at the 3'-side of the +
chain of the DNA coding for the amino acid sequence corresponding
to or near the sixth membrane-spanning domain of the G protein
coupled receptor protein; the DNA having a nucleotide sequence
represented by SEQ ID NO: 11 of the present invention can be
selectively and complementarily bonded (hybridized) with the
nucleotide sequence at the 3'-side of the + chain of the DNA coding
for the amino acid sequence corresponding to or near the third
membrane-spanning domain of the G protein coupled receptor protein;
and the DNA having a nucleotide sequence represented by SEQ ID NO:
13 of the present invention can be selectively and complementarily
bonded (hybridized) with the nucleotide sequence at the 3'-side of
the + chain of the DNA coding for the amino acid sequence
corresponding to or near the third membrane-spanning domain of the
G protein coupled receptor protein and, accordingly, said DNA is
also advantageously useful as a probe for screening DNA libraries
for DNA (or DNA fragment(s)) encoding part or all of the
polypeptide sequence of G protein coupled receptor proteins.
[0623] These screening methods for DNA (or DNA fragment(s))
encoding part or all of the polypeptide sequence of G protein
coupled receptor proteins from the DNA library by using as a
reagent, because it can be used as a probe the DNA of the present
invention may be carried out according to DNA cloning methods known
per se by those of skill in the art or methods similar thereto.
Especially when the DNA of the present invention is used as a DNA
primer for the PCR, both amplification and screening of the DNA (or
DNA fragment) coding for the G protein coupled receptor protein can
be conducted in a single step.
[0624] Thus, when the DNAs of the present invention are suitably
combined and used as the DNA primer for the PCR, said DNA primer(s)
is(are) bonded (hybridized) with RNA or DNA (or fragment(s) thereof
encoding the amino acid sequence of the first membrane-spanning
(transmembrane) domain, the second membrane-spanning domain, the
third membrane-spanning domain, the sixth membrane-spanning domain,
the seventh membrane-spanning domain or other membrane-spanning
domains of G protein coupled receptor proteins to amplify, for
example,
[0625] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of from the first membrane-spanning to the sixth
membrane-spanning domains of G protein coupled receptor
proteins,
[0626] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence of from the first membrane-spanning to the seventh
membrane-spanning domains of G protein coupled receptor
proteins,
[0627] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence of from the third membrane-spanning to the sixth
membrane-spanning domains of G protein coupled receptor
proteins,
[0628] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of from the third membrane-spanning to the seventh
membrane-spanning domains of G protein coupled receptor
proteins,
[0629] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of from the second membrane-spanning to the sixth
membrane-spanning domains of G protein coupled receptor proteins or
RNA or DNA (or fragment(s) thereof) coding for the amino acid
sequence of other domains thereof,
[0630] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of from the second membrane-spanning to the seventh
membrane-spanning domains of G protein coupled receptor
proteins,
[0631] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of from the first membrane-spanning to the third
membrane-spanning domains of G protein coupled receptor proteins
or
[0632] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence of other domains of G protein coupled receptor
proteins.
[0633] Through using the DNA primer according to the present
invention, therefore, selective amplifications of:
[0634] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence covering from the first membrane-spanning domain to the
sixth membrane-spanning domain of G protein coupled receptor
proteins;
[0635] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence covering from the first membrane-spanning domain to
the seventh membrane-spanning domain of G protein coupled receptor
proteins;
[0636] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence covering from the third membrane-spanning domain to the
sixth membrane-spanning domain of G protein coupled receptor
proteins;
[0637] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence covering from the third membrane-spanning domain to
the seventh membrane-spanning domain of G protein coupled receptor
proteins;
[0638] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence covering from the second membrane-spanning domain to
the sixth membrane-spanning domain of G protein coupled receptor
proteins or RNA or DNA (or fragment(s) thereof coding for the amino
acid sequence covering other areas thereof,
[0639] RNA or DNA (or fragment(s) thereof coding for the amino acid
sequence covering from the second membrane-spanning domain to the
seventh membrane-spanning domain of G protein coupled receptor
proteins;
[0640] RNA or DNA (or fragment(s) thereof) coding for the amino
acid sequence covering from the first membrane-spanning domain to
the third membrane-spanning domain of G protein coupled receptor
proteins; and the like, from DNA libraries can be successfully
achieved.
[0641] Among the DNA primers of the present invention, the
combination of
[0642] DNA primer having a nucleotide sequence represented by SEQ
ID NO: 1 or SEQ ID NO: 2; with
[0643] at least one DNA primer selected from the group consisting
of a DNA primer having a nucleotide sequence represented by SEQ ID
NO: 2, a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 4, a DNA primer having a nucleotide sequence represented by
SEQ ID NO: 8, a DNA primer having a nucleotide sequence represented
by SEQ ID NO: 9, a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 15, a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 17 and a DNA primer having a
nucleotide sequence represented by SEQ ID NO: 19;
[0644] is, unlike conventional primers, capable of selectively
amplifying a broad area covering from the first membranespanning
domain to the sixth membrane-spanning domain or other domains of G
protein coupled receptor proteins.
[0645] Among the DNA primers of the present invention, the
combination of
[0646] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 1 or SEQ ID NO: 12; with
[0647] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 11;
[0648] is, unlike conventional primers, capable of selectively
amplifying a broad area covering from the first membranespanning
domain to the seventh membrane-spanning domain or other domains of
G protein coupled receptor proteins.
[0649] Among the DNA primers of the present invention, the
combination of
[0650] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 10 or SEQ ID NO: 16; with
[0651] at least one DNA primer selected from the group consisting
of a DNA primer having a nucleotide sequence represented by SEQ ID
NO: 2, a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 4, a DNA primer having a nucleotide sequence represented by
SEQ ID NO: 8, a DNA primer having a nucleotide sequence represented
by SEQ ID NO: 9, a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 15, a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 17 and a DNA primer having a
nucleotide sequence represented by SEQ ID NO: 19;
[0652] is, unlike conventional primers, capable of selectively
amplifying a broad area covering from the second membranespanning
domain to the sixth membrane-spanning domain or other domains of G
protein coupled receptor proteins.
[0653] Among the DNA primers of the present invention, the
combination of
[0654] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 10 or SEQ ID NO: 16; with
[0655] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 11;
[0656] is, unlike conventional primers, capable of selectively
amplifying a broad area covering from the second membranespanning
domain to the seventh membrane-spanning domain or other domains of
G protein coupled receptor proteins.
[0657] Among the DNA primers of the present invention, the
combination of
[0658] at least one DNA primer selected from the group consisting
of a DNA primer having a nucleotide sequence represented by SEQ ID
NO: 3, a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 5, a DNA primer having a nucleotide sequence represented by
SEQ ID NO: 6, a DNA primer having a nucleotide sequence represented
by SEQ ID NO: 7, a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 14 a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 18; with
[0659] DNA primer having a nucleotide sequence represented by SEQ
ID NO: 11;
[0660] is, unlike conventional primers, capable of selectively
amplifying a broad area covering from the third membranespanning
domain to the seventh membrane-spanning domain or other domains of
G protein coupled receptor proteins.
[0661] Therefore, the protein hydrophobicity plotting of G protein
coupled receptor proteins and the homology at the amino acid level
or the nucleic acid level between G protein coupled receptor
proteins and other similar receptor proteins [said hydrophobicity
plotting and homology both serve as standards for determining
whether or not RNA or DNA (or fragment(s) thereof obtained
according to the present invention is(are) encoding part or all of
the amino acid sequence of G protein coupled receptor protein] can
now be more clearly calculated.
[0662] Among the DNA primers of the present invention, the
combination of
[0663] at least one DNA primer selected from the group consisting
of a DNA primer having a nucleotide sequence represented by SEQ ID
NO: 3, a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 5, a DNA primer having a nucleotide sequence represented by
SEQ ID NO: 6, a DNA primer having a nucleotide sequence represented
by SEQ ID NO: 7, a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 14 and a DNA primer having a nucleotide
sequence represented by SEQ ID NO: 18; with
[0664] at least one DNA primer selected from the group consisting
of a DNA primer having a nucleotide sequence represented by SEQ ID
NO: 2, a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 4, a DNA primer having a nucleotide sequence represented by
SEQ ID NO: 8, a DNA primer having a nucleotide sequence represented
by SEQ ID NO: 9, a DNA primer having a nucleotide sequence
represented by SEQ ID NO: 15, a DNA primer having a nucleotide
sequence represented by SEQ IS NO: 17 and a DNA primer having a
nucleotide sequence represented by SEQ ID NO: 19;
[0665] is capable of amplifying the areas covering from the third
membrane-spanning domain to the sixth membrane-spanning domain
thereof at once like the conventional DNA primers and, moreover, it
is capable of more selectively and efficiently amplifying DNA
coding for G protein coupled receptor proteins though it has not
been obtained through the conventional DNA primers.
[0666] Moreover, among the DNA primers of the present invention,
the combination of
[0667] at least one DNA primer selected from DNA primers having a
nucleotide sequence of SEQ ID NO: 1 and DNA primers having a
nucleotide sequence of SEQ ID NO: 12; with
[0668] DNA primer having a nucleotide sequence represented by SEQ
ID NO: 13;
[0669] is capable of amplifying the areas covering from the first
membrane-spanning domain to the third membrane-spanning domain
thereof at once.
[0670] Then (a) the amplified DNA (or fragment(s) thereof) coding
for the amino acid --sequence of from the first membranespanning
domain to the sixth membrane-spanning domain of G protein coupled
receptor protein, (b) the amplified DNA (or fragment(s) thereof
coding for the amino acid sequence of from the first
membrane-spanning domain to the seventh membrane-spanning domain of
G protein coupled receptor protein, (c) the amplified DNA (or
fragment(s) thereof) coding for the amino acid sequence of from the
third membrane-spanning domain to the sixth membrane-spanning
domain of G protein coupled receptor protein, (d) the amplified DNA
(or fragment(s) thereof coding for the amino acid sequence of from
the third membrane-spanning domain to the seventh membrane-spanning
domain of G protein coupled receptor protein, (e) the amplified DNA
(or fragment(s) thereof coding for the amino acid sequence of from
the second membrane-spanning domain to the sixth membrane-spanning
domain of G protein coupled receptor protein, (f) the amplified DNA
(or fragment(s) thereof coding for the amino acid sequence of from
the second membrane-spanning domain to the seventh
membrane-spanning domain of G protein coupled receptor protein, (g)
the amplified DNA (or fragment(s) thereof coding for the amino acid
sequence of from the first membrane-spanning domain to the third
membrane-spanning domain of G protein coupled receptor protein or
(h) the amplified DNA (or fragment(s) thereof) coding for the amino
acid sequence of other domains of G protein coupled receptor
protein may be used as a probe(s) to screen for full-length DNA
which completely encodes G protein coupled receptor proteins from
DNA libraries according to methods known per se by those of skill
in the art or methods similar thereto.
[0671] The DNA libraries used in the present invention include any
of genome DNA libraries, cDNA libraries and RNA libraries. The term
"DNA library" or "DNA libraries" as used herein refers to a DNA
library or DNA libraries including all of those libraries.
[0672] The present invention further provides screening methods for
target DNA (or fragment(s) thereof) coding for G protein coupled
receptor protein from the DNA library containing DNA (or
fragment(s) thereof) coding for receptor proteins, which comprise
employing the DNA of the present invention as a DNA primer for the
PCR.
[0673] One preferred embodiment of the present invention is a
method for cloning full-length DNA which completely encodes an
amino acid sequence of G protein coupled receptor protein from DNA
libraries which comprises the steps of
[0674] (i) using the DNA of the present invention as a DNA primer
for PCR;
[0675] (ii) carrying out PCR in the presence of a mixture of said
DNA primer with the DNA library to amplify and select (i.e. screen
for) a DNA fragment coding for the amino acid sequence of from the
first membrane-spanning domain to the sixth membrane-spanning
domain of G protein coupled receptor protein, a DNA fragment coding
for the amino acid sequence of from the first membrane-spanning
domain to the seventh membrane-spanning domain of G protein coupled
receptor protein, a DNA fragment coding for the amino acid sequence
of from the third membrane-spanning domain to the sixth
membrane-spanning domain of G protein coupled receptor protein, a
DNA fragment coding for the amino acid sequence of from the third
membrane-spanning domain to the seventh membrane-spanning domain of
G protein coupled receptor protein, a DNA fragment coding for the
amino acid sequence of from the second membrane-spanning domain to
the sixth membrane-spanning domain of G protein coupled receptor
protein, a DNA fragment coding for the amino acid sequence of from
the second membrane-spanning domain to the seventh
membrane-spanning domain of G protein coupled receptor protein, a
DNA fragment coding for the amino acid sequence of from the first
membrane-spanning domain to the third membrane spanning domain of G
protein coupled receptor protein or a DNA fragment coding for other
domains of G protein coupled receptor protein; and
[0676] (iii) cloning said full-length DNA from the DNA library
according to cloning methods known per se by those of skill in the
art or methods similar thereto by using, as a probe, the DNA
fragment obtained in the above step (ii).
[0677] Preferably, an embodiment of the present invention is a
screening method of DNA coding for G protein coupled receptor
proteins from DNA libraries, which comprises carrying out a
polymerase chain reaction in the presence of a mixture of
[0678] the DNA library,
[0679] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 3, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 5, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 6, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 7, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 10, DNA primers having a
nucleotide sequence represented by SEQ ID NO: 14, DNA primers
having a nucleotide sequence represented by SEQ ID NO: 16 and DNA
primers having a nucleotide sequence represented by SEQ ID NO: 18
and
[0680] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 11, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 15, DNA primers having a
nucleotide sequence represented by SEQ ID NO: 17 and DNA primers
having a nucleotide sequence represented by SEQ ID NO: 19 to
selectively amplify template DNA coding for G protein coupled
receptor protein contained in the DNA library.
[0681] More preferably, embodiments of the present invention
include:
[0682] (1) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof from a DNA library, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0683] the DNA library,
[0684] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0685] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19
[0686] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof) contained in the DNA library;
[0687] (2) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof from a DNA library, which comprises
carrying out a polymerase chain reaction in the presence of a
mixture of
[0688] the DNA library,
[0689] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0690] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11
[0691] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof) contained in the DNA library;
[0692] (3) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the second transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof) from a DNA library, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0693] the DNA library,
[0694] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 10 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 16 and
[0695] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19
[0696] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the second transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof contained in the DNA library;
[0697] (4) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the second transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof) from a DNA library, which comprises
carrying out a polymerase chain reaction in the presence of a
mixture of
[0698] the DNA library,
[0699] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 10 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 16 and
[0700] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11
[0701] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the second transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof) contained in the DNA library;
[0702] (5) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the third transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof) from a DNA library, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0703] the DNA library,
[0704] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 3, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 5, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 6, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 7, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 14 and DNA primers having a nucleotide
sequence represented by SEQ ID NO: 18 and
[0705] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 2, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 4, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 8, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 9, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 15, DNA primers having a nucleotide
sequence represented by SEQ ID NO: 17 and DNA primers having a
nucleotide sequence represented by SEQ ID NO: 19
[0706] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the third transmembrane domain to the
sixth transmembrane domain of G protein coupled receptor protein or
other domains thereof) contained in the DNA library;
[0707] (6) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the third transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof from a DNA library, which comprises
carrying out a polymerase chain reaction in the presence of a
mixture of
[0708] the DNA library,
[0709] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 3, DNA primers having a nucleotide sequence represented by SEQ
ID NO: 5, DNA primers having a nucleotide sequence represented by
SEQ ID NO: 6, DNA primers having a nucleotide sequence represented
by SEQ ID NO: 7, DNA primers having a nucleotide sequence
represented by SEQ ID NO: 14 and DNA primers having a nucleotide
sequence represented by SEQ ID NO: 18 and
[0710] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 11
[0711] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the third transmembrane domain to the
seventh transmembrane domain of G protein coupled receptor protein
or other domains thereof contained in the DNA library; and
[0712] (7) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
third transmembrane domain of G protein coupled receptor protein or
other domains thereof) from a DNA library, which comprises carrying
out a polymerase chain reaction in the presence of a mixture of
[0713] the DNA library,
[0714] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 1 and DNA primers having a nucleotide sequence represented by
SEQ ID NO: 12 and
[0715] at least one DNA primer selected from the group consisting
of DNA primers having a nucleotide sequence represented by SEQ ID
NO: 13
[0716] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein and the like (e.g.
the regions spanning from the first transmembrane domain to the
third transmembrane domain of G protein coupled receptor protein or
other domains thereof contained in the DNA library.
[0717] Particularly preferably, embodiments of the present
invention include:
[0718] (8) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein from a DNA library,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0719] the DNA library,
[0720] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 1 and
[0721] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 2
[0722] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein contained in the DNA
library;
[0723] (9) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein from a DNA library,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0724] the DNA library,
[0725] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 3 and
[0726] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 4
[0727] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein contained in the DNA
library;
[0728] (10) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein from a DNA library,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0729] the DNA library,
[0730] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 6 and
[0731] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 8
[0732] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein contained in the DNA
library; and
[0733] ( 11) a screening method of DNA coding for the amino acid
sequence of G protein coupled receptor protein from a DNA library,
which comprises carrying out a polymerase chain reaction in the
presence of a mixture of
[0734] the DNA library,
[0735] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 10 and
[0736] a DNA primer having a nucleotide sequence represented by SEQ
ID NO: 11
[0737] to selectively amplify the DNA coding for the amino acid
sequence of G protein coupled receptor protein contained in the DNA
library.
[0738] The cloned DNAs can be analyzed, usually by restriction
enzyme analysis and/or sequencing.
[0739] Target RNA or DNA (or fragment(s) thereof) coding for G
protein coupled receptor protein in the amplification and the
screening by the PCR techniques wherein the DNA of the present
invention is employed may include RNA, DNA or fragments thereof
coding for known (or prior art) G protein coupled receptor proteins
and RNA, DNA or fragments thereof coding for unknown (novel) G
protein coupled receptor proteins.
[0740] These target RNA or DNA (or fragment(s) thereof) may include
novel nucleotide sequences and even known nucleotide sequences.
[0741] Examples of such nucleotide sequences are RNA or DNA (or
fragment(s)) coding for a G protein coupled receptor protein, said
RNA or DNA (or fragment(s)) being derived from all cells and
tissues (e.g. pituitary gland, brain, pancreas, lung, adrenal
gland, etc.) of vertebrate animals (e.g. mice, rats, cats, dogs,
swines, cattle, horses, monkeys, human beings, etc.), insects or
other invertebrate animals (e.g. drosophilae, silkworms, Barathra
brassicae, etc.), plants (e.g. rice plant, wheat, tomato, etc.) and
cultured cell lines derived therefrom, etc.
[0742] Specific examples of the nucleotide sequences are RNA or DNA
(or fragment(s)) coding for G protein coupled receptor proteins
such as receptor proteins to angiotensin, bombesin, canavinoid,
cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y,
opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal
and related peptide), somatostatin, dopamine, motilin, amylin,
bradykinin, CGRP (calcitonin gene related peptide), adrenomedullin,
leukotriene, pancreastatin, .alpha., GRO.beta., GRO.gamma. NAP-2,
ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, 1-309, MIP1.alpha.,
MIP-1.beta., RANTES, etc.), endothelia, enterogastrin, histamine,
neurotensin, TRH, pancreatic polypeptide, galanin, family members
thereof, etc.
[0743] In the PCR amplification using the DNA of the present
invention, the DNA (or DNA fragment) acting as a template may
include any DNA so far as it is derived from the above-mentioned
tissues and cells. More specifically, the template DNA (or DNA
fragment) includes any of genome DNA, genome DNA libraries, cDNA
derived from the tissues and cells and cDNA libraries derived from
the tissues and cells. cDNA libraries derived from human tissues
and cells are particularly suitable. Vectors to be used in the DNA
library may include any of bacteriophages, plasmids, cosmids,
phagimids, etc. It is also possible to directly amplify the
template DNA (or DNA fragment) by reverse transcriptase polymerase
chain reaction (RT-PCR) techniques using mRNA fractions prepared
from the tissues and cells. The DNA which is to be a template may
be either DNA completely coding for G protein coupled receptor
proteins or DNA fragments (or segments) thereof.
[0744] Preferably, the RNA or DNA (or fragment(s) thereof) obtained
via the instant screening method for G protein coupled receptor
protein coding DNA wherein said method uses the DNA according to
the present invention is a G protein coupled receptor
protein-encoding RNA or DNA (or fragment(s) thereof) contained in
the used DNA library. More specifically, it is an RNA or DNA (or
RNA fragment(s) or DNA fragment(s) (hereinafter, may be often
abbreviated as just "DNA") coding for G protein coupled receptor
proteins such as angiotensin receptor, bombesin receptor,
canavinoid receptor, cholecystokinin receptor, glutamine receptor,
serotonin receptor, melatonin receptor, neuropeptide Y receptor,
opioid receptor, purine receptor, vasopressin receptor, oxytocin
receptor, VIP receptor (vasoactive intestinal and related peptide
receptor), somatostatin receptor, dopamine receptor, motilin
receptor, amylin receptor, bradykinin receptor, CGRP receptor
(calcitonin gene related peptide receptor), adrenomedullin
receptor, leukotriene receptor, pancreastatin receptor,
prostaglandin receptor, thromboxane receptor, adenosine receptor,
adrenaline receptor, .alpha.- and .beta.-chemokine receptor
(receptors to IL-8, GRO.alpha., GRO.beta., GRO.gamma., NAP-2,
ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3, 1-309, MIP1.alpha.,
MIP-1.beta., RANTES, etc.), endothelin receptor, enterogastrin
receptor, histamine receptor, neurotensin receptor, TRH receptor,
pancreatic polypeptide receptor, galanin receptor, their family
member receptors, etc.
[0745] When the DNA obtained by the screening method of the present
invention is the DNA fragment which partially codes for a G protein
coupled receptor protein, it is possible to isolate DNA completely
encoding said G protein coupled receptor protein from a suitable
DNA library according to cloning techniques known per se by using
said DNA fragment as a probe.
[0746] Means for cloning the DNA completely encoding G protein
coupled receptor proteins may include a PCR amplification employing
a synthetic DNA primer having the partial nucleotide sequence of
the DNA fragment partially coding for the G protein coupled
receptor protein and a selection of the target DNA via a
hybridization with DNA or synthetic DNA having part or all of the
region of said DNA fragments. The hybridization may be conducted,
for example, by the methods described in Molecular Cloning, 2nd
ed.; J. Sambrook et al., Cold Spring Harbor Lab-. Press, 1989. When
the commercially available library is used, it may be conducted
according to the manners described in the protocols attached
thereto.
[0747] The DNA completely encoding G protein coupled receptor
protein (full-length G protein coupled receptor protein DNA) may be
used, depending upon its object, either as it is or after digesting
with a restriction enzyme or after ligating with a linker if
desired. Said DNA may have ATG at the 5'-terminal as the
translation initiation codon and TAA, TGA or TAG at the 3' terminal
as the translation termination codon. These translation initiation
codons and translation termination codons may be added using a
suitable synthetic DNA adaptor. In addition, it is possible to
determine said receptor protein-expressing tissues/cells by
northern blottings using said DNA as a probe. It is also possible
to express target receptor proteins by introducing DNA having the
entire coding region of the receptor protein into animal cells
after binding with a suitable promoter.
[0748] The G protein coupled receptor protein according to the
present invention is a G protein coupled receptor protein encoded
by the G protein coupled receptor protein-encoding DNA obtained by
the screening method of the present invention. More specifically,
the G protein coupled receptor protein according to the present
invention includes G protein coupled receptor proteins such as
angiotensin receptor protein, bombesin receptor protein, canavinoid
receptor protein, cholecystokinin receptor protein, glutamine
receptor protein, serotonin receptor protein, melatonin receptor
protein, neuropeptide Y receptor protein, opioid receptor protein,
purine receptor protein, vasopressin receptor protein, oxytocin
receptor protein, VIP receptor protein (vasoactive intestinal and
related peptide receptor protein), somatostatin receptor protein,
dopamine receptor protein, motilin receptor protein, amylin
receptor protein, bradykinin receptor protein, CGRP receptor
protein (calcitonin gene related peptide receptor protein),
adrenomedullin receptor protein, leukotriene receptor protein,
pancreastatin receptor protein, prostaglandin receptor protein,
thromboxane receptor protein, adenosine receptor protein,
adrenaline receptor protein, .alpha.- and .beta.Q -chemokine
receptor protein (receptor protein responsive to IL-8, GRO.alpha.,
GRO.beta., GRO.gamma., NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1,
HC14, MCP-3, 1-309, MIP1.alpha., MIP-1.beta., RANTES, etc.),
endothelin receptor protein, enterogastrin receptor protein,
histamine receptor protein, neurotensin receptor protein, TRH
receptor protein, pancreatic polypeptide receptor protein, galanin
receptor protein, family members thereof, etc.
[0749] According to the present invention, novel G protein coupled
receptors proteins, peptide segments or fragments derived from the
G protein coupled receptor protein, modified derivatives or
analogues thereof, and salts thereof may be recognized, cloned,
produced, isolated or characterized.
[0750] These G protein coupled receptor proteins are those derived
from all cells and tissues (e.g. pituitary gland, pancreas, brain,
kidney, liver, gonad, thyroid gland, cholecyst, bone marrow,
adrenal, skin, muscle, lung, digestive duct, blood vessel, heart,
etc.) of warm-blooded animals (e.g. guinea pig, rat, mouse, swine,
sheep, cattle, monkey, human beings, rabbit, cat, dog, horse,
etc.), and any of proteins as long as they comprise an amino acid
sequence selected from the group consisting of an amino acid
sequence represented by SEQ ID NO: 24, an amino acid sequence
represented by SEQ ID NO: 25, an amino acid sequence represented by
SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27,
an amino acid sequence represented by SEQ ID NO: 28, an amino acid
sequence represented by SEQ ID NO: 34, an amino acid sequence
represented by SEQ ID NO: 35, an amino acid sequence represented by
SEQ ID NO: 38, an amino acid sequence represented by SEQ ID NO: 39,
an amino acid sequence represented by SEQ ID NO: 56, and
substantial equivalents to the amino acid sequence represented by
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 39,
and/or SEQ ID NO: 56.
[0751] In one embodiment of the present invention, G protein
coupled receptor proteins are those derived from all cells and
tissues (e.g. pituitary gland, pancreas, brain, kidney, liver,
gonad, thyroid gland, cholecyst, bone marrow, adrenal, skin,
muscle, lung, digestive duct, blood vessel, heart, etc.) of
warm-blooded animals (e.g. guinea pig, rat, mouse, swine, sheep,
cattle, monkey, human beings, cat, dog, horse, etc.), and any of
proteins as long as they comprise an amino acid sequence selected
from the group consisting of an amino acid sequence represented by
SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25,
an amino acid sequence represented by SEQ ID NO: 26, an amino acid
sequence represented by SEQ ID NO: 27, an amino acid sequence
represented by SEQ ID NO: 28, and substantial equivalents to the
amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. These G protein
coupled receptor proteins may include proteins having an amino acid
sequence selected from the group consisting of an amino acid
sequence represented by SEQ ID NO: 24, an amino acid sequence
represented by SEQ ID NO: 25, an amino acid sequence represented by
SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27
and an amino acid sequence represented by SEQ ID NO: 28, proteins
wherein the amino acid sequence thereof is about 90% to 99.9%
homologous to an amino acid sequence represented by SEQ ID NO: 24,
an amino acid sequence represented by SEQ ID NO: 25, an amino acid
sequence represented by SEQ ID NO: 26, an amino acid sequence
represented by SEQ ID NO: 27 or an amino acid sequence represented
by SEQ ID NO: 28 and the activity thereof is substantially
equivalent to the protein having an amino acid sequence represented
by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO:
25, an amino acid sequence represented by SEQ ID NO: 26, an amino
acid sequence represented by SEQ ID NO: 27 or an amino acid
sequence represented by SEQ ID NO: 28 and the like. The
substantially equivalent activity may include ligand binding
activity, signal information transmitting, etc. The term
"substantially equivalent" or "substantial equivalent" means that
the nature of the ligand binding activity and the like is
equivalent. Therefore, it is allowable that even differences among
grades such as ligand binding affinity grades and ligand binding
activity grades and quantitative factors such as molecular weights
of receptor proteins are present.
[0752] In another embodiment of the present invention, G protein
coupled receptor proteins include human pituitary gland-derived G
protein coupled receptor proteins comprising an amino acid sequence
selected from the group consisting of an amino acid sequence
represented by SEQ ID NO: 24, and/or an amino acid sequence
represented by SEQ ID NO: 25, mouse pancreas-derived G protein
coupled receptor proteins comprising an amino acid sequence
represented by SEQ ID NO: 27, mouse pancreas-derived G protein
coupled receptor proteins comprising an amino acid sequence
represented by SEQ ID NO: 28, etc. Examples of the human pituitary
gland-derived G protein coupled receptor protein comprising an
amino acid sequence selected from the group consisting of an amino
acid sequence represented by SEQ ID NO: 24, and an amino acid
sequence represented by SEQ ID NO: 25, are human pituitary
gland-derived G protein coupled receptor proteins comprising an
amino acid sequence represented by SEQ ID NO: 24, etc. These G
protein coupled receptor proteins may include proteins wherein one
or more amino acid residues (preferably from 2 to 30 amino add
residues, more preferably from 2 to 10 amino acid residues) are
deleted from the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:
25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, proteins wherein
one or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, proteins wherein one
or more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) in the
amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
SEQ ID NO: 27 or SEQ ID NO: 28, are substituted with one or more
amino acid residues, etc.
[0753] In yet another embodiment of the present invention, G
protein coupled receptor proteins include those derived from, all
cells and tissues (e.g. amygdaloid nucleus, pituitary gland,
pancreas, brain, kidney, liver, gonad, thyroid gland, cholecyst,
bone marrow, lung, digestive duct, blood vessel, heart, thymus,
spleen, leukocyte, etc.) of warm-blooded animals (e.g. guinea pig,
rat, mouse, pig, sheep, cattle, monkey, human beings, etc.), and
any of proteins as long as they comprise an amino acid sequence
selected from the group consisting of an amino acid sequence
represented by SEQ ID NO: 34 and/or an amino acid sequence
represented by SEQ ID NO: 35. These G protein coupled receptor
proteins may include proteins having an amino acid sequence
selected from the group consisting of an amino acid sequence
represented by SEQ ID NO: 34 or/and an amino acid sequence
represented by SEQ ID NO: 35, proteins wherein the amino acid
sequence thereof is about 90% to 99.9% homologous to an amino acid
sequence represented by SEQ ID NO: 34 or/and an amino acid sequence
represented by SEQ ID NO: 35 and the activity thereof is
substantially equivalent to the protein having an amino acid
sequence represented by SEQ ID NO: 34 and/or an amino acid sequence
represented by SEQ ID NO: 35, and the like. The substantially
equivalent activity may include ligand binding activity, signal
information transmitting, etc. The term "substantially equivalent"
or "substantial equivalent" means that the nature of the ligand
binding activity and the like is equivalent. Therefore, it is
allowable that even differences among grades such as ligand binding
affinity grades and ligand binding activity grades and quantitative
factors such as molecular weights of receptor proteins are present.
Examples of the G protein coupled receptor protein are human
amygdaloid nucleus-derived G protein coupled receptor proteins
having an amino acid sequence selected from the group consisting of
an amino acid sequence represented by SEQ ID NO: 34 and/or an amino
acid sequence represented by SEQ ID NO: 35, etc. These G protein
coupled receptor proteins may include proteins wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are deleted from
the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35, proteins
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) are added to the amino acid sequence of SEQ ID NO: 34 or
SEQ ID NO: 35, proteins wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) in the amino acid sequence of SEQ ID
NO: 34 or SEQ ID NO: 35, are substituted with one or more amino
acid residues, etc.
[0754] In still another embodiment of the present invention, these
G protein coupled receptor proteins are those derived from all
cells and tissues (e.g. amygdaloid nucleus, pituitary body,
pancreas, brain, kidney, liver, gonad, thyroid gland, cholecyst,
bone marrow, lung, digestive duct, blood vessel, heart, thymus,
leukocyte, etc.) of warm-blooded animals (e.g. guinea pig, rat,
mouse, swine, sheep, cattle, monkey, human beings, etc.), and any
of proteins as long as they comprise an amino acid sequence
represented by SEQ ID NO: 38, or substantial equivalents to the
amino acid sequence represented by SEQ ID NO: 38, preferably an
amino acid sequence represented by SEQ ID NO: 39, or substantial
equivalents to the amino acid sequence represented by SEQ ID NO:
39. These G protein coupled receptor proteins may include proteins
having an amino acid sequence represented by SEQ ID NO: 38,
proteins wherein the amino acid sequence thereof is about 90% to
99.9% homologous to an amino acid sequence represented by SEQ ID
NO: 38 and the activity thereof is substantially equivalent to the
protein having an amino acid sequence represented by SEQ ID NO: 38
and the like. These G protein coupled receptor proteins are
preferably proteins having an amino acid sequence represented by
SEQ ID NO: 39, proteins wherein the amino acid sequence thereof is
about 90% to 99.9% homologous to an amino acid sequence represented
by SEQ ID NO: 39 and the activity thereof is substantially
equivalent to the protein having an amino acid sequence represented
by SEQ ID NO: 39, etc. The substantially equivalent activity may
include ligand binding activity, signal information transmitting,
etc. The term "substantially equivalent" or "substantial
equivalent" means that the nature of the ligand binding activity
and the like is equivalent. Therefore, it is allowable that even
differences among grades such as ligand binding affinity grades and
ligand binding activity grades and quantitative factors such as
molecular sizes or weights of receptor proteins are present.
[0755] It is suggested by data that the mouse pancreatic a-cell
strain, MIN6-derived receptor protein of the present invention
(e.g., SEQ ID NO: 38 and SEQ ID NO: 39, or proteins encoded by
pMAH2-17) is a novel purihoceptor subtype which is clearly distinct
from prior art purinoceptors.
[0756] In another more specific embodiment of the present
invention, G protein coupled receptor proteins include mouse
pancreatic .beta.-cell line, MIN6, derived G protein coupled
receptor proteins comprising an amino acid sequence represented by
SEQ ID NO: 38, mouse pancreatic .beta.-cell line, MIN6, derived G
protein coupled receptor proteins wherein one or more amino acid
residues (preferably from 2 to 30 amino acid residues, more
preferably from 2 to 10 amino acid residues) are deleted from the
amino acid sequence of SEQ ID NO: 38, proteins wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are added to the
amino acid sequence of SEQ ID NO:38, proteins wherein one or more
amino acid residues (preferably from 2 to 30 amino acid residues,
more preferably from 2 to 10 amino acid residues) are substituted
with other amino acid residues in the amino acid sequence of SEQ ID
NO:38, etc. Further preferably these G protein coupled receptor
proteins include mouse pancreatic .beta.-cell line, MIN6, derived G
protein coupled receptor proteins comprising an amino acid sequence
represented by SEQ ID NO: 39, mouse pancreatic .beta.-cell line,
MIN6, derived G protein coupled receptor proteins wherein one or
more amino acid residues (preferably from 2 to 30 amino acid
residues, more preferably from 2 to 10 amino acid residues) are
deleted from the amino acid sequence of SEQ ID NO: 39, proteins
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) are added to the amino acid sequence of SEQ ID NO: 39,
proteins wherein one or more amino acid residues (preferably from 2
to 30 amino acid residues, more preferably from 2 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 39 are
substituted with other amino acid residues, etc.
[0757] In still another embodiment of the present invention, these
G protein coupled receptor proteins are those derived from all
cells and tissues (e.g. placenta, gonad, amygdaloid nucleus,
pituitary body, pancreas, brain, kidney, liver, thyroid gland,
cholecyst, bone marrow, lung, digestive duct, blood vessel, heart,
thymus, leukocyte, etc.) of human beings, and any of proteins as
long as they comprise an amino acid sequence represented by SEQ ID
NO: 56, or substantial equivalents to the amino acid sequence
represented by. SEQ ID NO: 56. These G protein coupled receptor
proteins may include proteins having an amino acid sequence
represented by SEQ ID NO: 56, proteins wherein the amino acid
sequence thereof is about 90% to 99.9% homologous to an amino acid
sequence represented by SEQ ID NO: 56 and the activity thereof is
substantially equivalent to the protein having an amino acid
sequence represented by SEQ ID NO: 56 and the like. The
substantially equivalent activity may include ligand binding
activity, signal information transmitting, etc. The term
"substantially equivalent" or "substantial equivalent" means that
the nature of the ligand binding activity and the like is
equivalent. Therefore, it is allowable that even differences among
grades such as ligand binding affinity grades and ligand binding
activity grades and quantitative factors such as molecular sizes or
weights of receptor proteins are present.
[0758] In another more specific embodiment of the present
invention, G protein coupled receptor proteins include G protein
coupled receptor proteins comprising an amino acid sequence
represented by SEQ ID NO: 56, G protein coupled receptor proteins
wherein one or more amino acid residues (preferably from 2 to 30
amino acid residues, more preferably from 2 to 10 amino acid
residues) are deleted from the amino acid sequence of SEQ ID NO:
56, proteins wherein one or more amino acid residues (preferably
from 2 to 30 amino acid residues, more preferably from 2 to 10
amino acid residues) are added to the amino acid sequence of SEQ ID
NO: 56, proteins wherein one or more amino acid residues
(preferably from 2 to 30 amino acid residues, more preferably from
2 to 10 amino acid residues) in the amino acid sequence of SEQ ID
NO: 56, are substituted with other amino acid residues, etc.
[0759] A portion of the amino acid sequence may be modified (e.g.
addition, deletion, substitution with other amino acids, etc.) in
the G protein coupled receptor proteins of the present
invention.
[0760] Furthermore, the G protein coupled receptor proteins of the
present invention includes those wherein N-terminal Met is
protected with a protecting group (e.g., C.sub.1-6 acyl group such
as formyl, acetyl, etc.), those wherein the N-terminal side of Glu
is cleaved in vivo to make said Glu pyroglutaminated, those wherein
the intramolecular side chain of amino acids is protected with a
suitable protecting group (e.g., C.sub.1-6 acyl group such as
formyl, acetyl, etc.), conjugated proteins such as so-called
"glycoproteins" wherein saccharide chains are bonded, etc.
[0761] The salt of said G protein coupled receptor protein of the
present invention includes preferably physiologically acceptable
acid addition salts. Examples of such salts are salts thereof with
inorganic acids (e.g. hydrochloric acid, phosphoric acid,
hydrobromic acid, sulfuric acid, etc.), salts thereof with organic
acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid,
maleic acid, succinic acid, tartaric acid, citric acid, malic acid,
oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic
acid, etc.), etc.
[0762] The G protein coupled receptor protein or its salt of the
present invention may be manufactured from the tissues or cells of
warm-blooded animals by purifying methods which are known per se by
those skilled in the art or methods similar thereto or may be
manufactured by culturing the transformant (or transfectant) (as
described herein below) containing G protein coupled receptor
protein encoding DNA. The protein or its salt of the present
invention may be manufactured by the peptide synthesis as described
herein below.
[0763] The G protein coupled receptor protein fragment (the partial
peptide of said G protein coupled receptor protein) may include,
for example, the site which is exposed outside cell membranes,
among the G protein coupled receptor protein molecule. Examples of
the fragment are peptides containing a region which is analyzed as
an extracellular area (hydrophilic region or site) in a hydrophobic
plotting analysis on the G protein coupled receptor protein
represented by any of FIGS. 24, 25, 28, 31, 32, 36, 38, 41, 44, 47,
50, 53, 57, 58, 59, 64, 70, 74, and 78. A peptide which partly
contains a hydrophobic region or site may be used as well. Further,
a peptide which separately contains each domain may be used too
although the partial peptide (peptide fragment) which contains
plural domains at the same time will be used as well.
[0764] The salt of said G protein coupled receptor protein fragment
(partial peptide thereof) includes preferably physiologically
acceptable acid addition salts. Examples of such salts are salts
thereof with inorganic acids (e.g. hydrochloric acid, phosphoric
acid, hydrobromic acid, sulfuric acid, etc.), salts thereof with
organic acids (e.g. acetic acid, formic acid, propionic acid,
fumaric acid, maleic acid, succinic acid, tartaric acid, citric
acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,
benzenesulfonic acid, etc.), etc.
[0765] The G protein coupled receptor protein fragment (the partial
peptide of the G protein coupled receptor protein) may be
manufactured by synthesizing methods for peptides which are known
per se by those skilled in the art or methods similar thereto or by
cleaving (digesting) G protein coupled receptor proteins by a
suitable peptidase. Methods of synthesizing peptide may be any of a
solid phase synthesis and a liquid phase synthesis. Thus, a partial
peptide (peptide fragment) or amino acids which can construct the
protein of the present invention is condensed with the residual
part thereof and, when the product has a protective group, said
protective group is detached whereupon a desired peptide can be
manufactured. Examples of the known methods for condensation and
for detachment of protective groups include the following
.quadrature. to .quadrature.:
[0766] M. Bodanszky and M. A. Ondetti: Peptide Synthesis,
Interscience Publishers, New York (1966).
[0767] Schroeder and Luebke: The Peptide, Academic Press, New York,
1965.
[0768] Nobuo Izumiya et al.: Fundamentals and Experiments of the
Peptide Synthesis, Maruzen KK, Japan (1975).
[0769] Haruaki Yajima and Shumpei Sakakibara: "Seikagaku Jikken
Koza 1"(Experiments of Biochemistry, Part 1), "Tanpakusitu No
Kagaku IV" (Chemistry of Protein, IV), p.205 (1977), Japan.
[0770] Haruaki Yajima (ed): Development of Pharmaceuticals (Second
Series), Vol. 14, Peptide Synthesis, Hirokawa Shoten, Japan.
[0771] After the reaction, conventional purifying techniques such
as salting-out, extraction with solvents, distillation, column
chromatography, liquid chromatography, electrophoresis,
recrystallization, etc. are optionally combined so that the protein
of the present invention can be purified and isolated. When the
protein obtained as such is a free compound, it may be converted to
a suitable salt by known methods while, when it is obtained as a
salt, the salt may be converted to a free compound or other salt
compounds by known methods.
[0772] Furthermore, the product may be manufactured by culturing
the transformant (transfectant) containing the DNA coding for said
partial peptide.
[0773] The G protein coupled receptor protein-encoding DNA obtained
by the above-mentioned screening method using the DNA of the
present invention and the G protein coupled receptor protein
encoded by said DNA or the peptide fragment (partial peptide
thereof encoded by said DNA may, for example, be used for the
determination of a ligand to said G protein coupled receptor
protein or for the screening of a compound which inhibits the
binding of said protein coupled receptor protein with a ligand.
[0774] In that case, an expression system for the G protein coupled
receptor protein-encoding DNA is at first constructed. Hosts for
said DNA may be any of animal cells, insect cells, yeasts, Bacillus
subtilis, Escherichia coli, etc. Promoters used therefor may be
anyone so far as it is suitable as a promoter for the host used for
gene expression. Incidentally, the utilization of enhancers for
expression is effective as well.
[0775] Then the expressing cells per se which constructed to
express the G protein coupled receptor protein or the cell membrane
fractions prepared therefrom by methods known per se by those
skilled in the art or methods similar thereto may be subjected to a
variety of receptor binding experiments. Ligands used therefor may
include any of compounds labeled by a commercially available
radioisotope, etc., culture supernatants and tissue extracts which
are directly labeled by a chloramine T method or by a
lactoperoxidase method. Separation of bonded or free ligands may be
carried out by a direct washing when cells adhered to substrates
are used, while, in the case of floating cells or cell membrane
fractions thereof, it may be carried out by means of centrifugal
separation or filtration. Nonspecific binding with container, etc.
may be estimated by addition of unlabeled ligands which are about
100 times as much concentrated relatively to the poured labeled
ligand.
[0776] The ligand which is obtained by such a receptor binding
experiment may be subjected to a discrimination of agonist versus
antagonist.
[0777] To be more specific, a natural substance or compound which
is presumed to be a ligand with the G protein coupled receptor
protein-expressing cell is cultured and, after that, the culture
supernatant liquid is collected or the cell is extracted. A change
in the components contained therein is measured by, for example, a
commercially available measuring kit (e.g. kits for cAMP,
diacylglycerol, cGMP, proteinkinase A, etc.). Alternatively, it is
possible to measure physiological responses such as liberation of
Pura-2, [.sup.3H]arachidonic acid and [.sup.3H]inositol phosphate
metabolites by methods known per se by those skilled in the art or
methods similar thereto. The compound or natural substance which is
obtained by such a screening is an agonist for said G protein
coupled receptor protein or an antagonist for said G protein
coupled receptor protein and is presumed to act on the tissues and
cells in which said receptor is distributed. Accordingly, it is
possible to check the pharmaceutical response (pharmaceutical
effect) more efficiently by referring to the distribution disclosed
(clarified) by a northern blotting or the like. Moreover, a
development of compounds having a novel pharmaceutical response
(pharmaceutical effect) in, for example, central nervous tissues,
circulatory system, kidney, pancreas, etc. is expected. An
efficient development of pharmaceuticals can be proceeded by
amplifying G protein coupled receptor protein-encoding DNA
selectively from tissues.
[0778] The G protein coupled receptor protein-encoding DNA of the
present invention may be any coding DNA as long as it contains a
nucleotide sequence coding for a G protein coupled receptor protein
which contains an amino acid sequence substantially equivalent to
the amino acid sequence having SEQ ID NO: 24 and/or which has an
activity substantially equivalent to the amino acid sequence having
SEQ ID NO: 24, a G protein coupled receptor protein which contains
an amino acid sequence substantially equivalent to the amino acid
sequence having SEQ ID NO: 25 and/or which has an activity
substantially equivalent to the amino acid sequence having SEQ ID
NO: 25, a G protein coupled receptor protein which contains an
amino acid sequence substantially equivalent to the amino acid
sequence having SEQ ID NO: 26 and/or which has an activity
substantially equivalent to the amino acid sequence having SEQ ID
NO: 26, a G protein coupled receptor protein which contains an
amino acid sequence substantially equivalent to the amino acid
sequence having SEQ ID NO: 27 and/or which has an activity
substantially equivalent to the amino acid sequence having SEQ ID
NO: 27, or a G protein coupled receptor protein which contains an
amino acid sequence substantially equivalent to the amino acid
sequence having SEQ ID NO: 28 and/or which has an activity
substantially equivalent to the amino acid sequence having SEQ ID
NO: 28.
[0779] Still the G protein coupled receptor protein-encoding DNA of
the present invention may be any coding DNA as long as it contains
a nucleotide sequence coding for a G protein coupled receptor
protein which contains an amino acid sequence substantially
equivalent to the amino acid sequence having SEQ ID NO: 34 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 34, or a G protein coupled receptor
protein which contains an amino acid sequence substantially
equivalent to the amino acid sequence having SEQ ID NO: 35 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 35.
[0780] Yet the G protein coupled receptor protein-encoding DNA of
the present invention may be any coding DNA as long as it contains
a nucleotide sequence coding for a G protein coupled receptor
protein which contains an amino acid sequence substantially
equivalent to the amino acid sequence having SEQ ID NO: 38 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 38, or preferably a G protein coupled
receptor protein which contains an amino acid sequence
substantially equivalent to the amino acid sequence having SEQ ID
NO: 39 and/or which has an activity substantially equivalent to the
amino acid sequence having SEQ ID NO: 39.
[0781] Yet the G protein coupled receptor protein-encoding DNA of
the present invention may be any coding DNA as long as it contains
a nucleotide sequence coding for a G protein coupled receptor
protein which contains an amino acid sequence substantially
equivalent to the amino acid sequence having SEQ ID NO: 56 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 56, or preferably a G protein coupled
receptor protein which contains an amino acid sequence
substantially equivalent to the amino acid sequence having SEQ ID
NO: 56 and/or which has an activity substantially equivalent to the
amino acid sequence having SEQ ID NO: 56.
[0782] The DNA of the present invention may be any one of a human
genome DNA, a human genome DNA library, a human tissue and
cell-derived cDNA, a human tissue and cell-derived cDNA library and
a synthetic DNA. The vector used for the library may include
bacteriophage, plasmid, cosmid, phagemid, etc. The DNA can be
further amplified directly by the reverse transcriptase polymerase
chain reaction (hereinafter briefly referred to as "RT-PCR") using
mRNA fractions prepared from tissues and cells.
[0783] In an embodiment, the DNA coding for the human pituitary
gland-derived G protein coupled receptor protein comprising the
amino acid sequence of SEQ ID NO: 24 includes DNA having a
nucleotide sequence represented by SEQ ID NO: 29, etc. The DNA
coding for the human pituitary gland-derived G protein coupled
receptor protein comprising the amino acid sequence of SEQ ID NO:
25 includes DNA having a nucleotide sequence represented by SEQ ID
NO: 30, etc. The DNA coding for the human pituitary gland-derived G
protein coupled receptor protein comprising the amino acid sequence
of SEQ ID NO: 26 includes DNA having a nucleotide sequence
represented by SEQ ID NO: 31, etc. The DNA coding for the mouse
pancreasderived G protein coupled receptor protein comprising the
amino acid sequence of SEQ ID NO: 27 includes DNA having a
nucleotide sequence represented by SEQ ID NO: 32, etc. The DNA
coding for the mouse pancreas-derived G protein coupled receptor
protein comprising the amino acid sequence of SEQ ID NO: 28
includes DNA having a nucleotide sequence represented by SEQ ID NO:
33, etc.
[0784] In another embodiment, the DNA coding for the human
amygdaloid nucleus-derived G protein coupled receptor protein
comprising the amino acid sequence of SEQ ID NO: 34 includes DNA
having a nucleotide sequence represented by SEQ ID NO: 36, etc. The
DNA coding for the human amygdaloid nucleus-derived G protein
coupled receptor protein comprising the amino acid sequence of SEQ
ID NO: 35 includes DNA having a nucleotide sequence represented by
SEQ ID NO: 37, etc. The DNA coding for the human amygdaloid
nucleus-derived G protein coupled receptor protein comprising the
amino acid sequence of SEQ ID NO: 34 or the amino acid sequence of
SEQ ID NO: 35 includes DNA having a nucleotide sequence represented
by SEQ ID NO: 36, DNA having a nucleotide sequence represented by
SEQ ID NO: 37, etc. Still in another embodiment, the DNA coding for
the mouse pancreatic .beta.-cell line, MIN6-derived G protein
coupled receptor protein comprising the amino acid sequence of SEQ
ID NO: 38 includes DNA having a nucleotide sequence represented by
SEQ ID NO: 40, etc. The DNA coding for the mouse pancreatic
.beta.-cell line, MIN6-derived G protein coupled receptor protein
comprising the amino acid sequence of SEQ ID NO: 39 includes DNA
having a nucleotide sequence represented by SEQ ID NO: 41, etc. Yet
in another embodiment, the DNA coding for the human-derived G
protein coupled receptor protein comprising the amino acid sequence
of SEQ ID NO: 56 includes DNA having a nucleotide sequence
represented by SEQ ID NO: 57, etc.
[0785] The DNA completely coding for the G protein coupled receptor
protein of the present invention can be cloned by (1) carrying out
the PCR amplification using a synthetic DNA primer having a partial
nucleotide sequence (nucleotide fragment) of the G protein coupled
receptor protein; or (2) effecting the selection of a DNA
constructed in a suitable vector, based on the hybridization with a
labeled DNA fragment having part or all of the region encoding a
human G protein coupled receptor protein or a labeled synthetic DNA
having part or all of the coding region thereof. The hybridization
is carried out according to methods as disclosed in, for example,
Molecular Cloning, 2nd Ed., J. Sambrook et al., Cold Spring Harbor
Lab. Press, 1989. When a DNA library commercially available in the
market is used, the hybridization is carried out according to
protocols manuals attached thereto.
[0786] The cloned/G protein coupled receptor proteinencoding DNA of
the present invention can be used as it is, or can be used, as
desired, after modifications including digestion with a restriction
enzyme or addition of a linker or adapter, etc. depending upon
objects. The DNA may have an initiation codon, ATG, on the 5'
terminal side and a termination codon, TAA, TGA or TAG, on the 3'
terminal side. These initiation and termination codons can be
ligated by using a suitable synthetic DNA adapter.
[0787] An expression vector for G protein coupled receptor proteins
can be produced by, for example, (a) cutting out a target DNA
fragment from the G protein coupled receptor protein-encoding DNA
of the present invention and (b) ligating the target DNA fragment
with the downstream site of a promoter in a suitable expression
vector.
[0788] The vector may include plasmids derived from Escherichia
coli (e.g., pBR322, pBR325, pUC12, pUC13, etc.), plasmids derived
from Bacillus subtilis (e.g., pUBilO, pTP5, pC194, etc.), plasmids
derived from yeasts (e.g., pSH19, pSH15, etc.), bacteriophages such
as .lambda.-phage, and animal virus such as retrovirus, vaccinia
virus and baculovirus.
[0789] According to the present invention, any promoter can be used
as long as it is compatible with a host which is used for
expressing a gene. When the host for the transformation is E. coli,
the promoters are preferably trp promoters, lac promoters, recA
promoters, .lambda..sub.PL promoters, lpp promoters, etc. When the
host for the transformation is the Bacillus, the promoters are
preferably SPO1 promoters, SP02 promoters, penP promoters, etc.
When the host is an yeast, the promoters are preferably PH05
promoters, PGK promoters, GAP promoters, ADH promoters, etc. When
the host is an animal cell, the promoters include SV40-derived
promoters, retrovirus promoters, metallothionein promoters, heat
shock promoters, cytomegalovirus promoters, SRa promoters, etc. An
enhancer can be effectively utilized for the expression.
[0790] As required, furthermore, a host-compatible signal sequence
is added to the N-terminal side of the G protein coupled receptor
protein. When the host is E. coli, the utilizable signal sequences
may include alkaline phosphatase signal sequences, OmpA signal
sequences, etc. When the host is the Bacillus, they may include a
-amylase signal sequences, subtilisin signal sequences, etc. When
the host is an yeast, they may include mating factor a signal
sequences, invertase signal sequences, etc. When the host is an
animal cell, they may include insulin signal sequences,
a-interferon signal sequences, antibody molecule signal sequences,
etc.
[0791] A transformant or transfectant is produced by using the
vector thus constructed, which carries the G protein coupled
receptor protein-encoding DNA of the present invention. The host
may be, for example, Escherichia microorganisms, Bacillus
microorganisms, yeasts, insect cells, animal cells, etc. Examples
of the Escherichia and Bacillus microorganisms include Escherichia
coli K12-DH1 [Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)],
JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221 [Journal
of Molecular Biology, Vol. 120, 517 (1978)], HB101 [Journal of
Molecular Biology, Vol. 41, 459 (1969)], C600 [Genetics, Vol. 39,
440 (1954)], etc. Examples of the Bacillus microorganism are, for
example, Bacillus subtilis MI 14 [Gene, Vol. 24, 255 (1983)],
207-21 (Journal of Biochemistry, Vol. 95, 87 (1984)], etc. The
yeast may be, for example, Saccharomyces cerevisiae AH22, AH22R,
NA87-11A, DKD-5D, 20B-12, etc. The insect-may include a silkworm
(Bombyx mori larva), [Maeda et al, Nature, Vol. 315, 592 (1985)]
etc. The host animal cell may be, for example, monkey-derived cell
line, COS-7, Vero, Chinese hamster ovary cell line (CHO cell), DHFR
gene-deficient Chinese hamster cell line (dhfr CHO cell), mouse L
cell, murine myeloma cell, human FL cell, etc.
[0792] Depending on the host cell used, transformation is done
using standard techniques appropriate to such cells. Transformation
of Escherichia microorganisms can be carried out in accordance with
methods as disclosed in, for example, Proc. Natl. Acad. Sci. USA,
Vol. 69, 2110 (1972), Gene, Vol. 17, 107 (1982), etc.
Transformation of Bacillus microorganisms can be carried out in
accordance with methods as disclosed in, for example, Molecular
& General Genetics, Vol. 168, 111 (1979), etc. Transformation
of the yeast can be carried out in accordance with methods as
disclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 75,
1929 (1978), etc. The insect cells can be transformed in accordance
with methods as disclosed in, for example, Bio/Technology, 6,
47-55, 1988. The animal cells can be transformed by methods as
disclosed in, for example, Virology, Vol. 52, 456, 1973, etc. The
transformants or transfectants which are transformed with
expression vectors containing,a G protein coupled receptor
protein-encoding DNA are produced according to the aforementioned
techniques.
[0793] Cultivation of the transformant (transfectant) in which the
host is Escherichia or Bacillus microorganism can be carried out
suitably in a liquid culture medium. The culture medium may
contains carbon sources, nitrogen sources, minerals, etc. necessary
for growing the transformant. The carbon source may include
glucose, dextrin, soluble starch, sucrose, etc. The nitrogen source
may include organic or inorganic substances such as ammonium salts,
nitrates, corn steep liquor, peptone, casein, meat extracts,
bean-cakes, potato extracts, etc. Examples of the minerals may
include calcium chloride, sodium dihydrogen phosphate, magnesium
chloride, etc. It is further allowable to add yeasts, vitamines,
growth-promoting factors, etc. It is desired that the culture
medium is pH from about 5 to about 8.
[0794] The Escherichia microorganism culture medium is preferably
an M9 medium containing, for example, glucose and casamino acid
(Miller, Journal of Experiments in Molecular Genetics), 431-433,
Cold Spring Harbor Laboratory, New York, 1972. Depending on
necessity, the medium may be supplemented with drugs such as
3,8-indolyl acrylic acid in order to improve efficiency of the
promoter. In the case of the Escherichia host, the cultivation is
carried out usually at about 15 to 43.degree. C. for about 3 to 24
hours. As required, aeration and stirring may be applied. In the
case of the Bacillus host, the cultivation is carried out usually
at about 30 to 40.degree. C. for about 6 to 24 hours. As required,
aeration and stirring may be also applied. In the case of the
transformant in which the host is an yeast, the culture medium used
may include, for example, a Burkholder minimum medium [Bostian, K.
L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)], an SD
medium containing 0.5% casamino acid [Bitter, G. A. et al., Proc.
Natl. Acad. Sci. USA, Vol. 81, 5330 (1984)], etc. It is preferable
that pH of the culture medium is adjusted to be from about 5 to
about 8. The cultivation is carried out usually at about 20 to
35.degree. C. for about 24 to 72 hours. As required, aeration and
stirring may be applied. In the case of the transformant in which
the host is an insect, the culture medium used may include those
obtained by suitably adding additives such as passivated (or
immobilized) 10% bovine serum and the like to the Grace's insect
medium (Grace, T. C. C., Nature, 195, 788 (1962)). It is preferable
that pH of the culture medium is adjusted to be about 6.2 to 6.4.
The cultivation is usually carried out at about 27.degree. C. for
about 3 to 5 days. As desired, aeration and stirring may be
applied. In the case of the transformant in which the host is an
animal cell, the culture medium used may include MEM medium
[Science, Vol. 122, 501 (1952)], DMEM medium [Virology, Vol. 8, 396
(1959)], RPMI 1640 medium [Journal of the American Medical
Association, Vol. 199, 519 (1967)], 199 medium [Proceedings of the
Society of the Biological Medicine, Vol. 73, 1 (1950)], etc. which
are containing, for example, about 5 to 20% of fetal calf serum. It
is preferable that the pH is from about 6 to about 8. The
cultivation is usually carried out at about 30 to 40.degree. C. for
about 15 to 60 hours. As required, aeration and stirring may be
applied.
[0795] Separation and purification of the G protein coupled
receptor protein from the above-mentioned cultures can be carried
out according to methods described herein below.
[0796] To extract G protein coupled receptor proteins from the
cultured microorganisms or cells, the microorganisms or cells are
collected by known methods after the cultivation, suspended in a
suitable buffer solution, disrupted by ultrasonic waves, lysozyme
and/or freezing and thawing, etc. and, then, a crude extract of the
G protein coupled receptor protein is obtained by centrifugation or
filtration. Other conventional extracting or isolating methods can
be applied. The buffer solution may contain a protein-denaturing
agent such as urea or guanidine hydrochloride or a surfactant such
as Triton X-100 (registered trademark, hereinafter often referred
to as "TM").
[0797] In case where G protein coupled receptor proteins are
secreted into culture media, supernatant liquids are separated from
the microorganisms or cells after the cultivation is finished and
the resulting supernatant liquid is collected by widely known
methods. The culture supernatant liquid and extract containing G
protein coupled receptor proteins can be purified by suitable
combinations of widely known methods for separation, isolation and
purification. The widely known methods of separation, isolation and
purification may include methods which utilizes solubility, such as
salting out or sedimentation with solvents methods which utilizes
chiefly a difference in the molecular size or weight, such as
dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide
gel electrophoresis, methods utilizing a difference in the electric
charge, such as ion-exchange chromatography, methods utilizing
specific affinity such as affinity chromatography, methods
utilizing a difference in the hydrophobic property, such as
inverse-phase high-performance liquid chromatography, and methods
utilizing a difference in the isoelectric point such as isoelectric
electrophoresis, etc.
[0798] In case where the G protein coupled receptor protein thus
obtained is in a free form, the free protein can be converted into
a salt thereof by known methods or method analogous thereto. In
case where the G protein coupled receptor protein thus obtained is
in a salt form vice versa, the protein salt can be converted into a
free form or into any other salt thereof by known methods or method
analogous thereto.
[0799] The G protein coupled receptor protein produced by the
transformant can be arbitrarily modified or a polypeptide can be
partly removed therefrom, by the action of a suitable
protein-modifying enzyme before or after the purification. The
protein-modifying enzyme may include trypsin, chymotrypsin, arginyl
endopeptidase, protein kinase, glycosidase, etc. The activity of
the G protein coupled receptor protein thus formed can be measured
by experimenting the coupling (or binding) with a ligand or by
enzyme immunoassays (enzyme linked immunoassays) using specific
antibodies.
[0800] The G protein coupled receptor protein-encoding DNA and the
G protein coupled receptor protein of the present invention can be
used for:
[0801] methods of determining ligands for the G protein coupled
receptor protein of the present invention,
[0802] obtaining an antibody and an antiserum,
[0803] constructing a system for expressing a recombinant receptor
protein,
[0804] developing a receptor-binding assay system using the above
developing system and screening pharmaceutical candidate
compounds,
[0805] designing drugs based upon the comparison with ligands and
receptors which have a similar or analogous structure,
[0806] preparing a probe in the analysis of genes and preparing a
PCR primer, and
[0807] gene manipulating therapy.
[0808] In particular, it is allowable to screen a G protein coupled
receptor agonist or antagonist specific to a warmblooded animal
such as human being by a receptor-binding assay system which uses a
system for expressing a recombinant G protein coupled receptor
protein of the present invention. The agonist or antagonist thus
screened or characterized permits various applications including
prevention and/or therapy of a variety of diseases.
[0809] Concretely described below are uses of G protein coupled
receptor proteins, partial peptide thereof (peptide fragment
thereof), G protein coupled receptor protein-encoding DNAs and
antibodies against the G protein coupled receptor protein according
to the present invention.
[0810] As hereunder, more detailed description will be made on the
usefulness of the G protein coupled receptor proteinencoding DNA
obtained by the screening method for G protein coupled receptor
protein-encoding DNAs according to the present invention, the G
protein coupled receptor proteins encoded by said DNA, peptide
fragments or segments thereof (including partial peptides thereof)
or salts thereof (hereinafter, those including their salts, will be
referred to as the "G protein coupled receptor protein or a peptide
fragment thereof"), cells or cell membrane fractions thereof each
containing the recombinant type G protein coupled receptor protein,
etc. Their various applications are also disclosed herein
below.
[0811] (1) Method for Determining Ligands to the G Protein Coupled
Receptor Protein
[0812] The G protein coupled receptor protein (or the peptide
segment thereof) is useful as a reagent for investigating or
determining a ligand to said G protein coupled receptor
protein.
[0813] According to the present invention, methods for determining
a ligand to the G protein coupled receptor protein which comprises
contacting the G protein coupled receptor protein or the peptide
segment or fragment thereof with the compound to be tested are
provided.
[0814] The compound to be tested may include not only known ligands
such as angiotensins, bombesins, canavinoids, cholecystokinins,
glutamine, serotonin, melatonins, neuropeptides Y, opioids, purine,
vasopressins, oxytocins, VIP (vasoactive intestinal and related
peptides), somatostatins, dopamine, motilins, amylins, bradykinins,
CGRP (calcitonin gene related peptides), adrenomedullins,
leukotrienes, pancreastatins, prostaglandins, thromboxanes,
adenosine, adrenaline, .alpha.- and .beta.-chemokines (IL-8,
GRO.alpha., GRO.beta., GRO.gamma., NAP-2, ENA-78, PF4, IP10, GCP-2,
MCP-1, HC14, MCP-3, 1-309, MIP1.alpha., MIP-1.beta., RANTES, etc.),
endothelins, enterogastrins, histamine, neurotensins, TRH,
pancreatic polypeptides, galanin, modified derivatives thereof,
analogues thereof, family members thereof and the like but also
tissue extracts, cell culture supernatants, etc. of warm-blooded
animals (such as mice, rats, swines, cattle, sheep, monkeys and
human being), etc. For example, said tissue extract, said cell
culture supernatant, etc. is added to the G protein coupled
receptor protein for measurement of the cell stimulating activity,
etc. and fractionated by relying on the measurements whereupon a
single ligand can be finally obtained.
[0815] In one specific embodiment of the present invention, said
method for determining the ligand includes a method for determining
a compound or a salt thereof capable of stimulating a target cell
which comprises binding said compound with the G protein coupled
receptor protein either in the presence of the G protein coupled
receptor protein or the peptide segment thereof or in a receptor
binding assay system in which the expression system for the
recombinant type receptor protein is constructed and used; and
measuring the receptor-mediated cell stimulating activity, etc.
Examples of said cell stimulating activities include promoting
activity or inhibiting activity on biological responses, e.g.
liberation of arachidonic acid, liberation of acetylcholine,
liberation of endocellular Ca.sup.2+, production of endocellular
cAMP, production of endocellular cGMP, production of inositol
phosphate, changes in the cell membrane potential, phosphorylation
of endocellular protein, activation of c-fos, lowering in pH,
activation of G protein, cell promulgation, etc. Examples of said
compound or salt capable of stimulating the cell via binding with
the G protein coupled receptor protein include peptides, proteins,
nonpeptidic compounds, synthetic compounds, fermented products,
etc.
[0816] In said method for determining the ligand, the
characteristic feature is that when the G protein coupled receptor
protein or the peptide segment thereof is contacted with the test
compound, for example, the binding amount, the cell stimulating
activity, etc. of the test compound to the G protein coupled
receptor protein or the peptide segment thereof is measured.
[0817] In more specific embodiments of the present invention, said
methods for determining the ligand includes:
[0818] a method of determining a ligand to a G protein coupled
receptor protein, which comprises contacting a labeled test
compound with a G protein coupled receptor protein or a peptide
segment thereof, and measuring the amount of the labeled test
compound binding with said protein or salt thereof or with said
peptide fragment or salt thereof;
[0819] a method of determining a ligand to a G protein coupled
receptor protein, which comprises contacting a labeled test
compound with cells containing the G protein coupled receptor
protein or the membrane fraction of said cell, and measuring the
amount of the labeled test compound binding with said cells or said
cell fraction;
[0820] a method of determining a ligand to a G protein coupled
receptor protein, which comprises contacting a labeled test
compound with the G protein coupled receptor protein expressed on
cell membranes by culturing transformants containing the DNA coding
for the G protein coupled receptor protein, and measuring the
amount of the labeled test compound binding with said G protein
coupled receptor protein;
[0821] a method of determining a ligand to a G protein coupled
receptor protein, which comprises contacting a test compound with
cells containing the G protein coupled receptor protein, and
measuring the cell stimulating activity (e.g. promoting or
inhibiting activity on biological responses such as liberation of
arachidonic acid, liberation of acetylcholine, liberation of
endocellular Ca.sup.2+production of endocellular cAMP, production
of endocellular cGMP, production of inositol phosphate, changes in
the cell membrane potential, phosphorylation of endocellular
protein, activation of c-fos, lowering in pH, activation of G
protein, cell promulgation, etc.) via the G protein coupled
receptor protein; and
[0822] a method of determining a ligand to the G protein coupled
receptor protein, which comprises contacting a test compound with
the G protein coupled receptor protein expressed on the cell
membrane by culturing transformants containing the DNA coding for
the G protein coupled receptor protein, and measuring the cell
stimulating activity (activity for promoting or inhibiting
physiological responses such as liberation of arachidonic acid,
liberation of acetylcholine, liberation of endocellular Ca.sup.2+,
production of endocellular cAMP, production of endocellular cGMP,
production of inositol phosphate, changes in the cell membrane
potential, phosphorylation of endocellular protein, activation of
c-fos, lowering in pH, activation of G protein, cell promulgation,
etc.) via the G protein coupled receptor protein.
[0823] Described below are specific explanations on the determining
method of ligands according to the present invention which are
provided only for illustrative purposes.
[0824] First, the G protein coupled receptor protein used for the
method for determining the ligand may include any material so far
as it contains a G protein coupled receptor protein or a peptide
fragment or segment thereof (including a partial peptide thereof or
a salt thereof although it is preferable to express a large amount
of G protein coupled receptor proteins in animal cells.
[0825] In the manufacture of the G protein coupled receptor
protein, the above-mentioned method can be used and it may be
carried out by expressing said protein encoding DNA in mammalian
cells or in insect cells. With respect to the DNA fragment coding
for the aimed region, complementary DNA may be used although it is
not limited thereto. For example, gene fragments or synthetic DNA
may be used as well.
[0826] In order to introduce the G protein coupled receptor
protein-encoding DNA fragment into host animal cells and to express
it efficiently, it is preferred that said DNA fragment is
incorporated into the downstream site of polyhedron promoters
derived from nuclear polyhedrosis virus belonging to baculovirus,
promoters derived from SV40, promoters derived from retrovirus,
metallothionein promoters, human heat shock promoters,
cytomegalovirus promoters, SRa promoters, etc. Examinations of the
quantity and the quality of the expressed receptor can be carried
out by methods per se known to those of skill in the art or methods
similar thereto. For example, they may be conducted by methods
described in publications such as Nambi, P. et al: The Journal of
Biochemical Society, vol.267, pages 19555-19559 (1992).
[0827] Accordingly, with respect to the determination of the
ligand, the material containing a G protein coupled receptor
protein or peptide segment thereof may include products containing
G protein coupled receptor proteins which are purified by methods
per se known to those of skill in the art or methods similar
thereto, peptide fragments of said G protein coupled receptor
protein, cells containing said G protein coupled receptor protein,
membrane fractions of the cell containing said protein, etc.
[0828] When the G protein coupled receptor proteincontaining cell
is used in the determining method of the ligand, said cell may be
immobilized with binding agents including glutaraldehyde, formalin,
etc. The immobilization may be carried out by methods per se known
to those of skill in the art or methods similar thereto.
[0829] The G protein coupled receptor protein-containing cells are
host cells expressing the G protein coupled receptor protein.
Examples of said host cells are microorganisms such as Escherichia
coli, Bacillus subtilis, yeasts, insect cells, animal cells,
etc.
[0830] The cell membrane fraction is a cell membrane-rich fraction
which is prepared by methods per se known to those of skill in the
art or methods similar thereto after disruption of cells. Examples
of cell disruption may include a method for squeezing cells using a
Potter-Elvejem homogenizer, a disruption by a Waring blender or a
Polytron (manufactured by Kinematica), a disruption by ultrasonic
waves, a disruption via blowing out cells from small nozzles
together with applying a pressure using a French press or the like,
etc. In the fractionation of the cell membrane, a fractionation
method by means of centrifugal force such as a fractional
centrifugal separation and a density gradient centrifugal
separation is mainly used. For example, disrupted cellular liquid
is centrifuged at a low speed (500 rpm to 3,000 rpm) for a short
period (usually, from about one to ten minutes), the supernatant
liquid is further centrifuged at a high speed (1,500 rpm to 3,000
rpm) usually for 30 minutes to two hours and the resulting
precipitate is used as a membrane fraction. Said membrane fraction
contains a lot of the expressed G protein coupled receptor protein
and a lot of membrane components such as phospholipids and membrane
proteins derived from the cells.
[0831] The amount of the G protein coupled receptor protein in the
membrane fraction cell containing said G protein coupled receptor
protein is preferably 10.sup.3-10.sup.8 molecules per cell or,
suitably, 10.sup.5 to 10.sup.7 molecules per cell. Incidentally,
the more the expressed amount, the higher the ligand binding
activity (specific activity) per membrane fraction whereby the
construction of a highly sensitive screening system becomes
possible and, moreover, it may enable us to measure the large
amount of samples within the same lot.
[0832] In conducting the above-mentioned methods .quadrature. to
.quadrature. wherein ligands capable of binding with the G protein
coupled receptor protein are determined, a suitable G protein
coupled receptor fraction and a labeled test compound are
necessary. The G protein coupled receptor fraction is preferably a
naturally occurring (natural type) G protein coupled receptor, a
recombinant type G protein coupled receptor having the activity
equivalent to that of the natural type. Here, the term "activity
equivalent to" means the equivalent ligand binding activity,
etc.
[0833] Suitable examples of the labeled test compound are
angiotensin, bombesin, canavinoid, cholecystokinin, glutamine,
serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin,
oxytocin, VIP (vasoactive intestinal and related peptides),
somatostatin, dopamine, motilin, amylin, bradykinin, CGRP
(calcitonin gene related peptides), adrenomedullin, leukotriene,
pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline,
.alpha.- and .beta.-chemokine (IL-8, GRO.alpha., GRO.beta.,
GRO.lambda., NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1, HC14, MCP-3,
1-309, MIP1.alpha., MIP-1.beta., RANTES, etc.), endothelin,
enterogastrin, histamine, neurotensin, TRH, pancreatic
polypeptides, galanin, an analogue derivative thereof, etc. which
are labeled with [.sup.3H], [.sup.125I], [.sup.14C], [.sup.35S],
etc.], etc.
[0834] Specifically, the determination of ligands capable of
binding with G protein coupled receptor proteins is carried out as
follows:
[0835] First, cells or cell membrane fractions containing the G
protein coupled receptor protein are suspended in a buffer suitable
for the determining method to prepare the receptor sample in
conducting the method of determining the ligand binding with the G
protein coupled receptor protein. The buffer may include any buffer
such as Tris-HCl buffer or phosphate buffer with pH 4-10
(preferably, pH 6-8), etc., as long as it does not inhibit the
binding of the ligand with the receptor. In addition,
surface-active agents such as CHAPS, Tween 80.TM. (Kao-Atlas,
Japan), digitonin, deoxycholate, etc. and various proteins such as
bovine serum albumin (BSA), gelatin, milk derivatives, etc. may be
added to the buffer with an object of decreasing the non-specific
binding. Further, a protease inhibitor such as PMSF, leupeptin,
E-64 (manufactured by Peptide Laboratory), pepstatin, etc. may be
added with an object of inhibiting the decomposition of the
receptor and the ligand by protease. A test compound labeled with a
predetermined (or certain) amount (5,000 cpm to 500,000 cpm) of
[.sup.3 H], [.sup.125I [.sup.14C], [.sup.35S], etc. is made
copresent in 0.01 ml to 10 ml of said receptor solution.
[0836] In order to know the non-specific binding amount (NSB), a
reaction tube to which a great excessive amount of the unlabeled
test compound is added is prepared as well. The reaction is carried
out at 0-50.degree. C. (preferably at 4-37.degree. C.) for 20
minutes to 24 hours (preferably 30 minutes to three hours). After
the reaction, it is filtered through a glass fiber filter or the
like, washed with a suitable amount of the same buffer and the
radioactivity remaining in the glass fiber filter is measured by
means of a liquid scintillation counter or a gamma-counter. The
test compound in which the count (B-NSB) obtained by subtracting
the non-specific binding amount (NSB) from the total binding amount
(B) is more than 0 cpm can be selected as a ligand to the G protein
coupled receptor protein of the present invention.
[0837] In conducting the above-mentioned methods .quadrature. to
.quadrature. wherein ligands capable of binding with the G protein
coupled receptor protein are determined, the cell stimulating
activity (e.g. the liberation of arachidonic acid, the liberation
of acetylcholine, endocellular Ca.sup.2+ liberation, endocellular
CAMP production, the production of insitol phosphate, changes in
the cell membrane potential, the phosphorylation of end-ocellular
protein, the activation of c-fos, lowering of pH, the activation of
G protein, cell promulgation, etc.) mediated by the G protein
coupled receptor protein may be measured by known methods or by the
use of commercially available measuring kits. To be more specific,
G protein coupled receptor protein-containing cells are at first
cultured in a multi-well plate or the like.
[0838] In conducting the determination of ligand, it is substituted
with a fresh medium or a suitable buffer which does not show
toxicity to the cells in advance of the experiment, and incubated
for certain period after adding a test compound, etc. thereto.
Then, the cells are extracted or the supernatant liquid is
recovered and the resulting product is determined by each of the
methods. When it is difficult to identify the production of the
substance (e.g. arachdonic acid) which is to be an index for the
cell stimulating activity due to the decomposing enzyme contained
in the cell, an assay may be carried out by adding an inhibitor
against said decomposing enzyme. With respect to the activity such
as an inhibitory action against cAMP production, it may be detected
as an inhibitory action against the production of the cells whose
fundamental production is increased by forskolin or the like.
[0839] The kit used for the method of determining the ligand
binding with the G protein coupled receptor protein includes a G
protein coupled receptor protein or a peptide fragment thereof,
cells containing the G protein coupled receptor protein, a membrane
fraction from the cells containing the G protein coupled receptor
protein, etc.
[0840] Examples of the kit for determining the ligand are as
follows:
[0841] 1. Reagent for Determining the Ligand.
[0842] Buffer for Measurement and Buffer for Washing.
[0843] The buffering product wherein 0.05% of bovine serum albumin
(manufactured by Sigma) is added to Hanks' Balanced Salt Solution
(manufactured by Gibco).
[0844] This product may be sterilized by filtration through a
membrane filter with a 0.45 u m pore size, and stored at 4.degree.
C. or may be formulated upon use.
[0845] G Protein Coupled Receptor Protein Sample.
[0846] CHO cells in which G protein coupled receptor proteins are
expressed are subcultured at the rate of 5.times.10.sup.5
cells/well in a 12-well plate and cultured at 37.degree. C. in a
humidified 5% CO.sub.2/95o air atmosphere for two days to prepare
the sample.
[0847] Labeled Test Compound.
[0848] The compound which is labeled with commercially available
.sup.13H], [.sup.125I], [.sup.14C], [.sup.35S], etc. or labeled
with a suitable method.
[0849] The product in a state of an aqueous solution is stored at
4.degree. C. or at -20.degree. C. and, upon use, diluted to 1 u M
with a buffer for the measurement. In the case of the test compound
which is hardly soluble in water, it is dissolved in
dimethylformamide, DMSO, methanol, etc.
[0850] Unlabeled Test Compound.
[0851] The same compound for the labeled one is prepared in a
concentration of 100 to 1,000-fold concentrated state.
[0852] 2. Method of Measurement.
[0853] G protein coupled receptor protein-expressing CHO cells
cultured in a 12-well tissue culture plate are washed twice with 1
ml of buffer for the measurement and then 490 u 1 of buffer for the
measurement is added to each well.
[0854] Five u 1 of the labeled test compound is added and the
mixture is made to react at room temperature for one hour. For
measuring the nonspecific binding amount, 5 u 1 of the unlabeled
test compound is added.
[0855] The reaction solution is removed from each well, which is
washed with 1 ml of a buffer for the measurement three times. The
labeled test compound which is binding with the cells is dissolved
in 0.2N NaOH-1% SDS and mixed with 4 ml of a liquid scintillator A
(manufactured by Wako Pure Chemical, Japan).
[0856] Radioactivity is measured using a liquid scintillation
counter (manufactured by Beckmann).
[0857] The ligand which can bind with the G protein coupled
receptor protein include substances occurring or existing, for
example, in brain, pituitary gland, pancreas, etc. Examples of the
ligand are angiotensin, bombesin, canavinoid, cholecystokinin,
glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine,
vasopressin, oxytocin, VIP (vasoactive intestinal and related
peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP
(calcitonin gene related peptide), adrenomedullin, leukotriene,
pancreastatin, prostaglandin, thromboxane, thromboxatin, adenosine,
adrenaline, .alpha.- and .beta.-chemokine (IL-8, GRO.alpha.,
GRO.beta., GRO.gamma., NAP-2, ENA-78, PF4, IP10, GCP-2, MCP-1,
HC14, MCP-3, 1-309, MIP1a, MIP-id, RANTES, etc.), endothelin,
enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide,
galanin, modified derivatives thereof, analogues thereof, etc.
[0858] Since the receptor protein encoded by pMAH2-17 is highly
homologous to prinoceptors, it is considered that there are strong
possibility of a subtype within prinoceptor families. All data
including electrophysiological measurements are supporting that the
mouse pancreatic .beta.-cell strain, MIN6-derived receptor protein
of the present invention (e.g., SEQ ID NO: 38 and SEQ ID NO: 39, or
proteins encoded by pMAH2-17) is a novel purinoceptor subtype. In
other words, it is suggested that the ligand capable of binding
with the mouse pancreatic .beta.-cell strain, MIN6-derived receptor
protein of the present invention (e.g., SEQ ID NO: 38 and SEQ ID
NO: 39, or proteins encoded by pMAH2-17) is a purine compound such
as ATP. Further, the receptor protein (e.g., SEQ ID NO: 56, or
proteins encoded by phAH2-17) is considered to be a novel human
type purinoceptor. It is presumed that it is advantageously useful
in efficiently screening for agonists or antagonists to receptor
proteins which control or regulate functions in the central nervous
system or immune system, related to purine compounds, and in
developing pharmaceuticals.
[0859] (2) Preventive and Therapeutic Agent for of G Protein
Conjugated Receptor Protein Deficiency Diseases
[0860] If a ligand to the G protein coupled receptor protein is
disclosed via the aforementioned method (1), the G protein coupled
receptor protein-encoding DNA can be used a preventive and/or
therapeutic agent for treating said G protein coupled receptor
protein deficiency diseases depending upon the action that said
ligand exerts.
[0861] For example, when there is a patient for whom the
physiological action of the ligand cannot be expected because of a
decrease in the G protein coupled receptor protein in vivo, the
amount of the G protein coupled receptor protein in the brain cells
of said patient can be increased whereby the action of the ligand
can be fully achieved by:
[0862] (a) administering the G protein coupled receptor
proteinencoding DNA to the patient to express it; or
[0863] (b) inserting the G protein coupled receptor
protein-encoding DNA into brain cells or the like to express it,
followed by transplanting said brain cells or the like to said
patient. Accordingly, the G protein coupled receptor
protein-encoding DNA can be used as a safe and less toxic
preventive and therapeutic agent for the G protein coupled receptor
protein deficiency diseases. In an embodiment, it is suggested that
the ligands capable of binding with the mouse pancreatic p -cell
strain, MIN6-derived receptor protein of the present invention
(e.g., SEQ ID NO: 38 and SEQ ID NO: 39, or proteins encoded by
pMAH2-17) and further with the human-derived receptor protein of
the present invention (e.g., SEQ ID NO: 56, or proteins encoded by
phAH2-17) are purine compounds such as ATP. Therefore, the disease
to be treated may include diseases or syndromes in connection with
purine ligand compounds. Examples of such diseases may include
cancer, immunodeficiency, autoimmune disease, rheumatoid arthritis,
rejection on internal organ transplant, hypertension, diabetes,
cystic fibrosis, hypotension, incontinence of urine, pain, etc.
[0864] (3) Preventive and Therapeutic Pharmaceutical Composition
for Human-Derived G Protein Conjugated Receptor Protein Deficiency
Diseases
[0865] If the human-derived G protein coupled receptor
protein-encoding DNA is screened and a ligand for said humanderived
G protein coupled receptor protein can be clarified using the
above-mentioned method (1), the human-derived G protein coupled
receptor protein-encoding DNA can be used as an agent for the
prevention or therapy of the deficiency diseases of said
human-derived G protein coupled receptor protein devendinq upon the
action that said ligand exhibits.
[0866] For example, when there is a patient for whom the
physiological action of the ligand cannot be expected because of a
decrease in the G protein coupled receptor protein in vivo, the
amount of the G protein coupled receptor protein in the brain cells
of said patient can be increased whereby the action of the ligand
can be fully achieved by:
[0867] (a) administering the G protein coupled receptor
protein-encoding DNA to the patient to express it; or
[0868] (b) inserting the G protein coupled receptor
protein-encoding DNA into brain cells or the like to express it,
followed by transplanting said brain cells or the like to said
patient. Accordingly, the G protein coupled receptor
protein-encoding DNA can be used as a safe and less toxic
preventive and therapeutic agent for the G protein coupled receptor
protein deficiency diseases.
[0869] When the G protein coupled receptor protein-encoding DNA is
used as the above-mentioned agent, said DNA may be used alone or
after inserting it into a suitable vector such as retrovirus
vector, adenovirus vector, adenovirus-associated virus vector, etc.
followed by subjecting the product vector to a conventional means.
Thus, it may be administered orally parenterally, by inhalation
spray, rectally, or topically as pharmaceutical compositions or
formulations. Oral formulations include tablets (sugar-coated if
necessary), capsules, elixirs, microcapsules, etc. Parenteral
formulations include injections such as an aseptic solution or a
suspension in water or in other pharmaceutically acceptable liquid.
For example, the DNA of the present invention is admixed in a unit
dose form which is required for preparing generally approved
pharmaceutical preparations together with a physiologically
acceptable carriers, flavoring agents, adjuvants, excipients,
diluents, fillers, vehicles, antiseptics, stabilizers, binders,
etc. whereupon the preparation can be manufactured. The amount of
the effective component in those preparations is to be in such an
extent that the suitable dose within an indicated range is
achieved.
[0870] Examples of the additives which can be admixed in the
tablets, capsules, etc. are binders such as gelatin, corn starch,
tragacanth and gum arabicum; fillers such as crystalline cellulose;
swelling agents such as corn starch, gelatin and alginic acid;
lubricating agents such as magnesium stearate; sweetening agents
such as sucrose, lactose and saccharine; and flavoring agents such
as pepper mint, akamono oil and cherry. When the unit dose form of
the preparation is a capsule, a liquid carrier such as fat/oil may
be further added in addition of the above-mentioned types of
materials. The aseptic composition for injection may be formulated
by conventional practices for the preparations such as that the
active substance in a vehicle such as water for injection is
dissolved or suspended in naturally occurring plant oil such as
sesame oil and palm oil.
[0871] Examples of an aqueous liquid for injection are a
physiological saline solution and isotonic solutions containing
glucose and other auxiliary agents (e.g. D-sorbitol, D-mannitol,
sodium chloride, etc.) wherein a suitable auxiliary solubilizers
such as alcohol (e.g. ethanol, etc.), polyalcohol (e.g. propylene
glycol polyethylene glycol, etc.), nonionic surface-active agent
(e.g. Polysorbate 80.TM., HCO-50, etc.), etc. may be jointly used.
Examples of an oily liquid include sesame oil, soybean oil, etc.
wherein benzyl benzoate, benzyl alcohol, etc. may be jointly used
as auxiliary solubilizers. In addition, buffers (e.g. phosphate
buffer, sodium acetate buffer, etc.), analgesic agents (e.g.
benzalkonium chloride, procaine hydrochloride, etc.), stabilizers
(e.g. human serum albumin, polyethylene glycol, etc.), stabilizers
(e.g. benzyl alcohol phenol, etc.), antioxidants, etc. may be
admixed therewith too. The prepared injection solution is filled in
suitable ampoules. The preparation prepared as such is safe and
less toxic and, therefore, it can be administered to warm-blooded
animals (e.g., rat, rabbit, sheep, swine, cattle, cat, dog, monkey,
human beings, etc.).
[0872] Specific dose levels of said DNA may vary depending upon a
variety of factors including the activity of drugs employed, the
age, body weight, general health, sex, diet, time of
administration, route of administration, drug combination, and the
severity of the symptom. In the case of oral administration, it is
usually about 0.1-100 mg, preferably about 1.0-50 mg or, more
preferably, about 1.0-20 mg per day for adults (as 60 kg). When it
is administered parenterally, its dose at a time may vary depending
upon the object (patient) to be administered, organs to be
administered, symptoms, administering methods, etc. but, in the
case of injections, it is usually convenient to give by an
intravenous route in an amount of about 0.01-30 mg, preferably
about 0.1-20 mg or, more preferably, about 0.1-10 mg per day to
adults (as 60 kg). In the case of other animals, the dose
calculated for 60 kg may be administered as well.
[0873] (4) Quantitative Determination of Ligand to the G Protein
Conjugated Receptor Protein of the Present Invention.
[0874] The G protein coupled receptor protein or a peptide fragment
thereof has a binding property to ligand and, therefore, it is
capable of determining quantitatively an amount of ligands in vivo
with good sensitivity.
[0875] This quantitative determination may be carried out by, for
example, combining with a competitive method. Thus, samples to be
determined is contacted with G protein coupled receptor proteins or
peptide fragments thereof so that the ligand concentration in said
sample can be determined. In one embodiment of the quantitative
determination, the protocols described in the following
.quadrature. and .quadrature. or the methods similar thereto may be
used:
[0876] (Hiroshi Irie (ed): "Radioimmunoassay" (Kodansha, Japan,
1974); and
[0877] Hiroshi Irie (ed): "Radioimmunoassay, Second Series"
(Kodansha, Japan, 1979).
[0878] (5) Screening of Compound Inhibiting the Binding of Ligand
with the G Protein Conjugated Receptor Protein of the Present
Invention.
[0879] G Protein coupled receptor proteins or peptide fragments
thereof are used. Alternatively, expression systems for recombinant
type G Protein coupled receptor proteins or peptide fragments
thereof are constructed and receptor binding assay systems using
said expression system are used. In these assay systems, it is
possible to screen compounds (e.g. peptides, proteins, nonpeptidic
compounds, synthetic compounds, fermented products, cell extracts,
plant extracts, animal tissue extracts, etc.) or salts thereof
which inhibits the binding of a ligand with the G protein coupled
receptor protein. Such a compound includes a compound exhibiting a
G protein coupled receptor-mediated cell stimulating activity (e.g.
activity of promoting or activity of inhibiting physiological
reactions including liberation of arachdonic acid, liberation of
acetylcholine, endocellular Ca.sup.2+ liberation, endocellular cAMP
production, endocellular cGMP production, production of inositol
phosphate, changes in cell membrane potential, phosphorylation of
endocellular proteins, activation of c-fos, lowering of pH,
activation of G protein, cell promulgation, etc.) (so-called "G
protein coupled receptor-agonist"), a compound free of such a cell
stimulating activity (so-called "G protein coupled
receptor-antagonist"), etc.
[0880] Thus, the present invention provides a method of screening a
compound which inhibits the binding of a ligand with a G protein
coupled receptor protein or a salt thereof, characterized in
comparing the following two cases:
[0881] (i) the case wherein the ligand is contacted with the G
protein coupled receptor protein or salt thereof, or a peptide
fragment thereof or a salt thereof; and
[0882] (ii) the case wherein the ligand is contacted with a mixture
of the G protein coupled receptor protein or salt thereof or the
peptide fragment or salt thereof and said test compound.
[0883] In said screening method, one characteristic feature of the
present invention resides in that the amount of the ligand bonded
with said G protein coupled receptor protein or the peptide
fragment thereof, the cell stimulating activity of the ligand, etc.
are measured in the case where (i) the ligand is contacted with G
protein coupled receptor proteins or peptide fragments thereof and
in the case where (ii) the ligand and the test compound are
contacted with the G protein coupled receptor protein or the
peptide fragment thereof, respectively and then compared
therebetween.
[0884] In one more specific embodiment of the present invention,
the following is provided:
[0885] a method of screening a compound or a salt thereof which
inhibits the binding of a ligand with a G protein coupled receptor
protein, characterized in that, when a labeled ligand is contacted
with a G protein coupled receptor protein or a peptide fragment
thereof and when a labeled ligand and a test compound are contacted
with a G protein coupled receptor protein or a peptide fragment
thereof, the amounts of the labeled ligand bonded with said protein
or peptide fragment thereof or salt thereof are measured and
compared;
[0886] a method of screening a compound or a salt thereof which
inhibits the binding of a ligand with a G protein coupled receptor
protein, characterized in that, when a labeled ligand is contacted
with cells containing G protein coupled receptor proteins or a
membrane fraction of said cells and when a labeled ligand and a
test compound are contacted with cells containing G protein coupled
receptor proteins or a membrane fraction of said cells, the amounts
of the labeled ligand binding with said protein or peptide fragment
thereof or salt thereof are measured and compared;
[0887] a method of screening a compound or a salt thereof which
inhibits the binding of a ligand with a G protein coupled receptor
protein, characterized in that, when a labeled ligand is contacted
with G protein coupled receptor proteins expressed on the cell
membrane by culturing a transformant containing a G protein coupled
receptor protein encoding DNA and when a labeled ligand and a test
compound are contacted with G protein coupled receptor proteins
expressed on the cell membrane by culturing a transformant
containing a G protein coupled receptor protein encoding DNA, the
amounts of the labeled ligand binding with said G protein coupled
receptor protein are measured and compared;
[0888] a method of screening a compound or a salt thereof which
inhibits the binding of a ligand with a G protein coupled receptor
protein, characterized in that, when a G protein coupled receptor
protein-activating compound (e.g. a ligand to the G protein coupled
receptor protein) is contacted with cells containing G protein
coupled receptor proteins and when the G protein coupled receptor
protein-activating compound and a test compound are contacted with
cells containing G protein coupled receptor proteins, the resulting
G protein coupled receptor protein-mediated cell stimulating
activities (e.g. activities of promoting or activities of
inhibiting physiological responses including liberation of
arachdonic acid, liberation of acetylcholine, endocellular
Ca.sup.2+ liberation, endocellular cAMP production, endocellular
cGMP production, production of inositol phosphate, changes in cell
membrane potential, phosphorylation of endocellular proteins,
activation of c-fos, lowering of pH, activation of G protein, cell
promulgation, etc.) are measured and compared; and
[0889] a method of screening a compound or a salt thereof which
inhibits the binding of a ligand with a G protein coupled receptor
protein, characterized in that, when a G protein coupled receptor
protein-activating compound (e.g. a ligand to the G protein coupled
receptor protein) is contacted with G protein coupled receptor
proteins expressed on cell membranes by culturing transformants
containing G protein coupled receptor protein-encoding DNA and when
a G protein coupled receptor protein-activating compound and a test
compound are contacted with the G protein coupled receptor protein
expressed on the cell membrane by culturing the transformant
containing the G protein coupled receptor protein-encoding DNA, the
resulting G protein coupled receptor protein-mediated cell
stimulating activities (activities of promoting or activities of
inhibiting physiological responses such as liberation of arachdonic
acid, liberation of acetylcholine, endocellular Ca.sup.2+
liberation, endocellular cAMP production, endocellular cGMP
production, production of inositol phosphate, changes in cell
membrane potential, phosphorylation of endocellular proteins,
activation of c-fos, lowering of pH, activation of G protein, and
cell promulgation) are measured and compared.
[0890] Before the G protein coupled receptor protein of the present
invention was obtained, the G protein coupled receptor agonist or
antagonist had to be screened by, first, obtaining a candidate
compound by using G protein coupled receptor protein-containing
cells, tissues or cell membrane fractions derived from rat or the
like (primary screening) and, then, making sure whether the
candidate compound really inhibits the binding between human G
protein coupled receptor proteins and ligands (secondary
screening). Other receptor proteins inevitably exist when the
cells, the tissues or the cell membrane fractions are used as they
are, whereby they intrinsically make it difficult to screen
agonists or antagonists to the desired receptor proteins. By using
the human-derived G protein coupled receptor protein, however,
there is no need of effecting the primary screening, whereby it is
allowable to efficiently screen a compound that inhibits the
binding between a ligand and a G protein coupled receptor. Besides,
it is allowable to evaluate whether the compound that is screened
is a G protein coupled receptor agonist or a G protein coupled
receptor antagonist.
[0891] Specific explanations of the screening method will be given
as hereunder.
[0892] First, with respect to the G protein coupled receptor
protein used for the screening method of the present invention, any
product may be used so far as it contains G protein coupled
receptor proteins or peptide fragment thereof although the use of a
membrane fraction of mammalian organs is suitable. However, human
organs is extremely hardly available and, accordingly, G protein
coupled receptor proteins which are expressed in a large amount
using a recombinant are suitable for the screening.
[0893] In the manufacture of the G protein coupled receptor
protein, the above-mentioned method can be used and it may be
carried out by expressing the DNA coding for said protein in
mammalian cells or in insect cells. With respect to the DNA
fragment coding for the target region, complementary DNA may be
used although it is not limited thereto. Thus, for example, gene
fragments or synthetic DNA may be used as well.
[0894] In order to introduce the G protein coupled receptor
protein-encoding DNA fragment into host animal cells and to express
it efficiently, it is preferred that said DNA fragment is
incorporated into the downstream of polyhedron promoter of nuclear
polyhedrosis virus belonging to baculovirus, promoter derived from
SV40, promoter of retrovirus, metallothionein promoter, human heat
shock promoter, cytomegalovirus promoter, SR.alpha. promoter, etc.
Examinations of the quantity and the quality of expressed receptors
can be carried out by known methods per se or modified methods
substantially analogous thereto. For example, they may be conducted
by the method described in publications such as Nambi, P. et al.:
The Journal of Biochemical Society, vol.267, pages 19555-19559
(1992).
[0895] Accordingly, in the screening method, the substance
containing a G protein coupled receptor protein or a peptide
fragment thereof may be a G protein coupled receptor protein which
is purified by known methods per se or a G protein coupled receptor
protein fragment which is purified by known methods per se, or a
cell containing said protein or a cell membrane fraction of the
cell containing said protein, etc.
[0896] When the G protein coupled receptor protein-containing cells
are used in the screening method, said cells may be immobilized
with glutaraldehyde, formalin, etc. The immobilization may be
carried out by known methods per se or modified methods
substantially analogous thereto.
[0897] The G protein coupled receptor protein-containing cells are
host cells expressing the G protein coupled receptor protein.
Examples of said host cells may include Escherichia coli Bacillus
subtilis, yeasts, insect cells, animal cells such as CHO cell and
COS cell, etc.
[0898] Cell membrane fractions are fractions which contain a lot of
cell membranes prepared by known methods per se or modified methods
substantially analogous thereto after disrupting or crushing the
cells. Examples of disruptions of the cell may include methods by
squeezing the cells with a Potter-Elvejem homogenizer, disrupting
or crushing by a Waring blender or a Polytron (manufactured by
Kinematica), disrupting or crushing by means of ultrasonic wave,
disrupting by blowing out the cells from small nozzles together
with applying a pressure with a French press or the like, etc.
Fractionation of the cell membrane is carried out mainly by
fractionation techniques by means of centrifugal force such as a
fractional centrifugal separation and a density gradient
centrifugal separation. For example, disrupted liquid of cells is
centrifuged at a low speed (500 rpm to 3,000 rpm) for a short
period (usually, from about one to ten minutes), the supernatant
liquid is further centrifuged at a high speed (1,500 rpm to 3,000
rpm) usually for 30 minutes to two hours and the resulting
precipitate is used as a membrane fraction. Said membrane fraction
contains a lot of expressed G protein coupled receptor proteins and
membrane components such as phospholipids and membrane proteins
derived from the cells.
[0899] The amount of the G protein coupled receptor protein in the
G protein coupled receptor protein-containing cell and in the cell
membrane fraction obtained from the cell is preferably
10.sup.3-10.sup.8 molecules per cell or, suitably, 10.sup.5 to
10.sup.7 molecules per cell. Incidentally, the more the expressed
amount, the higher the ligand binding activity (specific activity)
per membrane fraction whereby the construction of a highly
sensitive screening system is possible and, moreover, it is
possible to measure the large amount of samples in the same
lot.
[0900] In conducting the above-mentioned methods .quadrature. to
.quadrature. for screening the compound capable of inhibiting the
binding of the ligand with the G protein coupled receptor protein,
a suitable G protein coupled receptor fraction and a labeled ligand
are necessary. With respect to the G protein coupled receptor
fraction, it is preferred to use naturally occurring G protein
coupled receptors (natural type G protein coupled receptors) or
recombinant type G protein coupled receptor fractions with the
activity equivalent to that of the natural type G protein coupled.
Here the term "activity equivalent to" means the same ligand
binding activity, or the substantially equivalent ligand binding
activity.
[0901] With respect to the labeled ligand, it is possible to use
labeled ligands, labeled ligand analogized compounds, etc. For
example, ligands labeled with [3 .sub.H], [125.sub.I] [14 .sub.C],
[35.sub.S], etc. and other labeled substances may be utilized.
[0902] Specifically, G protein coupled receptor protein-containing
cells or cell membrane fractions are first suspended in a buffer
which is suitable for the determining method to prepare the
receptor sample in conducting the screening for a compound which
inhibits the binding of the ligand with the G protein coupled
receptor protein. With respect to the buffer, any buffer such as
Tris-HCl buffer or phosphate buffer of pH 4-10 (preferably, pH 6-8)
which does not inhibit the binding of the ligand with the receptor
may be used.
[0903] In addition, a surface-active agent such as CHAPS, Tween
80.TM. (Kao-Atlas, Japan), digitonin, deoxycholate, etc. and/or
various proteins such as bovine serum albumin (BSA), gelatine, etc.
may be added to the buffer with an object of decreasing the
nonspecific binding. Further, a protease inhibitor such as PMSF,
leupeptin, E-64 (manufactured by Peptide Laboratory, Japan),
pepstatin, etc. may be added with an object of inhibiting the
decomposition of the receptor and the ligand by protease. A labeled
ligand in a certain amount (5,000 cpm to 500,000 cpm) is added to
0.01 ml to 10 ml of said receptor solution and, at the same time,
10.sup.-4 M to 10.sup.-10 M of a test compound is made copresent.
In order to determine the nonspecific binding amount (NSB), a
reaction tube to which a great excessive amount of unlabeled test
compounds is added is prepared as well.
[0904] The reaction is carried out at 0-50.degree. C. (preferably
at 4-37.degree. C.) for 20 minutes to 24 hours (preferably 30
minutes to three hours). After the reaction, it is filtered through
a glass fiber filter, a filter paper, or the like, washed with a
suitable amount of the same buffer and the radioactivity retained
in the glass fiber filter, etc. is measured by means of a liquid
scintillation counter or a gamma-counter. Supposing that the count
(B.sub.0-NSB) obtained by subtracting the nonspecific binding
amount (NSB) from the total binding amount (B.sub.0) wherein an
antagonizing substance is not present is set at 100%, the test
compound in which the specific binding amount (B-NSB) obtained by
subtracting the nonspecific binding amount (NSB) from the total
binding amount (B) is, for example, less than 50% may be selected
as a candidate ligand to the G protein coupled receptor protein of
the present invention.
[0905] In conducting the above-mentioned methods .quadrature. to
.quadrature. for screening the compound which inhibits the binding
of the ligand with the G protein coupled receptor protein, the G
protein coupled receptor protein-mediated cell stimulating activity
(e.g. activities of promoting or activities of inhibiting
physiological responses such as liberation of arachidonic acid,
liberation of acetylcholine, endocellular CA.sup.2+ liberation
endocellular CAMP production, production of insitol phosphate,
changes in the cell membrane potential, phosphorylation of
endocellular proteins, activation of c-fos, lowering of pH,
activation of G protein and cell promulgation, etc.) may be
measured by known methods or by the use of commercially available
measuring kits. To be more specific, G protein coupled receptor
protein-containing cells are at first cultured in a multiwell plate
or the like.
[0906] In conducting the screening, it is substituted with a
suitable buffer which does not show toxicity to fresh media or
cells in advance, incubated for a certain period after adding a
test compound, etc. thereto. The resultant cells are extracted or
the supernatant liquid is recovered and the resulting product is
determined, preferably quantitatively, by each of the methods. When
it is difficult to identify the production of the index substance
(e.g. arachidonic acid, etc.) which is to be an index for the cell
stimulating activity due to the presence of decomposing enzymes
contained in the cell, an assay may be carried out by adding an
inhibitor against said decomposing enzyme. With respect to the
activities such as an inhibitory action against cAMP production, it
may be detected as an inhibitory action against the CAMP production
in the cells whose fundamental production has been increased by
forskolin or the like.
[0907] In conducting a screening by measuring the cell stimulating
activity, cells in which a suitable G protein coupled receptor
protein is expressed are necessary. Preferred G protein coupled
receptor protein-expressing cells are naturally occurring G protein
coupled receptor protein (natural type G protein coupled receptor
protein)-containing cell lines or strains (e.g. mouse pancreatic R
cell line, MIN6, etc.), the above-mentioned recombinant type G
protein coupled receptor protein-expressing cell lines or strains,
etc.
[0908] Examples of the test compound includes peptides, proteins,
non-peptidic compounds, synthesized compounds, fermented products,
cell extracts, plant extracts, animal tissue extracts, serum,
blood, body fluid, etc. Those compounds may be novel or known.
[0909] A kit for screening the compound which inhibits the binding
of the ligand with the G protein coupled receptor protein or a salt
thereof of the present invention comprises a G protein coupled
receptor protein or a peptide fragment thereof, or G protein
coupled receptor protein-containing cells or cell membrane fraction
thereof.
[0910] Examples of the screening kit include as follows: 1. Reagent
for Determining Ligand.
[0911] Buffer for Measurement and Buffer for Washing.
[0912] The product wherein 0.05% of bovine serum albumin
(manufactured by Sigma) is added to Hanks' Balanced Salt Solution
(manufactured by Gibco).
[0913] This may be sterilized by filtration through a membrane
filter with a 0.45 .mu.m pore size, and stored at 4.degree. C. or
may be prepared upon use.
[0914] Sample of G Protein Conjugated Receptor Protein.
[0915] CHO cells in which a G protein coupled receptor protein is
expressed are subcultured at the rate of 5.times.10.sup.5
cells/well in a 12-well plate and cultured at 37.degree. C. with a
5% CO.sub.2 and 95% air atomosphere for two days to prepare the
sample.
[0916] Labeled Ligand.
[0917] The ligand which is labeled with commercially available
[3.sub.HJ, [125I], [.sup.14 C], [.sup.35SJ, etc.
[0918] The product in a state of an aqueous solution is stored at
4.degree. C. or at -20.degree. C. and, upon use, diluted to 1 .mu.M
with a buffer for the measurement.
[0919] Standard Ligand Solution.
[0920] Ligand is dissolved in PBS containing 0.1% of bovine serum
albumin (manufactured by Sigma) to make 1 mM and stored at
-20.degree. C.
[0921] 2. Method of the Measurement.
[0922] CHO cells are cultured in a 12-well tissue culture plate to
express G protein coupled receptor proteins. The G protein coupled
receptor protein-expressing CHO cells are washed with 1 ml of
buffer for the measurement twice. Then 490 u 1 of buffer for the
measurement is added to each well.
[0923] Five .mu.l of a test compound solution of 10-3 to 10-10 M is
added, then 5 .mu.l of a labeled ligand is added and is made to
react at room temperature for one hour. For knowing the
non-specific binding amount, 5 .mu.l of the ligand of 10.sup.-3 M
is added instead of the test compound.
[0924] The reaction solution is removed from the well, which is
washed with 1 ml of buffer for the measurement three times. The
labeled ligand binding with the cells is dissolved in 0.2N NaOH-1%
SDS and mixed with 4 ml of a liquid scintillator A (manufactured by
Wako Pure Chemical, Japan).
[0925] {circle over (R)} Radioactivity is measured using a liquid
scintillation counter (manufactured by Beckmann) and PMB (percent
of maximum binding) is calculated by the following expression:
[0926] PMB=[(B-NSB)/(B.sub.0-NSB)].times.100
[0927] PMB: Percent of maximum binding
[0928] B: Value when a sample is added
[0929] NSB: Nonspecific binding
[0930] B.sub.0: Maximum binding
[0931] The compound or a salt thereof obtained by the screening
method or by the screening kit is a compound which inhibits the
binding of a ligand with a G protein coupled receptor protein and,
more particularly, it is a compound having a cell stimulating
activity mediated via a G protein coupled receptor or a salt
thereof (so-called "G protein coupled receptor agonist") or a
compound having no said stimulating activity (so-called "G protein
coupled receptor antagonist"). Examples of said compound are
peptides, proteins, non-peptidic compounds, synthesized compounds,
fermented products, etc. and the compound may be novel or
known.
[0932] Said G protein coupled receptor agonist has the same
physiological action as the ligand to the G protein coupled
receptor protein has and, therefore, it is useful as a safe and
less toxic pharmaceutical composition depending upon said ligand
activity.
[0933] On the other hand, said G protein coupled receptor
antagonist is capable of inhibiting the physiological activity of
the ligand to the G protein coupled receptor protein and, there
fore, it is useful as a safe and less toxic pharmaceutical
composition for inhibiting said ligand activity.
[0934] It is also strongly suggested that agonists and/or
antagonists related to the receptor encoded by pMAH2-17 obtained in
Example 19 and/or the receptor encoded by phAH2-17 obtained in
Example 21 would be useful in therapeutic or prophylactic treatment
of diseases or syndromes in connection with purine ligand compounds
or related analogues. It is expected that the agonists of the
receptor encoded by pMAH2-17 and/or of the receptor encoded by
phAH2-17 are useful as an immunomodulator or an antitumor agent, in
addition they are useful in therapeutically or prophylactically
treating hypertension, diabetes, cystic fibrosis, etc. It is still
expected that the antagonists of the receptor encoded by pMAH2-17
and/or of the receptor encoded by phAH2-17 are useful as
hypotensive agents, analgesics, agents for therapeutically or
prophylactically treating incontinence of urine, etc. With regard
to purinoceptors, the mutation of conserved basic amino acid
residues in the 6th or 7th putative transmembrane domain of
purinoceptors introduces alteration into the receptor's responses
to ATP (J. Biol. Chem., Vol.270(9), pp. 4185-4188 (1995)). It is
suggested that ATP is related to blood pressure control and
circular systems via receptors (Circulation Research, Vol. 58(3),
pp. 319-330 (1986)) and that ATP and purinoceptors are closely
related (Am. Phys. Soc., ppC577-C606 (1993).
[0935] When the compound or the salt thereof obtained by the
screening method or by the screening kit is used as the
above-mentioned pharmaceutical composition, a conventional means
may be applied therefor. The compound or the salt thereof may be
orally, parenterally, by inhalation spray, rectally, or topically
administered as pharmaceutical compositions or formulations (e.g.
powders, granules, tablets, pills, capsules, injections, syrups,
emulsions, elixirs, suspensions, solutions, etc.). For example, it
may be used by an oral route as tablets (sugar-coated if
necessary), capsules, elixiers, microcapsules, etc. or by a
parenteral route as injections such as an aseptic solution or a
suspension in water or in other pharmaceutically acceptable liquid.
The pharmaceutical compositions or formulations may comprise at
least one such compound alone or in admixture with pharmaceutically
acceptable carriers, adjuvants, vehicles, excipients and/or
diluents. The pharmaceutical compositions cam be formulated in
accordance with conventional methods. For example, said compound or
the salt thereof is mixed in a unit dose form which is required for
preparing a generally approved pharmaceutical preparations together
with a physiologically acceptable carriers, flavoring and/or
perfuming agents (fragrances), fillers, vehicles, antiseptics,
stabilizers, binders, etc. whereupon the preparation can be
manufactured. An amount of the effective component in those
preparations is to be in such an extent that the suitable dose
within an indicated range is achieved.
[0936] Examples of the additives which can be admixed in the
tablets, capsules, etc. are binders such as gelatin, corn starch,
tragacanth and gum arabicum; fillers such as crystalline cellulose;
swelling agents such as corn starch, gelatin and alginic acid;
lubricants such as magnesium stearate; sweetening agents such as
sucrose, lactose and saccharine; preservatives such as parabens and
sorbic acid; antioxidants such as ascorbic acid, .alpha.-tocopherol
and cysteine; fragrances such as peppermint, akamono oil and
cherry; disintegrants; buffering agents; etc. Other additives may
include mannitol, maltitol, dextran, agar, chitin, chitosan,
pectin, collagen, casein, albumin, synthetic or semi-synthetic
polymers, glyceride, lactide, etc. When the unit form of the
preparation is a capsule, a liquid carrier such as fat/oil may be
further added besides the above-mentioned types of materials. The
aseptic composition for injection may be formulated by a
conventional technique or practice for the preparations such as
that the active substance in a vehicle such as water for injection
is dissolved or suspended in a naturally occurring plant oil such
as sesame oil and palm oil.
[0937] Examples of an aqueous liquid for the injection are a
physiological saline solution and isotonic solutions containing
glucose and other auxiliary agents (e.g. D-sorbitol, D-mannitol,
sodium chloride, etc.) wherein a suitable auxiliary solubilizers
such as alcohol (e.g. ethanol, etc.), polyalcohol (e.g. propylene
glycol, polyethylene glycol, etc.), nonionic surface-active agent
(e.g. Polysorbate 80.TM., HCO-50, etc.), etc. may be jointly used.
In the case of the oily liquid, sesame oil, soybean oil, etc. may
be exemplified wherein benzyl benzoate, benzyl alcohol, etc. may be
jointly used as auxiliary solubilizers.
[0938] In addition, buffers (e.g. phosphate buffer, sodium acetate
buffer, etc.), analgesic agents (e.g. benzalkonium chloride,
procaine hydrochloride, etc.), stabilizers (e.g. human serum
albumin, polyethylene glycol, etc.), stabilizers (e.g. benzyl
alcohol, phenol, etc.), antioxidants, etc. may be compounded
therewith too. The prepared injection solution is filled in
suitable ampoules. The formulation prepared as such is safe and
less toxic and, therefore, it can be administered to warm-blooded
mammals such as rats, rabbits, sheep, swines, cattle, cats, dogs,
monkeys, human being, etc.
[0939] Dose levels of said compound or the salt thereof may vary
depending upon the symptom. Specific dose levels for any particular
patient will be employed depending upon a variety of factors
including the activity of specific compounds employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination, and
the severity of the particular disease undergoing therapy. In the
case of oral administration, it is usually about 0.1-100 mg,
preferably about 1.0-50 mg or, more preferably, about 1.0-20 mg per
day for adults (as 60 kg). When it is administered parenterally,
its dose at a time may vary depending upon the object to be
administered, organs to be administered, symptoms, administering
methods, etc. The term "parenteral" as used herein includes
subcutaneous injections, intravenous, intramuscular,
intraperitoneal injections, or infusion techniques. In the case of
injections, it is usually convenient to give by an intraveous route
in an amount of about 0.01-30 mg, preferably about 0.1-20 mg or,
more preferably, about 0.1-10 mg per day to adults (as 60 kg). In
the case of other animals, the dose calculated for 60 kg may be
administered as well.
[0940] (6) Manufacture of Antibody or Antiserum against the G
Protein Coupled Receptor Protein of the Present Invention, Its
Peptide Fragment or Its Salt.
[0941] Antibodies (e.g. polyclonal antibody and monoclonal
antibody) and antisera against the G protein coupled receptor
protein or salt thereof of the present invention or against the
peptide fragment of the G protein coupled receptor protein or salt
thereof of the present invention may be manufactured by antibody or
antiserum-manufacturing methods per se known to those of skill in
the art or methods similar thereto, using the G protein coupled
receptor protein or its-salt of the present invention or the
peptide fragment of the G protein coupled receptor protein or its
salt of the present invention. For example, monoclonal antibodies
can be manufactured by the method as given below.
[0942] [Preparation of Monoclonal Antibody]
[0943] (a) Preparation of Monoclonal Antibody-Producing Cells.
[0944] The G protein coupled receptor protein of the present
invention or its salt or the peptide fragment of the G protein
coupled receptor protein of the present invention or its salt
(hereinafter, may be abbreviated as the "G protein coupled receptor
protein") is administered to warm-blooded animals either solely or
together with carriers or diluents to the site where the production
of antibody is possible by the administration. In order to
potentiate the antibody productivity upon the administration,
complete Freund's adjuvants or incomplete Freund's adjuvants may
be. administered. The administration is usually carried out once
every two to six weeks and two to ten times in total. Examples of
the applicable warm-blooded animals are monkeys, rabbits, dogs,
guinea pigs, mice, rats, sheep, goats and chickens and the use of
mice and rats is preferred.
[0945] In the preparation of the cells which produce monoclonal
antibodies, an animal wherein the antibody titer is noted is
selected from warm-blooded animals (e.g. mice) immunized with
antigens, then spleen or lymph node is collected after two to five
days from the final immunization and antibody-producing cells
contained therein are fused with myeloma cells to give monoclonal
antibody-producing hybridomas. Measurement of the antibody titer in
antisera may, for example, be carried out by reacting a labeled G
protein coupled receptor protein (which will be mentioned later)
with the antiserum followed by measuring the binding activity of
the labeling agent with the antibody. The operation for fusing may
be carried out, for example, by a method of Koehler and Milstein
(Nature, 256, 495, 1975). Examples of the fusion accelerator are
polyethylene glycol (PEG), Sendai virus, etc. and the use of PEG is
preferred.
[0946] Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1,
etc. and the use of P3U 1 is preferred. The preferred fusion ratio
of the numbers of antibody-producing cells used (spleen cells) to
the numbers of myeloma cells is within a range of about 1:1 to
20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added in a
concentration of about 10-80% followed by incubating at
20-40.degree. C. (preferably, at 30-37.degree. C.) for one to ten
minutes, an efficient cell fusion can be carried out.
[0947] Various methods may be applied for screening a hybridoma
which produces anti-G protein coupled receptor antibody. For
example, a supernatant liquid of hybridoma culture is added to a
solid phase (e.g. microplate) to which the G protein coupled
receptor protein antigen is adsorbed either directly or with a
carrier, then anti-immunoglobulin antibody (anti-mouse
immunoglobulin antibody is used when the cells used for the cell
fusion are those of mouse) which is labeled with a radioactive
substance, an enzyme or the like, or protein A is added thereto and
then anti-G protein coupled receptor monoclonal antibodies bound on
the solid phase are detected; or a supernatant liquid of the
hybridoma culture is added to the solid phase to which
anti-immunoglobulin or protein A is adsorbed, then the G protein
coupled receptor labeled with a radioactive substance or an enzyme
is added and anti-G protein coupled receptor monoclonal antibodies
bonded with the solid phase is detected.
[0948] Selection and cloning of the anti-G protein coupled receptor
monoclonal antibody-producing hybridoma may be carried out by
methods per se known to those of skill in the art or methods
similar thereto. Usually, it is carried out in a medium for animal
cells, containing HAT (hypoxanthine, aminopterin and thymidine).
With respect to a medium for the selection, for the cloning and for
the growth, any medium may be used so far as hybridoma is able to
grow therein. Examples of the medium are an RPMI 1640 medium
(Dainippon Pharmaceutical Co., Ltd., Japan) containing 1-20%
(preferably 10-20%) of fetal calf serum (FCS), a GIT medium (Wako
Pure Chemical, Japan) containing 1-20% of fetal calf serum and a
serum-free medium for hybridoma culturing (SFM-101; Nissui Seiyaku,
Japan). The culturing temperature is usually 20-40.degree. C. and,
preferably, about 37.degree. C. The culturing time is usually from
five days to three weeks and, preferably, one to two weeks. The
culturing is usually carried out in 5% carbon dioxide gas. The
antibody titer of the supernatant liquid of the hybridoma culture
may be measured by the same manner as in the above-mentioned
measurement of the antibody titer of the anti-G protein coupled
receptor in the antiserum.
[0949] The cloning can be usually carried out by methods known per
se such as techniques in semi-solid agar and limiting dilution. The
cloned hybridoma is preferably cultured in modern serum-free
culture media to obtain optimal amounts of antibody in
supernatants. The target monoclonal antibody is also preferably
obtained from ascitic fluid derived from a mouse, etc. injected
intraperitoneally with live hybridoma cells. (b) Purification of
the Monoclonal Antibody.
[0950] Like in the separation/purification of conventional
polyclonal antibodies, the separation/purification of the anti-G
protein coupled receptor monoclonal antibody may be carried out by
methods for separating/purifying immunoglobulin (such as
salting-out, precipitation with an alcohol, isoelectric
precipitation, electrophoresis, adsorption/deadsorption using ion
exchangers such as DEAE, ultracentrifugation, gel filtration,
specific purifying methods in which only an antibody is collected
by treatment with an active adsorbent (such as an antigen-binding
solid phase, protein A or protein G) and the bond is dissociated
whereupon the antibody is obtained.
[0951] The G protein coupled receptor antibody of the present
invention which is manufactured by the aforementioned method (a) or
(b) is capable of specifically recognizing G protein coupled
receptors and, accordingly, it can be used for a quantitative
determination of the G protein coupled receptor in test liquid
samples and particularly for a quantitative determination by
sandwich immunoassays.
[0952] Thus, the present invention provides, for example, the
following methods:
[0953] (i) a quantitative determination of a G protein coupled
receptor in a test liquid sample, which comprises
[0954] (a) competitively reacting the test liquid sample and a
labeled G protein coupled receptor with an antibody which reacts
with the G protein coupled receptor of the present invention,
and
[0955] (b) measuring the ratio of the labeled G protein coupled
receptor binding with said antibody; and
[0956] (ii) a quantitative determination of a G protein coupled
receptor in a test liquid sample, which comprises
[0957] (a) reacting the test liquid sample with an antibody
immobilized on an insoluble carrier and a labeled antibody
simultaneously or continuously, and
[0958] (b) measuring the activity of the labeling agent on the
insoluble carrier wherein one antibody is capable of recognizing
the N-terminal region of the G protein coupled receptor while
another antibody is capable of recognizing the C-terminal region of
the G protein coupled receptor.
[0959] When the monoclonal antibody of the present invention
recognizing a G protein coupled receptor (hereinafter, may be
referred to as "anti-G protein coupled receptor antibody") is used,
G protein coupled receptors can be measured and, moreover, can be
detected by means of a tissue staining, etc. as well. For such an
object, antibody molecules per se may be used or F(ab').sub.2, Fab'
or Fab fractions of the antibody molecule may be used too. There is
no particular limitation for the measuring method using the
antibody of the present invention and any measuring method may be
used so far as it relates to a method in which the amount of
antibody, antigen or antibody-antigen complex, depending on or
corresponding to the amount of antigen (e.g. the amount of G
protein coupled receptor, etc.) in the liquid sample to be
measured, is detected by a chemical or a physical means and then
calculated using a standard curve prepared by a standard solution
containing the known amount of antigen. For example, nephrometry,
competitive method, immunometric method and sandwich method are
suitably used and, in terms of sensitivity and specificity, the
sandwich method which will be described herein later is
particularly preferred.
[0960] Examples of the labeling agent used in the measuring method
using the labeling substance are radioisotopes, enzymes,
fluorescent substances, luminescent substances, colloids, magnetic
substances, etc. Examples of the radioisotope are [.sup.125I],
[.sup.131I, [.sup.3H] and .sup.14C]; preferred examples of the
enzyme are those which are stable and with big specific activity,
such as .beta.-galactosidase, .beta.-glucosidase, alkali
phosphatase, peroxidase and malate dehydrogenase; examples of the
fluorescent substance are fluorescamine, fluorescein
isothiocyanate, etc.; and examples of the luminescent substance are
luminol, luminol derivatives, luciferin, lucigenin, etc. Further, a
biotin-avidin system may also be used for binding an antibody or
antigen with a labeling agent.
[0961] In an insolubilization (immobilization) of antigens or
antibodies, a physical adsorption may be used or a chemical binding
which is usually used for insolubilization or immobilization of
proteins or enzymes may be used as well. Examples of the carrier
are insoluble polysaccharides such as agarose, dextran and
cellulose; synthetic resins such as polystyrene, polyacrylamide and
silicone; glass; etc.
[0962] In a sandwich (or two-site) method, the test liquid is made
to react with an insolubilized anti-G protein coupled receptor
antibody (the first reaction), then it is made to react with a
labeled anti-G protein coupled receptor antibody (the second
reaction) and the activity of the labeling agent on the insoluble
carrier is measured whereupon the amount of the G protein coupled
receptor in the test liquid can be determined. The first reaction
and the second reaction may be conducted reversely or
simultaneously or they may be conducted with an interval. The type
of the labeling agent and the method of insolubilization
(immobilization) may be the same as those mentioned already herein.
In the immunoassay by means of a sandwich method, it is not always
necessary that the antibody used for the labeled antibody and the
antibody for the solid phase is one type or one species but, with
an object of improving the measuring sensitivity, etc., a mixture
of two or more antibodies may be used too.
[0963] In the method of measuring G protein coupled receptors by
the sandwich method of the present invention, the preferred anti-G
protein coupled receptor antibodies used for the first and the
second reactions are antibodies wherein their sites binding to the
G protein coupled receptors are different each other. Thus, the
antibodies used in the first and the second reactions are those
wherein, when the antibody used in the second reaction recognizes
the C-terminal region of the G protein coupled receptor, then the
antibody recognizing the site other than C-terminal regions, e.g.
recognizing the N-terminal region, is preferably used in the first
reaction.
[0964] The anti-G protein coupled receptor antibody of the present
invention may be used in a measuring system other than the sandwich
method such as a competitive method, an immunometric method and a
nephrometry. In a competitive method, an antigen in the test
solution and a labeled antigen are made to react with an antibody
in a competitive manner, then an unreacted labeled antigen (F) and
a labeled antigen binding with an antibody (B) are separated (i.e.
B/F separation) and the labeled amount of any of B and F is
measured whereupon the amount of the antigen in the test solution
is determined. With respect to a method for such a reaction, there
are a liquid phase method in which a soluble antibody is used as
the antibody and the B/F separation is conducted by polyethylene
glycol, a second antibody to the above-mentioned antibody, etc.;
and a solid phase method in which an immobilized antibody is used
as the first antibody or a soluble antibody is used as the first
antibody while an immobilized antibody is used as the second
antibody.
[0965] In an immunometric method, an antigen in the test solution
and an immobilized antigen are subjected to a competitive reaction
with a certain amount of a labeled antibody followed by separating
into solid and liquid phases; or the antigen in the test solution
and an excess amount of labeled antibody are made to react, then a
immobilized antigen is added to bind an unreacted labeled antibody
with the solid phase and separated into solid and liquid phases.
After that, the labeled amount of any of the phases is measured to
determine the antigen amount in the test solution.
[0966] In a nephrometry, the amount of insoluble sediment which is
produced as a result of the antigen-antibody reaction in a gel or
in a solution is measured. Even when the antigen amount in the test
solution is small and only a small amount of the sediment is
obtained, a laser nephrometry wherein scattering of laser is
utilized can be suitably used.
[0967] In applying each of those immunological measuring methods
(immunoassays) to the measuring method of the present invention, it
is not necessary to set up any special condition, operation, etc.
therefor. A measuring system (assay system) for G protein coupled
receptor may be constructed taking the technical consideration of
the persons skilled in the art into consideration in the
conventional conditions and operations for each of the methods.
With details of those conventional technical means, a variety of
reviews, reference books, etc. may be referred to. They are, for
example, Hiroshi Irie (ed): "Radioimmunoassay" (Kodansha, Japan,
1974); Hiroshi Irie (ed): "Radioimmunoassay; Second Series"
(Kodansha, Japan, 1979); Eiji Ishikwa et al. (ed): "Enzyme
Immunoassay" (Igaku Shoin, Japan, 1978); Eiji Ishikawa et al. (ed):
"Enzyme Immunoassay" (Second Edition) (Igaku Shoin, Japan, 1982);
Eiji Ishikawa et al. (ed): "Enzyme Immunoassay" (Third Edition)
(Igaku Shoin, Japan, 1987); "Methods in Enzymology" Vol.70
(Immuochemical Techniques (Part A)); ibid. Vol. 73 (Immunochemical
Techniques (Part B)); ibid. Vol. 74 (Immunochemical Techniques
(Part C)); ibid. Vol. 84 (Immunochemical Techniques (Part D:
Selected Immunoassays)); ibid. Vol. 92 (Immunochemical Techniques
(Part E: Monoclonal Antibodies and General Immunoassay Methods));
ibid. Vol. 121 (Immunochemical Techniques (Part I:Hybridoma
Technology and Monoclonal Antibodies)) (Academic Press); etc.
[0968] (7) Preparation of Animals Having the G Protein Coupled
Receptor Protein-Encoding DNA of the Present Invention.
[0969] It is possible to prepare transgenic animals expressing G
protein coupled receptors using G protein coupled receptor
protein-encoding DNA. Examples of the animals are warm-blooded
mammals such as rats, rabbit, sheep, swines, cattle, cats, dogs and
monkeys.
[0970] In transferring the G protein coupled receptor
protein-encoding DNA to the aimed animal, it is generally
advantageous that said DAN is used by ligating with a site at the
downstream of a promoter which is capable of expressing in animal
cells. For example, when G protein coupled receptor protein DNA is
to be transferred to a rabbit, a gene construct ligated with a site
at the downstream of various promoters which are capable of
expressing the G protein coupled receptor protein DNA derived from
an animal compatible to the animal in animal host cells is
subjected to a microinjection to the fertilized ovum (oosperm) of
the aimed animal (e.g. fertilized ovum (embryo) of rabbit)
whereupon the transgenic animal which produces the G protein
coupled receptor protein in a high amount can be prepared.
[0971] Examples of the promoters used are promoters derived from
virus and ubiquitous expression promoters such as metallothionein
promoters may be used but, preferably, enolase gene promoters and
NGF gene promoters capable of specifically expressing in brain are
used.
[0972] Transfer of the G protein coupled receptor protein DNA at a
fertilized ovum cell stage is secured in order that the DNA can be
present in all of embryonal cells and body somatic cells of an
aimed animal. The fact that the G protein coupled receptor protein
is present in the fertilized ovum cells of the produced transgenic
animal after the DNA transfer means that all progeny of the
produced transgenic animal have the G protein coupled receptor
protein in all of their embryonal cells and somatic cells.
Descendants (offsprings) of the animal of this type which inherited
the gene have the G protein coupled receptor protein in all of
their embryonal cells and somatic cells.
[0973] The transgenic animal to which the G protein coupled
receptor protein DNA is transferred can be subjected to a mating
and a breeding for generations under a common breeding circumstance
as the animal holding said DNA after confirming that the gene can
be stably retained. Moreover, male and female animals having the
desired DNA are mated to give a homozygote having the transduced
gene in both homologous chromosomes and then those male and female
animals are mated whereby it is possible to breed for generations
so that all descendants have said DNA.
[0974] The animal to which the G protein coupled receptor protein
DNA is transferred highly expresses the G protein coupled receptor
protein and, accordingly, it is useful as the animal for screening
for an agonist or an antagonist to said G protein coupled receptor
protein.
[0975] The DNA-transferred animal can be used as a cell source for
a tissue culture. For example, DNA or RNA in the tissue of the
DNA-transferred mouse is directly analyzed or protein tissues
expressed by gene are analyzed whereupon the G protein coupled
receptor protein can be analyzed. Cells of the G protein coupled
receptor protein-containing tissue are cultured by standard tissue
culture techniques whereupon it is possible to study the function
of the cells which are usually difficult to culture (e.g. those
derived from brain and peripheral tissues) using the resulting
culture. By using said cells, it is also possible to select the
pharmaceuticals which can potentiate, for example, the functions of
various tissues. Moreover, if a cell strain with a high expression
is available, it is possible to separate and purify G protein
coupled receptor proteins therefrom.
[0976] As such, the amount of G protein coupled receptor proteins
can now be determined with a high precision using the anti-G
protein coupled receptor antibody of the present invention.
[0977] (8) Antisense Oligonucleotides Capable of Inhibiting
Replication of G Protein Coupled Receptor Protein Gene
[0978] In another aspect of the present invention, antisense
oligonucleotides (nucleic acids) capable of inhibiting the
replication or expression of G protein coupled receptor protein
gene may be designed and synthesized based on information on the
nucleotide sequences of cloned and determined G protein coupled
receptor protein-encoding DNAs. Such an antisense oligonucleotide
(nucleic acid) is capable of hybridizing with RNA of G protein
coupled receptor protein genes to inhibit the synthesis or function
of said RNA or of modulating the expression of a G protein coupled
receptor protein gene via interaction with G protein coupled
receptor protein-related RNA. Oligonucleotides complementary to,
and specifically hybridizable with, selected sequences of G protein
coupled receptor protein-related RNA are useful in controlling or
modulating the expression of a G protein coupled receptor protein
gene in vitro and in vivo, and in treating or diagnosing disease
states of suspected animals. The term "corresponding" means
homologous to or complementary to a particular sequence of the
nucleotide sequence or nucleic acid including the gene. As between
nucleotides (nucleic acids) and peptides (proteins),
"corresponding" usually refers to amino acids of a peptide
(protein) in an order derived from the sequence of a nucleotides
(nucleic acids) or its complement. The G protein coupled receptor
protein gene 5' end hairpin loop, 5' end 6-base-pair repeats, 5'
end untranslated region, polypeptide translation initiation codon,
protein coding region, ORF translation initiation codon, 3'
untranslated region, 3' end palindrome region, and 3' end hairpin
loop may be selected as preferred targets though any region may be
a target among G protein coupled receptor protein genes. The
relationship between the target and oligonucleotides complementary
to at least a portion of the target, specifically hybridizable with
the target, is denoted as "antisense". The antisense
oligonucleotides may be polydeoxynucleotides containing
2-deoxy-D-ribose, polyribonucleotides containing D-ribose, any
other type of polynucleotide which is an N-glycoside of a purine or
pyrimidine base, or other polymers containing nonnucleotide
backbones (e.g., protein nucleic acids and synthetic
sequence-specific nucleic acid polymers commercially available) or
nonstandard linkages, providing that the polymers contain
nucleotides in a configuration which allows for base pairing and
base stacking such as is found in DNA and RNA. They may include
double- and single-stranded DNA, as well as double- and
single-stranded RNA and DNA:RNA hybrids, and also include, as well
as unmodified forms of the polynucleotide or oligonucleotide, known
types of modifications, for example, labels which are known to
those skilled in the art, "caps", methylation, substitution of one
or more of the naturally occurring nucleotides with analogue,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.) and with charged linkages or
sulfur-containing linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (including nucleases, nuclease
inhibitors, toxins, antibodies, signal peptides, poly-L-lysine,
etc.) and saccharides (e.g., monosaccharides, etc.), those with
intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, those with modified
linkages (e.g., alpha anomeric nucleic acids, etc.). The terms
"nucleoside", "nucleotide" and "nucleic acid" will include those
moieties which contain not only the known purine and pyrimidine
bases, but also other heterocyclic bases which have been modified.
Such modifications include methylated purines and pyrimidines,
acylated purines and pyrimidines, or other heterocycles. Modified
nucleosides or nucleotides will also include modifications on the
sugar moiety, e.g., wherein one or more of the hydroxyl groups are
replaced with halogen, aliphatic groups, or are functionalized as
ethers, amines, or the like.
[0979] The antisense nucleic acid of the present invention is RNA,
DNA or a modified nucleic acid. Examples of modified nucleic acid
are, but not limited to, degradation-resistant sulfurized and
thiophosphate derivatives of nucleic acids, and poly- or
oligonucleoside amides. Preferred design modifications of the
antisense nucleic acids of the present invention are modifications
that are designed to:
[0980] (1) increase the intracellular stability of the nucleic
acid;
[0981] (2) increase the cellular permeability of the nucleic
acid;
[0982] (3) increase the affinity of the nucleic acid for the target
sense strand; or
[0983] (4) decrease the toxicity (if any) of the nucleic acid.
[0984] Many such modifications are known to those skilled in the
art, as described in J. Kawakami et al., Pharm Tech Japan, Vol. 8,
pp.247, 1992; Vol. 8, pp.395, 1992; S. T. Crooke et al. ed.,
Antisense Research and Applications, CRC Press, 1993; etc. The
nucleic acids may contain altered or modified sugars, bases or
linkages, be delivered in specialized systems such as liposomes,
microspheres or by gene therapy, or may have attached moieties.
Such attached moieties include polycationic moieties such as
polylysine that act as charge neutralizers of the phosphate
backbone, or hydrophobic moieties such as lipids (e.g.,
phospholipids, cholesterols, etc.) that enhance interaction with
cell membranes or increase uptake of the nucleic acid. Preferred
lipids that may attached are cholesterols or derivatives thereof
(e.g., cholesteryl chloroformate, cholic acid, etc.). The moieties
may be attached at the 3' or 5' ends of the nucleic acids, and also
may be attached through a base, sugar, or internucleoside linkage.
Other moieties may be capping groups specifically placed at the 3'
or 5' ends of the nucleic acids to prevent degradation by nuclease
such as exonuclease, RNase, etc. Such capping groups include, but
are not limited to, hydroxyl protecting groups known to those
skilled in the art, including glycols such as polyethylene glycols,
tetraethylene glycol and the like.
[0985] The inhibitory activity of antisense nucleic acids can be
examined using the transformant (or transfectant) of the present
invention, the in vitro and in vivo gene expression system of the
present invention, or the in vitro and in vivo translation system
of G protein coupled receptor proteins. The nucleic acid can be
placed in the cell through any number of ways known per se.
[0986] In the specification and drawings of the present
application, the abbreviations used for bases (nucleotides), amino
acids and so forth are those recommended by the IUPAC-IUB
Commission on Biochemical Nomenclature or those conventionally used
in the art. Examples thereof are given below. Amino acids for which
optical isomerism is possible are, unless otherwise specified, in
the L form.
[0987] DNA: Deoxyribonucleic acid
[0988] cDNA: Complementary deoxyribonucleic acid
[0989] A: Adenine
[0990] T: Thymine
[0991] G: Guanine
[0992] C: Cytosine
[0993] RNA: Ribonucleic acid
[0994] mRNA: Messenger ribonucleic acid
[0995] dATP: Deoxyadenosine triphosphate
[0996] dTTP: Deoxythymidine triphosphate
[0997] dGTP: Deoxyguanosine triphosphate
[0998] dCTP: Deoxycytidine triphosphate
[0999] ATP: Adenosine triphosphate
[1000] EDTA: Ethylenediamine tetraacetic acid
[1001] SDS: Sodium dodecyl sulfate
[1002] EIA: Enzyme Immunoassay
[1003] G, Gly: Glycine (or Glycyl)
[1004] A, Ala: Alanine (or Alanyl)
[1005] V, Val: Valine (or Valyl)
[1006] L, Leu: Leucine (or Leucyl)
[1007] I, Ile: Isoleucine (or Isoleucyl)
[1008] S, Ser: Serine (or Seryl)
[1009] T, Thr: Threonine (or Threonyl)
[1010] C, Cys: Cysteine (or Cysteinyl)
[1011] M, Met: Methionine (or Methionyl)
[1012] ,.E, Glu: Glutamic acid (or Glutamyl)
[1013] D, Asp: Aspartic acid (or Aspartyl)
[1014] K, Lys: Lysine (or Lysyl)
[1015] R, Arg: Arginine (or Arginyl)
[1016] H, His: Histidine (or Histidyl)
[1017] F, Phe: Pheylalanine (or Pheylalanyl)
[1018] Y, Tyr: Tyrossine (or Tyrosyl)
[1019] W, Trp: Tryptophan (or Tryptophanyl)
[1020] P, Pro: Proline (or Prolyl)
[1021] N, Asn: Asparagine (or Asparaginyl)
[1022] Q, Gin: Glutamine (or Glutaminyl)
[1023] NVal: Norvaline (or Norvalyl).
[1024] pGlu: Pyroglutamic acid (or Pyroglutamyl)
[1025] Bic: 7 -Butyrolacton-7 -carbonyl
[1026] Kpc: 2-Ketopiperidinyl-6-carbonyl
[1027] Otc: 3-Oxoperhydro-1,4-thiazin-5-carbonyl
[1028] Me: Methyl
[1029] Et: Ethyl
[1030] Bu: Butyl
[1031] Ph: Phenyl
[1032] TC: Thiazolidinyl-4 (R)-carboxamide
[1033] The transformant Escherichia coli, designated INV.alpha.
F'/p 19P2, which is obtained in the Example 3 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
Aug. 9, 1994, with the National Institute of Bioscience and
Human-Technology (NIBH), Agency of Industrial Science and
Technology, Ministry of International Trade and Industry, Japan and
has been assigned the Accession Number FERM BP-4776. It is also on
deposit from Aug. 22, 1994 with the Institute for Fermentation,
Osaka, Japan (IFO) and has been assigned the Accession Number IFO
15739.
[1034] The transformant Escherichia coli, designated INV.alpha.
F'/pG3-2, which is obtained in the Example 4 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
August 9, 1994, with NIBH and has been assigned the Accession
Number FERM BP-4775. It is also on deposit from Aug. 22, 1994 with
IFO and has been assigned the Accession Number IFO 15740.
[1035] The transformant Escherichia coli, designated INV.alpha.
F'/p63A2, which is obtained in the Example 5 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
Aug. 9, 1994, with NIBH and has been assigned the Accession Number
FERM BP-4777. It is also on deposit from Aug. 22, 1994 with IFO and
has been assigned the Accession Number IFO 15738.
[1036] The transformant Escherichia coli, designated JM 109/phGR3,
which is obtained in the Example 6 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from September 27,
1994, with NIBH and has been assigned the Accession Number FERM
BP-4807. It is also on deposit from Sep. 22, 1994 with IFO and has
been assigned the Accession Number IFO 15748.
[1037] The transformant Escherichia coli, designated JM
109/p3H2-17, which is obtained in the Example 7 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
September 27, 1994, with NIBH and has been assigned the Accession
Number FERM BP-4806. It is also on deposit from Sep. 22, 1994 with
IFO and has been assigned the Accession Number IFO 15747.
[1038] The transformant Escherichia coli, designated JM
109/p3H2-34, which is obtained in the Example 8 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
October 12, 1994, with NIBH and has been assigned the Accession
Number FERM BP-4828. It is also on deposit from Oct. 12, 1994 with
IFO and has been assigned the Accession Number IFO 15749.
[1039] The transformant Escherichia coli, designated JM 109/pMD4,
which is obtained in the Example 9 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from November 11,
1994, with NIBH and has been assigned the Accession Number FERM
BP-4888. It is also on deposit from Nov. 17, 1994 with IFO and has
been assigned the Accession Number IFO 15765.
[1040] The transformant Escherichia coli, designated JM 109/pMGR20,
which is obtained in the Example 10 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Dec. 15, 1994,
with NIBH and has been assigned the Accession Number FERM BP-4937.
It is also on deposit from December 14, 1994 with IFO and has been
assigned the Accession Number IFO 15773.
[1041] The transformant Escherichia coli, designated JM 109/pMJ 10,
which is obtained in the Example 12 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Dec. 15, 1994,
with NIBH and has been assigned the Accession Number FERM BP-4936.
It is also on deposit from Dec. 16, 1994 with IFO and has been
assigned the Accession Number IFO 15784.
[1042] The transformant Escherichia coli, designated JM 109/pMH28,
which is obtained in the Example 14 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Jan. 13, 1995,
with NIBH and has been assigned the Accession Number FERM BP-4970.
It is also on deposit from Jan. 20, 1995 with IFO and has been
assigned the Accession Number IFO 15791.
[1043] The transformant Escherichia coli, designated JM 109/pMN7,
which is obtained in the Example 16 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Feb. 22, 1995,
with NIBH and has been assigned the Accession Number FERM BP-5011.
It is also on deposit from Feb. 27, 1995 with IFO and has been
assigned the Accession Number IFO 15803.
[1044] The transformant Escherichia coli, designated JM 109/p5S38,
which is obtained in the Example 17 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Oct. 27, 1994,
with NIBH and has been assigned the Accession Number FERM BP-4856.
It is also on deposit from Oct. 25, 1994 with IFO and has been
assigned the Accession Number IFO 15754.
[1045] The transformant Escherichia coli, designated JM
109/pMAH2-17, which is obtained in the Example 19 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
Apr. 7, 1995, with NIBH and has been assigned the Accession Number
FERM BP-5073. It is also on deposit from Mar. 31, 1995 with IFO and
has been assigned the Accession Number IFO 15813.
[1046] The transformant Escherichia coli, designated JM 109/pMN128,
which is obtained in the Example 20 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Mar. 17, 1995,
with NIBH and has been assigned the Accession Number FERM BP-5039.
It is also on deposit from Mar. 22, 1995 with IFO and has been
assigned the Accession Number IFO 15810.
[1047] The transformant Escherichia coli, designated JM
109/phAH2-17, which is obtained in the Example 21 mentioned herein
below, is on deposit under the terms of the Budapest Treaty from
Jul. 20, 1995, with NIBH and has been assigned the Accession Number
FERM BP-5168. It is also on deposit from Jul. 14, 1995 with IFO and
has been assigned the Accession Number IFO 15856.
[1048] Each SEQ ID NO set forth in the SEQUENCE LISTING of the
specification refers to the following sequence:
[1049] [SEQ ID NO: 24] is a partial amino acid sequence of the
human pituitary gland-derived G protein coupled receptor protein
encoded by the human pituitary gland-derived G protein coupled
receptor protein cDNA fragment included in p19P2,
[1050] [SEQ ID NO: 25] is a partial amino acid sequence of the
human pituitary gland-derived G protein coupled receptor protein
encoded by the human pituitary gland-derived G protein coupled
receptor protein cDNA fragment included in p19P2,
[1051] [SEQ ID NO: 26] is an entire amino acid sequence of the
human pituitary gland-derived G protein coupled receptor protein
encoded by the human pituitary gland-derived G protein coupled
receptor protein cDNA fragment included in phGR3,
[1052] [SEQ ID NO: 27] is a partial amino acid sequence of the
mouse pancreatic 3 -cell line, MIN6-derived G protein coupled
receptor protein encoded by the mouse pancreatic .beta.-cell line,
MIN6-derived G protein coupled receptor protein cDNA fragment
having a nucleotide sequence, (SEQ ID NO: 32), derived based upon
the nucleotide sequences of the mouse pancreatic .beta.-cell line,
MIN6-derived G protein coupled receptor protein cDNA fragments each
included in pG3-2 and pG1-10,
[1053] [SEQ ID NO: 28] is a partial amino acid sequence of the
mouse pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein encoded by p5S38,
[1054] [SEQ ID NO: 29] is a nucleotide sequence of the human
pituitary gland-derived G protein coupled receptor protein cDNA
fragment included in p 19P2,
[1055] [SEQ ID NO: 30] is a nucleotide sequence of the human
pituitary gland-derived G protein coupled receptor protein cDNA
fragment included in p 19P2,
[1056] [SEQ ID NO: 31] is an entire nucleotide sequence of the
human pituitary gland-derived G protein coupled receptor protein
cDNA included in phGR3,
[1057] [SEQ ID NO: 32] is a nucleotide sequence of the mouse
pancreatic S-cell line, MIN6-derived G protein coupled receptor
protein cDNA, derived based upon the nucleotide sequences of the
mouse pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein cDNA fragments each included in pG3-2 and
pG1-10,
[1058] [SEQ ID NO: 33] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived G protein cDNA included
in p5S38,
[1059] [SEQ ID NO: 34] is a partial amino acid sequence of the
human amygdaloid nucleus-derived G protein coupled receptor protein
encoded by the cDNA fragment included in p63A2,
[1060] [SEQ ID NO: 35] is a partial amino acid sequence of the
human amygdaloid nucleus-derived G protein coupled receptor protein
encoded by the cDNA fragment included in p63A2,
[1061] [SEQ ID NO: 36] is a nucleotide sequence of the human
amygdaloid nucleus-derived G protein coupled receptor protein cDNA
fragment included in p63A2,
[1062] [SEQ ID NO: 37] is a nucleotide sequence of the human
amygdaloid nucleus-derived G protein coupled receptor protein cDNA
fragment included in p63A2,
[1063] [SEQ ID NO: 38] is a partial amino acid sequence encoded by
the mouse pancreatic .beta.-cell line, MIN6-derived G protein
coupled receptor protein cDNA included in p3H2-17,
[1064] [SEQ ID NO: 39] is a full-length amino acid sequence encoded
by the open reading frame of the mouse pancreatic .beta.-cell line,
MIN6-derived G protein coupled receptor protein cDNA included in
pMAH2-17,
[1065] [SEQ ID NO: 40] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein cDNA included in p3H2-17,
[1066] [SEQ ID NO: 41] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein cDNA included in pMAH2-17,
[1067] [SEQ ID NO: 42] is a partial amino acid sequence encoded by
the mouse pancreatic .beta.-cell line, MIN6-derived G protein
coupled receptor protein cDNA included in p3H2-34,
[1068] [SEQ ID NO: 43] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein cDNA fragment included in p3H2-34,
[1069] (SEQ ID NO: 44] is a partial amino acid sequence encoded by
the rabbit gastropyrolic part smooth muscle-derived G protein
coupled receptor protein cDNA included in pMD4,
[1070] [SEQ ID NO: 45] is a nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in pMD4,
[1071] [SEQ ID NO: 46] is an entire amino acid sequence encoded by
the mouse pancreatic .beta.-cell line, MIN6-derived G protein
coupled receptor protein cDNA included in pMGR20,
[1072] [SEQ ID NO: 47] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived G protein coupled
receptor protein cDNA included in pMGR20,
[1073] (SEQ ID NO: 48] is a partial amino acid sequence encoded by
the rabbit gastropyrolic part smooth muscle-derived G protein
coupled receptor protein cDNA included in pMJ10,
[1074] [SEQ ID NO: 49] is a nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in pMJ10,
[1075] [SEQ ID NO: 50] is a partial amino acid sequence encoded by
the rabbit gastropyrolic part smooth muscle-derived G protein
coupled receptor protein cDNA included in pMH28,
[1076] [SEQ ID NO: 51] is a nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in pMH28,
[1077] [SEQ ID NO: 52] is a partial amino acid sequence encoded by
the rabbit gastropyrolic part smooth muscle-derived G protein
coupled receptor protein cDNA included in pMN7,
[1078] [SEQ ID NO: 53] is a nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in pMN7,
[1079] (SEQ. ID NO: 54] is a partial amino acid sequence encoded by
the rabbit gastropyrolic part smooth muscle-derived G protein
coupled receptor protein cDNA included in pMN128,
[1080] [SEQ ID NO: 55] is a nucleotide sequence of the rabbit
gastropyrolic part smooth muscle-derived G protein coupled receptor
protein cDNA fragment included in pMN128,
[1081] [SEQ ID NO: 56] is a full-length amino acid sequence of the
human-derived G protein coupled receptor protein encoded by the
human-derived G protein coupled receptor protein cDNA included in
phAH2-17, and
[1082] [SEQ ID NO: 57] is a nucleotide sequence of the
human-derived G protein coupled receptor protein cDNA included in
phAH2-17.
EXAMPLES
[1083] Described below are working examples of the present
invention which are provided only for illustrative purposes, and
not to limit the scope of the present invention. In light of the
present disclosure, numerous embodiments within the scope of the
claims will be apparent to those of ordinary skill in the art.
Example 1
[1084] Preparation of Synthetic DNA Primer for Amplifying DNA
Coding for G Protein Coupled Receptor Protein
[1085] A comparison of deoxyribonucleotide sequences coding for the
known amino acid sequences corresponding to or near the first
membrane-spanning domain each of human-derived TRH receptor protein
(HTRHR), human-derived RANTES receptor protein (L10918, HUMRANTES),
human Burkitt's lymphoma-derived unknown ligand receptor protein
(X68149, HSBLR1A), human-derived somatostatin receptor protein
(L14856, HUMSOMAT), rat-derived .mu.-opioid receptor protein
(U02083, RNU02083), rat-derived .kappa..sup.- opioid receptor
protein (U00442, U00442), human-derived neuromedin B receptor
protein (M73482, HUMNMBR), human-derived muscarinic acetylcholine
receptor protein (X15266, HSHM4), rat-derived adrenaline
.alpha..sub.1B receptor protein (L08609, RATAADRE01), human-derived
somatostatin 3 receptor protein (M96738, HUMSSTR3X), human-derived
C.sub.5a receptor protein (HUMC5AAR), human-derived unknown ligand
receptor protein (HUMRDC1A), human-derived unknown ligand receptor
protein (M84605, HUMOPIODRE) and rat-derived adrenaline a2B
receptor protein (M91466, RATA2BAR) was made. As a result, highly
homologous regions or parts were found (FIG. 1).
[1086] Further, a comparison of deoxynucleotide sequences coding
for the known amino acid sequences corresponding to or near the
sixth membrane-spanning domain each of mouse-derived unknown ligand
receptor protein (M80481, MUSGIR), human-derived bombesin receptor
protein (L08893, HUMBOMB3S), human-derived adenosine A2 receptor
protein (S46950, S46950), mouse-derived unknown ligand receptor
protein (D21061, MUSGPCR), mousederived TRH receptor protein
(S43387, S43387), rat-derived neuromedin K receptor protein
(J05189, RATNEURA), rat-derived adenosine Al receptor protein
(M69045, RATA1ARA), human-derived neurokinin A receptor protein
(M57414, HUMNEKAR), rat-derived adenosine A3 receptor protein
(M94152, RATADENREC), humanderived somatostatin 1 receptor protein
(M81829, HUMSRI1A), human-derived neurokinin 3 receptor protein
(S86390, S86371S4), rat-derived unknown ligand receptor protein
(X61496, RNCGPCR), human-derived somatostatin 4 receptor protein
(L07061, HUMSSTR4Z) and rat-derived GnRH receptor protein (M31670,
RATGNRHA) was made. As a result, highly homologous regions or parts
were found (FIG. 2).
[1087] The aforementioned abbreviations in the parentheses are
identifiers (reference numbers) which are indicated when
GenBank/EMBL Data Bank is retrieved by using DNASIS Gene/Protein
Sequencing Data Base (CDO19, Hitachi Software Engineering, Japan)
and are usually called "Accession Numbers" or "Entry Names". HTRHR
is, however, the sequence as disclosed in Japanese Unexamined
Patent Publication No. 286986/1993 (EPA 638645).
[1088] Specifically, it was planned to incorporate mixed bases
relying upon the base regions that were in agreement with cDNAs
coding for a large number of receptor proteins in order to enhance
base agreement of sequences with as many receptor cDNAs as possible
even in other regions. Based upon these sequences, the degenerate
synthetic DNA having a nucleotide sequence represented by SEQ ID
NO: 1 which is complementary to the homologous nucleotide sequence
of FIG. 1 and the degenerate synthetic DNA having a nucleotide
sequence represented by SEQ ID NO: 2 which is complementary to the
homologous nucleotide sequence of FIG. 2 were produced. Nucleotide
synthesis was carried out by a DNA synthesizer.
[1089] [Synthetic DNAs]
1 5'-CGTGG (G or C) C (A or C) T (G or C) (G or C) TGGGCAAC (A, G,
C or T) (C or T) (SEQ ID NO: 1) CCTG-3' 5'-GT(A, G, C or T) G (A or
T) (A or G) (A or G) GGCA (A, G, C or T) CCAGCAGA (G or T) (SEQ ID
NO: 2) GGCAAA-3'
[1090] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis.
Example 2
[1091] Isolation of Human Somatostatin Receptor Protein-Encoding
DNA, Human D5 Dopamine Receptor Protein-Encoding DNA, and Rat
Somatostatin Receptor Protein-Encoding DNA
[1092] (1) Amplification of DNA by Polymerase Chain Reaction
(PCR)
[1093] cDNAs (QuickClone, CLONTECH Laboratories, Inc.) prepared
from human brain amygdaloid nucleus, human pituitary gland and rat
brain each in an amount of 1 ng as templates, the synthetic DNA
primers prepared in Example 1 each in an amount of 1 .mu.M, 2.5 mM
dNTPs (deoxyribonucleoside triphosphates), and 2.5 units of Taq DNA
polymerase (Takara Shuzo Co., Japan) were mixed together with a
buffer attached to the enzyme kit such that the total amount was
100 .mu.1. The polymerase chain reaction was carried out by using a
Thermal Cycler manufactured by Perkin-Elmer Co. One cycle was set
to include 96.degree. C. for 30 sec., 45.degree. C. for 1 min. and
60.degree. C. for 3 min.. Totally this one cycle was repeated 30
times to amplify DNAs. Amplification of DNAs was confirmed by 1.2%
agarose electrophoresis [FIG. 17).
[1094] (2) Isolation of Amplified DNA and Analysis of DNA
Sequence
[1095] By using a TA Cloning Kit (Invitrogen Co.), the DNA
amplified by the PCR was inserted into a plasmid vector, pCR.TM.I.
The DNA was transfected into E. coli attached to the kit to form an
amplified DNA library. Colonies formed by the transformants were
selected under guidance based on the activity of
.beta.-galactosidase on X-gal
(5-bromo-4-chloro-3indolyl-.beta.-D-galactoside)-added LB
(Luria-Bertani) plates in order to separate only white colonies in
which DNA fragments are inserted. They were cultured in an LB
culture medium to which ampicillin was added and plasmid DNAs were
prepared with an automatic plasmid extracting machine (Kurabo Co.,
Japan).
[1096] An aliquot of the DNA thus prepared was further digested
with EcoRI to confirm DNA fragments that were inserted, and a DNA
yield each of clones was compared with a marker. An aliquot of the
plasmid DNA thus prepared was treated with RNase, extracted with
phenol/chloroform, precipitated in ethanol, and the resulting
product was then reacted for sequencing by using a DyeDeoxy
terminator cycle sequencing kit (Applied Biosystems Co.).
[1097] Sequencing was carried out by using a 370A fluorescent
automatic sequencer manufactured by Applied Biosystems Co. The
nucleotide sequences obtained were analyzed by using DNASIS
(Hitachi Software Engineering, Japan). The nucleotide sequences
obtained are shown in FIGS. 18, 19, 20 and 21. From these Figures
and the results of homology retrieval, it was learned that the DNAs
obtained were DNAs encoding human somatostatin receptor protein
[FIGS. 18 and 19], human D5 dopamine receptor protein [FIG. 20] and
rat somatostatin receptor protein [FIG. 21] that can be classified
each into a group of G protein coupled receptor proteins.
[1098] In FIG. 18 as described herein, the nucleotide sequence of
the DNA is in agreement with the nucleotide sequence encoding
somatostatin receptor (HUMSOMAT) and the clone, A58, is a human
somatostatin receptor cDNA. The underlined part represents the 5'
side synthetic DNA primer used for the PCR. Thus, even when parts
of the nucleotide sequence are mismatched, amplification is
effected to a sufficient degree by the PCR.
[1099] It will be understood from FIG. 19 that the clone, A58 is in
good agreement with the nucleotide sequence coding for the human
somatostatin receptor (HUMSOMAT) even when the sequencing is
carried out from the opposite side. The underlined part represents
the 3' side synthetic DNA primer used for the PCR. In this figure,
the nucleotide sequences are mismatched to some extent even in the
portions other than the primer portion presumably due to base
substitution at the time of PCR or due to partial deviation in the
sequencing reaction. It can be confirmed via sequencing of chains
complementary thereto as required.
[1100] In FIG. 20 as described herein, the nucleotide sequence of
the DNA is in good agreement with a nucleotide sequence coding for
the human D5 dopamine receptor (HUMDRD5A) except the primer portion
(underlined). It was learned that the clone, 57-A-2, is a human D5
dopamine receptor cDNA.
[1101] In FIG. 21 as described herein, the DNA is in good agreement
with a nucleotide sequence coding for the rat somatostatin receptor
(RNU04738) except the primer portion (underlined). It was learned
that the clone, B54, is a rat somatostatin receptor cDNA.
Example 3
[1102] Isolation of Human Pituitary Gland-Derived G Protein Coupled
Receptor Protein-Encoding DNA
[1103] (1) Amplification of Receptor cDNA by PCR Using Human
Pituitary Gland-Derived cDNA
[1104] By using human pituitary gland-derived cDNA (QuickClone,
CLONTECH Laboratories, Inc.) as a template, PCR amplification using
the DNA primers synthesized in Example 1 was carried out. The
composition of the reaction solution consisted of the synthetic DNA
primers (SEQ; 5' primer sequence and 3' primer sequence) each in an
amount of 1 .mu.M, 1 ng of the template cDNA, 0.25 mM dNTPs, 1
.mu.l of Taq DNA polymerase and a buffer attached to the enzyme
kit, and the total amount of the reaction solution was made to be
100 .mu.l. The cycle for amplification including 95.degree. C. for
1 min., 55.degree. C. for 1 min. and 72.degree. C. for 1 min. was
repeated 30 times by using a Thermal Cycler (Perkin-Elmer Co.).
Prior to adding Taq DNA polymerase, the remaining reaction solution
was mixed and was heated at 95.degree. C. for 5 minutes and at
65.degree. C. for 5 minutes. The amplified products were confirmed
relying upon 1.2% agarose gel electrophoresis and ethidium bromide
staining.
[1105] (2) Subcloning of PCR Product into Plasmid Vector and
[1106] Selection of Novel Receptor Candidate Clone via Decoding
Nucleotide Sequence of Inserted cDNA Region
[1107] The PCR products were separated by using a 0.8% low-melting
temperature agarose gel, the band parts were excised from the gel
with a razor blade, and were heat-melted, extracted with phenol and
precipitated in ethanol to recover DNAs. According to the protocol
attached to a TA Cloning Kit (Invitrogen Co.), the recovered DNAs
were subcloned into the plasmid vector, pCR.TM.II (TM represents
registered trademark). The recombinant vectors were introduced into
E. coli INV.alpha. F' competent cells (Invitrogen Co.) to produce
transformants. Then, transformant clones having a cDNA-inserted
fragment were selected in an LB agar culture medium containing
ampicillin and X-gal. Only transformant clones exhibiting white
color were picked with a sterilized toothstick to obtain
transformant Escherichia coli INV.alpha. F'/p19P2.
[1108] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNA thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNA was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan). The underlined portions represent
regions corresponding to the synthetic primers [FIGS. 22 and
23].
[1109] Homology retrieval was carried out based upon the determined
nucleotide sequences [FIGS. 22 and 23]. As a result, it was learned
that a novel G protein coupled receptor protein was encoded by the
cDNA fragment insert in the plasmid, p 19P2, possessed by the
transformant Escherichia coli INV.alpha. F' p19P2. To further
confirm this fact, by using DNASIS (Hitachi System Engineering Co.,
Japan) the nucleotide sequences were converted into amino acid
sequences [FIGS. 22 and 23], and homology retrieval was carried out
in view of hydrophobicity plotting (FIGS. 24 and 25] and at the
amino acid sequence level to find homology relative to neuropeptide
Y receptor proteins [FIG. 26].
Example 4
[1110] Isolation of Mouse Pancreas-Derived G Protein Coupled
Receptor Protein-Encoding DNA
[1111] (1) Preparation of Poly(A) RNA Fraction from Mouse
Pancreatic
[1112] .beta.-Cell Strain, MIN6 and Synthesis of cDNA
[1113] A total RNA was prepared from the mouse pancreatic
[1114] .beta.-cell strain, MIN6 (Jun-ichi Miyazaki et al.,
Endocrinology, Vol. 127, No. 1, p.126-132) according to the
guanidine thiocyanate method (Kaplan B. B. et al., Biochem. J.,
183, 181-184 (1979)) and, then, poly(A).sup.+ RNA fractions were
prepared with a mRNA purifying kit (Pharmacia Co.). Next, to 5
.mu.g of the poly(A).sup.+ RNA fraction was added a random DNA
hexamer (BRL Co.) as a primer, and the resulting mixture was
subjected to reaction with mouse Moloney Leukemia virus (MMLV)
reverse transcriptase (BRL Co.) in the buffer attached to the MMLV
reverse transcriptase kit to synthesize complementary DNAs. The
reaction product was extracted with phenol/chloroform (1:1),
precipitated in ethanol, and was then dissolved in 30 .mu.l of TE
buffer (10 mM Tris-HCl at pH8.0, 1 mM EDTA at pH8.0).
[1115] (2) Amplification of Receptor cDNA by PCR Using MIN6-Derived
cDNA and Sequencing
[1116] By using, as a template, 5 .mu.l of cDNA prepared
[1117] from the mouse pancreatic .beta.-cell strain, MIN6 in the
above step (1), PCR amplification using the DNA primers synthesized
in Example 1 was carried out under the same conditions as in
Example 3 (2). The resulting PCR product was subcloned into the
plasmid vector, pCR.TM.II, in the same manner as in Example 2 to
obtain a plasmid, pG3-2. The plasmid pG3-2 was transfected into E.
coli INV.alpha. F' to obtain transformed Escherichia coli
INV.alpha. F'/pG3-2.
[1118] By using, as a template, 5 .mu.l of the cDNA prepared from
the mouse pancreatic .beta.-cell strain, MIN6, PCR amplification
using DNA primers as disclosed in Libert F. et al., "Science,
244:569-572, 1989", i,e., a degenerate synthetic primer represented
by the following sequence:
2 5'-CTGTG (C or T) G (C or T) (G or C) AT (C or T) GCIIT (G or T)
GA (C or T) (A or C) (SEQ ID NO: 60) G (G or C) TAC-3'
[1119] wherein I is inosine; and
[1120] a degenerate synthetic primer represented by the following
sequence:
3 5'-A (G or T) G (A or T) AG (A or T) AGGGCAGCCAGCAGAI (G or C) (A
or G) (C or T) (SEQ ID NO: 61) GAA-3'
[1121] wherein I is inosine,
[1122] was carried out under the same conditions as in working
Example 1. The resulting R product was subcloned into the plasmid
vector, pCR.TM.II, in the same manner as described in Example 3(2)
to obtain a plasmid, pG1-10.
[1123] The reaction for determining the nucleotide sequence
(sequencing) was carried out with a DyeDeoxy terminator cycle
sequencing kit (ABI Co.), the DNA was decoded with the fluorescent
automatic sequencer (ABI Co.), and the data of the nucleotide
sequence obtained were analyzed with DNASIS (Hitachi System
Engineering Co., Japan).
[1124] FIG. 27 shows a mouse pancreatic .beta.-cell strain
MIN6-derived G protein coupled receptor protein-encoding DNA and an
amino acid sequence encoded by the isolated DNA based upon the
nucleotide sequences of plasmids pG3-2 and pG1-10 which are held by
the transformant Escherichia coli INV.alpha. F'/pG3-2. The
underlined portions represent regions corresponding to the
synthetic primers.
[1125] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 27]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment obtained. To further confirm this fact, by using DNASIS
(Hitachi System Engineering Co., Japan) the nucleotide sequence was
converted into an amino acid sequence [FIG. 27], hydrophobicity
plotting was carried out to confirm the presence of six hydrophobic
regions [FIG. 28]. Upon comparing the amino acid sequence with that
of p19P2 obtained in Example 3, furthermore, a high degree of
homology was found as shown in [FIG. 61]. As a result, it is
strongly suggested that the G protein coupled receptor proteins
encoded by pG3-2 and pG1-10 recognize the same ligand as the G
protein coupled receptor protein encoded by p19P2 does while the
animal species from which the receptor proteins encoded by pG3-2
and pG1-10 are derived is different from that from which the
receptor protein encoded by p19P2 is.
Example 5
[1126] Isolation of Human Amygdaloid Nucleus-Derived G Protein
Coupled Receptor Protein-Encoding DNA
[1127] (1) Amplification of Receptor cDNA by PCR Using Human
[1128] Amygdaloid Nucleus-Derived cDNA
[1129] By using an amplified human amygdala-derived cDNA
(QuickClone, CLONTECH Laboratories, Inc.) as a template, PCR
amplification using the DNA primers synthesized in Example 1 was
carried out. The composition of the reaction solution consisted of
the synthetic DNA primers (SEQ: 5' primer sequence and 3' primer
sequence) each in an amount of 1 .mu.M, 1 ng of the template cDNA,
0.25 mM dNTPs, 1 .mu.l of Taq DNA polymerase and a buffer attached
to the enzyme kit, and the total amount of the reaction solution
was made to be 100 .mu.l. The cycle for amplification including
95.degree. C. for 1 min., 55.degree. C. for 1 min. and 72.degree.
C. for 1 min. was repeated 30 times by using a Thermal Cycler
(Perkin-Elmer Co.). Prior to adding Taq DNA polymerase, the
remaining reaction solution was mixed and was heated at 95.degree.
C. for 5 minutes and at 65.degree. C. for 5 minutes. The amplified
products were confirmed relying upon 1.2% agarose gel
electrophoresis and ethidium bromide staining.
[1130] (2) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1131] The PCR products were separated by using a 0.8% low-melting
temperature agarose gel, the band parts were excised from the gel
with a razor blade, and were heat-melted, extracted with phenol and
precipitated in ethanol to recover DNAs. According to the protocol
attached to a TA Cloning Kit (Invitrogen Co.), the recovered DNAs
were subcloned to the plasmid vector, pCR.TM.II. The recombinant
vectors were introduced into E. coli INV.alpha. F' competent cells
(Invitrogen Co.) to produce transformants. Then, transformant
clones having a cDNA-inserted fragment were selected in an LB agar
culture medium containing ampicillin and X-gal. Only transformant
clones exhibiting white color were picked with a sterilized
toothstick to obtain transformant Escherichia coli INV.alpha.
F'/p63A2.
[1132] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNA thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNA was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan).
[1133] Homology retrieval was carried out based upon the determined
nucleotide sequences [FIGS. 29 and 30]. As a result, it was learned
that a novel G protein coupled receptor protein was encoded by the
cDNA fragment insert in the plasmid, p63A2 possessed by the
transformant Escherichia coli INV.alpha. F'/p63A2. To further
confirm this fact, by using DNASIS (Hitachi System Engineering Co.,
Japan) the nucleotide sequences were converted into amino acid
sequences [FIGS. 29 and 30], and homology retrieval was carried out
in view of hydrophobicity plotting [FIGS. 31 and 32] and at the
amino acid sequence level to find homology relative to mouse GIR
FIG. 33].
Example 6
[1134] Cloning of Human Pituitary Gland-Derived G Protein Coupled
Receptor Protein cDNA
[1135] (1) Cloning of cDNA Comprising Whole Coding Regions for
Receptor Protein from Human Pituitary Gland-Derived cDNA
Library
[1136] The DNA library constructed by Clontech Co. wherein .lambda.
gt11 phage vector is used (CLONTECH Laboratories, Inc.; CLH L1139b)
was employed as a human pituitary gland-derived cDNA library. The
human pituitary gland cDNA library (2.times.10.sup.6 pfU (plaque
forming units)) was mixed with E. coli Y1090 treated with magnesium
sulfate, and incubated at 37.degree. C. for 15 minutes followed by
addition of 0.5% agarose (Pharmacia Co.) LB. The E. coli was plated
onto a 1.5% agar (Wako-Junyaku Co.) LB plate (containing 50
.mu.g/ml of ampicillin). A nitrocellulose filter was placed on the
plate on which plaques were formed and the plaque was transferred
onto the filter. The filter was denatured with an alkali and then
heated at 80.degree. C. for 3 hours to fix DNAs.
[1137] The filter was incubated overnight at 42.degree. C. together
with the probe mentioned herein below in a buffer containing 50%
formamide, 5.times.SSPE (20.times.SSPE (pH 7.4) is 3 M NaCl, 0.2 M
NaH.sub.2PO.sub.4 H.sub.20, 25 mM EDTA), 5.times. Denhardt's
solution (Nippon Gene, Japan), 0.1% SDS and 100 .mu.g/ml of salmon
sperm DNA for hybridization.
[1138] The probe used was obtained by cutting the DNA fragment
inserted in the plasmid, p19P2, obtained in Working Example 3, with
EcoRI, followed by recovery and labelling by incorporation of [
.sup.32 P]dCTP (Dupont Co.) with a random prime DNA labelling kit
(Amasham Co.).
[1139] It was washed with 2.times.SSC (20.times.SSC is 3 M NaCl,
0.3 M sodium citrate), 0.1% SDS at 55.degree. C. for 1 hour and,
then, subjected to an autoradiography at -80.degree. C. to detect
hybridized plaques.
[1140] In this screening, hybridization signals were recognized in
three independent plaques. Each DNA was prepared from the three
clones. The DNAs digested with EcoRI were subjected to an agarose
electrophoresis and were analyzed by the southern blotting using
the same probe as the one used in the screening. Hybridizing bands
were identified at about 0.7 kb, 0.8 kb and 2.0 kb, respectively.
Among them, the DNA fragment corresponding to the band at about 2.0
kb (.lambda. hGR3) was selected. The .lambda. hGR3-derived EcoRI
fragment with a hybridizable size was subcloned to the EcoRI site
of the plasmid, pUC18, and E. coli JM109 was transformed with the
plasmid to obtain transformant E. coli JM109/phGR3. A restriction
enzyme map of the plasmid, phGR3, was prepared relying upon a
restriction enzyme map deduced from the nucleotide sequence as
shown in Example 3. As a result, it was learned that it carried a
full-length receptor protein-encoding DNA which was predicted from
the receptor protein-encoding DNA as shown in Example 3. (2)
Sequencing of Human Pituitary Gland-Derived Receptor Protein
cDNA.
[1141] Among the EcoRI fragments inserted in the plasmid, phGR3,
obtained in the above step (1), the from EcoRI to NheI nucleotide
sequence with about 1330 bp that is considered to be a receptor
protein-coding region was sequenced. Concretely speaking, by
utilizing restriction enzyme sites that exist in the EcoRI
fragments, unnecessary parts were removed or necessary fragments
were subcloned in order to prepare template plasmids for analyzing
the nucleotide sequence.
[1142] The reaction for determining the nucleotide sequence
(sequencing) was carried out with a DyeDeoxy terminator cycle
sequencing kit (ABI Co.), the DNA was decoded with the fluorescent
automatic sequencer (ABI Co.), and the data of the nucleotide
sequence obtained were analyzed with DNASIS (Hitachi System
Engineering Co., Japan).
[1143] FIG. 34 shows a nucleotide sequence of from immediate after
the EcoRI site up to the NheI site encoded by phGR3. The nucleotide
sequence of the human pituitary glandderived receptor
protein-encoding DNA corresponds to the nucleotide sequence of from
118th to 123rd nucleotides [FIG. 34). An amino acid sequence of the
receptor protein that is encoded by the nucleotide sequence is
shown in FIG. 34. FIG. 36 shows the results of hydrophobicity
plotting based upon the amino acid sequence.
[1144] (3) Northern Hybridization with Human Pituitary
Gland-Derived Receptor Protein-Encoding phGR3
[1145] Northern blotting was carried out in order to detect the
expression of phGR3-encoded human pituitary gland-derived receptor
proteins in the pituitary gland at a mRNA level. Human pituitary
gland mRNA (2.5 .mu.g, Clontech Co.) was used as a template mRNA
and the same as the probe used in Working Example 5 was used as a
probe. Nylon membrane (Pall Biodyne, U.S.A.) was used as a filter
for northern blotting and migration of the mRNA and adsorption
(sucking) thereof with the blotting filter was carried out
according to the method as disclosed in Molecular Cloning, Cold
Spring Harbor Laboratory Press, 1989.
[1146] The hybridization was effected by incubating the
above-mentioned filter and probe in a buffer containing 50%
formamide, 5.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS and
100 .mu.g/ml of salmon sperm DNA overnight at 42.degree. C. The
filter was washed with 0.1.times.SSC, 0.1% SDS at 50.degree. C.
and, after drying with an air, was exposed to an X-ray film (XAR5,
Kodak) for three days at -80.degree. C. The results were as shown
in FIG. 35 from which it is considered that the receptor gene
encoded by phGR3 is expressed in the human pituitary gland.
Example 7
[1147] Cloning of Mouse Pancreatic 4 -Cell Strain, MIN6-Derived G
Protein Coupled Receptor Protein cDNA
[1148] (1) Preparation of Poly(A).sup.+ RNA Fraction from Mouse
Pancreatic .beta.-Cell Strain, MIN6 and Synthesis of cDNA
[1149] A total RNA was prepared from the mouse pancreatic
[1150] .beta.-cell strain, MIN6 (Jun-ichi Miyazaki et al.,
Endocrinology, Vol. 127, No. 1, p. 126-132) according to the
guanidine thiocyanate method (Kaplan B. B. et al., Biochem. J.,
183, 181-184 (1979)) and, then, poly(A).sup.+ RNA fractions were
prepared with a mRNA purifying kit (Pharmacia Co.). Next, to 5
.mu.g of the poly(A) RNA fraction was added a random DNA hexamer
(BRL Co.) as a primer, and the resulting mixture was subjected to
reaction with MMLV reverse transcriptase (BRL Co.) in the buffer
attached to the MMLV reverse transcriptase kit to synthesize
complementary DNAs. The reaction product was extracted with
phenol/chloroform (1:1), precipitated in ethanol, and was then
dissolved in 30 .mu.l of TE.
[1151] (2) Amplification of Receptor cDNA by PCR Using MIN6-Derived
cDNA and Sequencing
[1152] By using, as a template, 5 .mu.l of cDNA prepared from the
mouse pancreatic .beta.-cell strain, MIN6 in the above step (1),
PCR amplification using the DNA primers synthesized in Example 1
was carried out. A reaction solution was composed of the synthetic
DNA primers (SEQ: 5' primer sequence and 3' primer sequence) each
in an amount of 100 pM, 0.25 mM dNTPs, 1 .mu.l of Taq DNA
polymerase and 10 u l of buffer attached to the enzyme kit, and the
total amount of the reaction solution was made to be 100 .mu.l. The
cycle for amplification including 96.degree. C. for 30 sec.,
45.degree. C. for 1 min. and 60.degree. C. for 3 min. was repeated
30 times by using a Thermal Cycler (Perkin-Elmer Co.). Prior to
adding Taq DNA polymerase, the remaining reaction solution was
mixed and was heated at 95.degree. C. for 5 minutes and at
65.degree. C. for 5 minutes. The amplified products were confirmed
relying upon 1.2% agarose gel electrophoresis and ethidium bromide
staining.
[1153] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1154] The PCR products obtained in the above step (2) were
separated by using a 0.8% low-melting temperature agarose gel, the
band parts were excised from the gel with a razor blade, and were
heat-melted, extracted with phenol and precipitated in ethanol to
recover DNAs. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAs were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG (isopropylthio-R -D-galactoside) and
X-gal. Only transformant clones exhibiting white color were picked
with a sterilized toothstick to obtain transformant Escherichia
coli JM 109/p3H2-17.
[1155] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNAs was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan).
[1156] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 37]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM 109/p3H2-17. To further confirm this fact, by
using DNASIS (Hitachi System Engineering Co., Japan) the nucleotide
sequence were converted into an amino acid sequence [FIG. 371, and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 38] and at the amino acid sequence level to find
homology relative to chicken ATP receptor (P34996), human
somatostatin receptor subtype 3 (A46226), human somatostatin
receptor subtype 4 (JN0605) and bovine neuropeptide Y receptor
(S28787) [FIG. 39]. Abbreviations in parentheses are reference
numbers assigned when they are registered as data to
NBRF-PIR/Swiss-PROT and are usually called "Accession Numbers".
Example 8
Cloning of Mouse Pancreatic i9 -Cell Strain, MIN6-Derived G Protein
Coupled Receptor Protein cDNA
[1157] (1) Preparation of Poly(A) RNA Fraction from Mouse
Pancreatic .beta.-Cell Strain, MIN6 and Synthesis of cDNA
[1158] A total RNA was prepared from the mouse pancreatic
[1159] .beta.-cell strain, MIN6 (Jun-ichi Miyazaki et al.,
Endocrinology, Vol. 127, No. 1, p.126-132) according to the
guanidine thiocyanate method (Kaplan B. B. et al., Biochem. J.,
183, 181-184 (1979)) and, then, poly(A).sup.+ RNA fractions were
prepared with a mRNA purifying kit (Pharmacia Co.). Next, to 5
.mu.g of the poly(A) RNA fraction was added a random DNA hexamer
(BRL Co.) as a primer, and the resulting mixture was subjected to
reaction with MMLV reverse transcriptase (BRL Co.) in the buffer
attached to the MMLV reverse transcriptase kit to synthesize
complementary DNAs. The reaction product was extracted with
phenol/chloroform (1:1), precipitated in ethanol, and was then
dissolved in 30 .mu.l of TE.
[1160] (2) Amplification of Receptor cDNA by PCR Using MIN6-Derived
cDNA and Sequencing
[1161] By using, as a template, 5 .mu.l of cDNA prepared from the
mouse pancreatic .beta.-cell strain, MIN6, in the above step (1),
PCR amplification using the DNA primers synthesized in Example 1
was carried out. A reaction solution was composed of the synthetic
DNA primers (SEQ: 5' primer sequence and 3' primer sequence) each
in an amount of 100 pM, 0.25 mm dNTPs, 1 .mu.l of Taq DNA
polymerase and 10 .mu.l of 10.times. buffer attached to the enzyme
kit, and the total amount of the reaction solution was made to be
100 .mu.. The cycle for amplification including 96.degree. C. for
30 sec., 45.degree. C. for 1 min. and 60.degree. C. for 3 min. was
repeated 30 times by using a Thermal Cycler (Perkin-Elmer Co.).
Prior to adding Taq DNA polymerase, the remaining reaction solution
was mixed and was heated at 95.degree. C. for 5 minutes and at
65.degree. C. for 5 minutes. The amplified products were confirmed
relying upon 1.2% agarose gel electrophoresis and ethidium bromide
staining.
[1162] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1163] The PCR products obtained in the above step (2) were
separated with a 0.8% low-melting temperature agarose gel, the band
parts were excised from the gel with a razor blade, and were
heat-melted, extracted with phenol and precipitated in ethanol to
recover DNAs. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAs were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized toothstick to
obtain transformant Escherichia coli JM 109/p3H2-34.
[1164] The, individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNAs was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan).
[1165] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 40]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM 109/p3H2-34. To further confirm this fact, by
using DNASIS (Hitachi System Engineering Co., Japan) the nucleotide
sequence were converted into an amino acid sequence [FIG. 40], and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 411 and at the amino acid sequence level to find
homology relative to human somatostatin receptor subtype 2 (B41795)
and rat-derived ligand unknown receptor (A39297) [FIG. 42].
Abbreviations in parentheses are reference numbers assigned when
they are registered as data to NBRF-PIR/Swiss-PROT and are usually
called "Accession Numbers" or "Entry Names".
Example 9
Cloning of Rabbit Gastropyrolic Part Smooth Muscle-Derived G
Protein Coupled Receptor Protein cDNA
[1166] (1) Preparation of Poly(A).sup.+ RNA Fraction from Rabbit
Gastropyrolic Part Smooth Muscle and Synthesis of cDNA
[1167] A total RNA was prepared from rabbit gastropyrolic part
smooth muscles by the guanidine thiocyanate method (Kaplan B. B. et
al., Biochem. J. 183, 181-184 (1979)) and, then, poly(A) RNA
fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
Next, to 5 .mu.g of the poly(A).sup.+ RNA fraction was. added a
random DNA hexamer (BRL Co.) as a primer, and the resulting mixture
was subjected to reaction with MMLV reverse transcriptase (BRL Co.)
in the buffer attached to the MMLV reverse transcriptase kit to
synthesize complementary DNAs. The reaction product was extracted
with phenol/chloroform. (1: 1), precipitated in ethanol, and was
then dissolved in 30 .mu.l of TE (Tris-EDTA solution).
[1168] (2) Amplification of Receptor cDNA by PCR Using Rabbit
Gastropyrolic Part Smooth Muscle-Derived cDNA and Sequencing
[1169] By using, as a template, 1 .mu.l of cDNA prepared from the
rabbit gastropyrolic part smooth muscle in the above step (1), PCR
amplification using the DNA primers synthesized in Example 1 was
carried out. A reaction solution was composed of the synthetic DNA
primers (SEQ: 5' primer sequence and 3' primer sequence) each in an
amount of 100 pM, 0.25 mM dNTPs, 1 .mu.l of Taq DNA polymerase and
10 .mu.l of buffer attached to the enzyme kit, and the total amount
of the reaction solution was made to be 100 l. The cycle for
amplification including 96.degree. C. for 30 sec., 45.degree. C.
for 1 min. and 60.degree. C. for 3 min. was repeated 25 times by
using a Thermal Cycler (Perkin-Elmer Co.). The amplified products
were confirmed relying upon 1.2% agarose gel electrophoresis and
ethidium bromide staining.
[1170] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1171] The PCR products obtained in the above step (2) were
separated with a 1.0% low-melting temperature agarose gel, the band
parts were excised from the gel with a razor blade, and were
heat-melted, extracted with phenol and precipitated in ethanol to
recover DNAS. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAS were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized toothstick to
obtain transformant Escherichia coli JM 109/pMD4.
[1172] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNAs was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan). The determined nucleotide sequence
was as shown in FIG. 43. It was learned from FIG. 43 that the
cloned cDNA fragment was amplified from both sides with only the
synthetic DNA primer having a nucleotide sequence represented by
SEQ ID NO: 1 as synthesized in Example 1.
[1173] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 43]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM 109/pMD4. To further confirm this fact, by
using DNASIS (Hitachi System Engineering Co., Japan) the nucleotide
sequence were converted into an amino acid sequence [FIG. 43], and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 44] and at the amino acid sequence level to find
homology relative to rat ligand-unknown receptor protein (A35639)
[FIG. 45]. Abbreviations in parentheses are reference numbers
assigned when they are registered as data to NBRF-PIR/ Swiss-PROT
and are usually called "Accession Numbers".
Example 10
Cloning of cDNA Comprising Whole Coding Regions for Receptor
Protein from Mouse Pancreatic .beta.-Cell Strain, MIN6-Derived cDNA
Library
[1174] (1) Cloning of cDNA Comprising Whole Coding Regions for
Receptor Protein from Mouse Pancreatic .beta.-Cell Strain,
MIN6-Derived cDNA Library
[1175] Superscript.TM. Lambda System (BRL, Cat. 8256) distributed
by BRL Co. and Glgapack II Gold (Stratagene, Cat. 200215)
distributed by Stratagene Co. were used to construct MIN6-derived
cDNA libraries. By using the above kits, a MIN6 cDNA library with
2.2.times.10.sup.6 pfu (plaque forming units) was constructed from
10 .mu.g of MIN6 poly(A)+RNA. The cDNA library was mixed with E.
coli Y1090 treated with magnesium sulfate, and incubated at
37.degree. C. for 15 minutes followed by addition of 0.5% agarose
(Pharmacia Co.) LB. The E. coli was plated onto a 1.5% agar
(Wako-Junyaku Co.) LB plate (containing 50 .mu.g/ml of ampicillin).
A nitrocellulose filter was placed on the plate on which plaques
were formed and the plaque was transferred onto the filter. The
filter was denatured with an alkali and then heated at 80.degree.
C. for 3 hours to fix DNAs.
[1176] The filter was incubated overnight at 42.degree. C. together
with the probe mentioned herein below in a buffer containing 50%
formamide, 5.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS and
100 u g/ml of salmon sperm DNA for hybridization.
[1177] The probe used was obtained by cutting the DNA fragment
inserted in the plasmid, p3H2-34, obtained in Working Example 8,
with EcoRI, followed by recovery and labeling by incorporation of
(.sup.32P]dCTP (Dupont Co.) with a random prime DNA labelling kit
(Amasham Co.).
[1178] It was washed with 2.times.SSC (150 mM NaCl and 15 mM sodium
citrate), 0.1% SDS at 55.degree. C. for 1 hour and, then, subjected
to an autoradiography at -80.degree. C. to detect hybridized
plaques.
[1179] In this screening, hybridization signals were recognized in
two independent plaques. Each DNA was prepared from the two clones.
The DNAs digested with SalI and NotI were subjected to an agarose
electrophoresis and were analyzed. Inserted fragments were
identified at about 2.0 kb and 3.0 kb, respectively. Between them,
the DNA fragment corresponding to the band at about 3.0 kb
(.lambda. No.20) was selected. The .lambda. No.20-derived NotI-SalI
fragment with about 3.0 kb was subcloned into the NotI-SalI site of
the plasmid, pBluescript.TM.II SK(+), and E. coli JM 109 was
transformed with the plasmid to obtain a transformant E. coli JM
109/pMGR20. A restriction enzyme map of the plasmid, pMGR20, was
prepared relying upon a restriction enzyme map deduced from the
nucleotide sequence as shown in Working Example 8. As a result, it
was learned that it carried a full-length receptor protein-encoding
DNA which was predicted from the receptor protein-encoding DNA as
shown in Working Example 8.
[1180] (2) Sequencing of MINE-Derived Receptor Protein Full-Length
cDNA
[1181] Among the NotI-SalI fragments inserted in the plasmid,
pMGR20, obtained in the above step (1), the nucleotide sequence
with total 1607 bp, including not only a region that is considered
to be a receptor protein-coding region (ORF) but also a neighboring
region thereof was sequenced. Concretely speaking, by utilizing
restriction enzyme sites that exist in the NotI-SalI fragments,
unnecessary parts were removed or necessary fragments were
subcloned in order to prepare template plasmids for analyzing the
nucleotide sequence thereof. As for the nucleotide sequences of
part of the regions, primers for sequencing were synthesized based
upon the nucleotide sequences that were determined already and used
to make confirmation.
[1182] The reaction for determining the nucleotide sequence
(sequencing) was carried out with a DyeDeoxy terminator cycle
sequencing kit (ABI Co.), the DNA was decoded with the fluorescent
automatic sequencer (ABI Co.), and the data of the nucleotide
sequence obtained were analyzed with DNASIS (Hitachi System
Engineering Co., Japan).
[1183] FIG. 46 shows a nucleotide sequence around an open reading
frame (ORF) of a mouse galanin receptor protein encoded by the cDNA
insert in pMGR20. The nucleotide sequence of mouse galanin receptor
protein-encoding DNA corresponds to from the 481st to 1525th
nucleotides of the nucleotide sequence in FIG. 46. The nucleotide
sequence was converted into an amino acid sequence [FIG. 46] and
hydrophobicity plotting was carried out [FIG. 47]. Since the amino
acid sequence [FIG. 46] has 92% homology to the human-derived
galanin receptor protein at the amino acid sequence level [FIG.
48], it was learned that the cDNA insert in the pMGR20 is a
mouse-derived galanin receptor protein-encoding cDNA.
Example 11
Preparation of Synthetic DNA Primer for Amplifying G Protein
Coupled Receptor Protein-Encoding DNA
[1184] Highly homologous parts were found by comparing nucleotide
sequences corresponding to or near the third membrane-spanning
domain [3C and 3D in FIG. 4] and the sixth membrane-spanning domain
[6C of FIG. 6] among known G protein coupled receptors, i.e.,
rat-derived angiotensin II receptor protein (L32840), rat-derived
angiotensin Ib receptor protein (x64052), rat-derived angiotensin
receptor protein subtype (M90065), human-derived angiotensin Ia
receptor protein (M91464), rat-derived cholecystokinin.sub.A
receptor protein (M88096), rat-derived cholecystokinin.sub.B
receptor protein (M99418), human-derived cholecystokinin.sub.B
receptor protein (L04473), mouse-derived low-affinity interleukin 8
receptor protein (M73969), human-derived high-affinity interleukin
8 receptor protein (X65858), mouse-derived C5a anaphylatoxin
receptor protein (S46665), human-derived N-formyl peptide receptor
protein (M60626), etc.
[1185] The aforementioned abbreviations in parentheses are
reference numbers that are indicated when the GenBank/EMBL data
base is retrieved, and are usually called "Accession Numbers".
[1186] It was planned to incorporate mixed bases relying upon the
base regions that were in agreement with a large number of receptor
protein cDNAs in order to enhance base agreement of sequences with
as many receptor cDNAs as possible even in other regions. Based
upon these sequences, the degenerate synthetic DNA (3D of FIG. 4)
having a nucleotide sequence represented by SEQ ID NO: 3 which is
complementary to the homologous nucleotide sequence-of FIG. 4 and
the degenerate synthetic DNA (nucleotide sequence complementary to
6C of FIG. 6) having a nucleotide sequence represented by SEQ ID
NO: 4 were produced. Nucleotide synthesis was carried out by a DNA
synthesizer.
[1187] [Synthetic DNAs]
4 5'-CTCGC (G or C) GC (C or T) (A or C) TI (A or G) G (C or T)
ATGGA (C or T) CGITAT-3' (SEQ ID NO:3) 5'-CATGT (A or G) G (T or A)
AGGGAAICCAG (G or C) A (A or C) AI (A or G) A (A or G)(A or (SEQ ID
NO:4) G) AA-3'
[1188] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis, provided that I
denotes inosine.
Example 12
Cloning of Rabbit Gastropyrolic Part Smooth Muscle-Derived G
Protein Coupled Receptor Protein cDNA
[1189] (1) Preparation of Poly(A) RNA Fraction from Rabbit
Gastropyrolic Part Smooth Muscle and Synthesis of cDNA
[1190] A total RNA was prepared from rabbit gastropyrolic part
smooth muscles by the guanidine thiocyanate method (Kaplan B. B. et
al., Biochem. J. 183, 181-184 (1979)) and, then, poly(A).sup.+ RNA
fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
Next, to 5 .mu.g of the poly(A) RNA fraction was added a random DNA
hexamer (BRL Co.) as a primer, and the resulting mixture was
subjected to reaction with MMLV reverse transcriptase (BRL Co.) in
the buffer attached to the MMLV reverse transcriptase kit to
synthesize complementary DNAs. The reaction product was extracted
with phenol/chloroform (1:1), precipitated in ethanol, and was then
dissolved in 30 .mu.l of TE.
[1191] (2) Amplification of Receptor cDNA by PCR Using Rabbit
Gastropyrolic Part Smooth Muscle-Derived cDNA and Sequencing
[1192] By using, as a template, 1 .mu.l of cDNA prepared from the
rabbit gastropyrolic part smooth muscle in the above step (1), PCR
amplification using the DNA primer having a nucleotide sequence
represented by SEQ ID NO: 3 and the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 4 synthesized in Example 11 was
carried out. A reaction solution was composed of the synthetic DNA
primers (SEQ: 5' primer sequence and 3' primer sequence) each in an
amount of 100 pM, 0.25 mM dNTPs, 1 .mu.l of Taq DNA polymerase and
10 .mu.l of buffer attached to the enzyme kit, and the total amount
of the reaction solution was made to be 100 .mu.l. The cycle for
amplification including 96.degree. C. for 30 sec., 45.degree. C.
for 1 min. and 60.degree. C. for 3 min. was repeated 25 times by
using a Thermal Cycler (Perkin-Elmer Co.). The amplified products
were confirmed relying upon 1.2% agarose gel electrophoresis and
ethidium bromide staining.
[1193] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1194] The PCR products obtained in the above step (2) were
separated with a 1.0% low-melting temperature agarose gel, the band
parts were excised from the gel with a razor blade, and were
heat-melted, extracted with phenol and precipitated in ethanol to
recover DNAs. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAs were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized toothstick to
obtain transformant Escherichia coli JM109/pMJ10.
[1195] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNAs was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan). The determined nucleotide sequence
was as shown in FIG. 49.
[1196] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 49]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM109/pMJ10. To further confirm this fact, by
using DNASIS (Hitachi System Engineering Co., Japan) the nucleotide
sequence were converted into an amino acid sequence [FIG. 49], and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 50] and at the amino acid sequence level to find
homology relative to human ligand unknown receptor protein
(B42009), human N-formyl peptide receptor protein (JC2014), rabbit
N-formyl peptide receptor protein (A46520), mouse C5a anaphylatoxin
receptor protein (A46525) and bovine neuropeptide Y receptor
protein (S28787) [FIG. 51]. Abbreviations in parentheses are
reference numbers assigned when they are registered as data to
NBRF-PIR/Swiss-PROT and are usually called "Accession Numbers".
Example 13
Preparation of Synthetic DNA Primer for Amplifying G Protein
Coupled Receptor Protein-Encoding DNA
[1197] A comparison of nucleotide sequences coding for regions
corresponding to or near the third membrane-spanning domain among
known G protein coupled receptors, i.e., mouse-derived
.quadrature.-opioid receptor protein (L11064), mouse-derived
.delta.-opioid receptor protein (L11065), rat-derived .mu.-opioid
receptor protein (D 16349), mouse-derived bradykinin B2 receptor
protein (X69676), rat-derived bradykinin B2 receptor protein
(M599967), mouse-derived bombesin receptor protein (M35328),
human-derived neuromedin B receptor protein (M73482), human-derived
gastrin releasing peptide receptor protein (M73481), human-derived
bombesin receptor protein subtype 3 (L08893), mouse-derived
substance K receptor protein (X62933), mouse-derived substance P
receptor protein (X62934), rat-derived neurokinin 3 receptor
protein (J05189), rat-derived endothelin receptor protein (M60786),
rat-derived ligand unknown receptor proteins (L04672), (X61496),
(x59249) and (L09249), mouse-derived ligand unknown receptor
protein (P30731), human-derived ligand unknown receptor proteins
(M31210) and (U03642), etc. was made. In particular, the degenerate
DNA primer having a nucleotide sequence (3B in FIG. 3; SEQ ID NO:
6) with highly common bases (highly homologous nucleotides) was
synthesized to enhance base agreement of sequences with as many
receptor cDNAs as possible even in other regions on the basis of
nucleotide sequence regions that were in agreement with a large
number of receptor cDNAs. Nucleotide synthesis was carried out by a
DNA synthesizer.
[1198] The nucleotide sequence represented by SEQ ID NO 6 is:
5 5'-CTGAC (C or T) G (C or T) TCTI (A or G)(G or C) I (A or G)(C
or T) TGAC (A or C) G (A, C or G) TAT-3'
[1199] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis, provided that I
denotes inosine.
[1200] Furthermore, a comparison of nucleotide sequences coding for
regions corresponding to or near the sixth membrane-spanning domain
among known G protein coupled receptors, i.e., mouse-derived
.quadrature.-opioid receptor protein (L11064), mouse-derived
.delta.-opioid receptor protein (L11065), rat-derived .mu.-opioid
receptor protein (D16349), mouse-derived bradykinin B2 receptor
protein (X69676), rat-derived bradykinin B2 receptor protein
(M59967), mouse-derived bombesin receptor protein (M35328),
human-derived neuromedin B receptor protein (M73482), human-derived
gastrin releasing peptide receptor protein (M73481), human-derived
bombesin receptor protein subtype 3 (L08893), mouse-derived
substance K receptor protein (X62933), mouse-derived substance P
receptor protein (X62934), rat-derived neurokinin 3 receptor
protein (J05189), rat-derived endothelin receptor protein (M60786),
rat-derived ligand unknown receptor proteins (L04672), (X61496),
(X59249) and (L09249), mouse-derived ligand unknown receptor
protein (P30731), human-derived ligand unknown receptor proteins
(M31210) and (U03642), etc. was made. In particular, the degenerate
DNA primer having a nucleotide sequence (SEQ ID NO: 8) which is
complementary to the nucleotide sequence (6A in FIG. 5) with highly
common bases (highly homologous nucleotides) was synthesized to
enhance base agreement of sequences with as many receptor cDNAs as
possible even in other portions on the basis of base regions that
are in agreement with a large number of receptor cDNAs.
[1201] The nucleotide sequence represented by SEQ ID NO: 8 is:
6 5'-GATGTG (A or G) TA (A or G) GG (G or C) (A or G) ICCAACAGAIG
(A or G) (C or T) AAA-3'
[1202] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis, provided that I
denotes inosine.
[1203] The aforementioned abbreviations in parentheses are
reference numbers indicated when the GenBank/EMBL data base is
retrieved and are usually called "Accession Numbers".
Example 14
Cloning of Rabbit Gastropyrolic Part Smooth Muscle-Derived G
Protein Coupled Receptor Protein cDNA
[1204] (1) Preparation of Poly(A).sup.+ RNA Fraction from Rabbit
Gastropyrolic Part Smooth Muscle and Synthesis of cDNA
[1205] A total RNA was prepared from rabbit gastropyrolic part
smooth muscles by the guanidine thiocyanate method (Kaplan B. B. et
al., Biochem. J. 183, 181-184 (1979)) and, then, poly(A).sup.+ RNA
fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
Next, to 5 ,u g of the poly(A).sup.+ RNA fraction was added a
random DNA hexamer (BRL Co.) as a primer, and the resulting mixture
was subjected to reaction with MMLV reverse transcriptase (BRL Co.)
in the buffer attached to the MMLV reverse transcriptase kit to
synthesize complementary DNAs. The reaction product was extracted
with phenol/chloroform (1:1), precipitated in ethanol, and was then
dissolved in 30 .mu.l of TE.
[1206] (2) Amplification of Receptor cDNA by PCR Using Rabbit
Gastropyrolic Part Smooth Muscle-Derived cDNA and Sequencing
[1207] By using, as a template, 1 .mu.l of cDNA prepared from the
rabbit gastropyrolic part smooth muscle in the above step (1), PCR
amplification using the DNA primer having a nucleotide sequence
represented by SEQ ID NO: 6 and the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 8 synthesized in Example 13 was
carried out. A reaction solution was composed of the synthetic DNA
primers (SEQ: 5' primer sequence and 3' primer sequence) each in an
amount of 100 pM, 0.25 mM dNTPs, 1 .mu.l of Taq DNA polymerase and
10 .mu.l of buffer attached to the enzyme kit, and the total amount
of the reaction solution was made to be 100 ,u 1. The cycle for
amplification including 96.degree. C. for 30 sec., 45.degree. C.
for 1 min. and 60.degree. C. for 3 min. was repeated 25 times by
using a Thermal Cycler (Perkin-Elmer Co.). The amplified products
were confirmed relying upon 1.2% agarose gel electrophoresis and
ethidium bromide staining.
[1208] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1209] The PCR products obtained in the above step (2) were
separated by using a 1.0% low-melting temperature agarose gel, the
band parts were excised from the gel with a razor blade, and were
heat-melted, extracted with phenol and precipitated in ethanol to
recover DNAs. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAs were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized toothstick to
obtain transformant Escherichia coli JM 109/pMH28.
[1210] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with an automatic
plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid
DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI to
confirm the size of the cDNA fragment that was inserted. An aliquot
of the remaining DNAs was further processed with RNase, extracted
with phenol/chloroform, and precipitated in ethanol so as to be
condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer, and the data of the
nucleotide sequences obtained were read by using DNASIS (Hitachi
System Engineering Co., Japan). The determined nucleotide sequence
was as shown in FIG. 52.
[1211] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 52]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM109/pMH28. To further confirm this fact, by
using DNASIS (Hitachi System Engineering Co., Japan) the nucleotide
sequence were converted into an amino acid sequence [FIG. 52], and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 53] and at the amino acid sequence level to find
homology relative to mouse IL-8 receptor protein (P35343), human
somatostatin receptor protein 1 (A41795) and human somatostatin
receptor protein 4 (A47457) [FIG. 54]. The aforementioned
abbreviations in parentheses are reference numbers assigned when
they are registered as data to NBRF-PIR or SWISS-PROT and are
usually called "Accession Numbers".
Example 15
Preparation of Synthetic DNA Primer for Amplifying G Protein
Coupled Receptor Protein-Encoding DNA
[1212] A comparison of nucleotide sequences coding for regions
corresponding to or near the second membrane-spanning domain among
known G protein coupled receptors, i.e., human-derived galanin
receptor (HUMGALAREC), rat-derived .alpha.-1B-adrenergic receptor
(RATADR1B), human-derived .beta.-1-adrenergic receptor (HUMADRB1),
rabbit-derived IL-8 receptor (RABIL8RSB), human-derived opioid
receptor (HUMOPIODRE), bovine-derived substance K receptor (BTSKR),
human-derived somatostatin receptor-2 (HUMSTRI2A), human-derived
somatostatin receptor-3 (HUMSSTR3Y), human-derived gastrin receptor
(HUMGARE), human-derived cholecystokinin A receptor (HUMCCKAR),
human-derived dopamine receptor-D5 (HUMD1B), human-derived
serotonin receptor 5HT1E (HUM5HT1E), human-derived dopamine
receptor D4 (HUMD4C), mouse-derived serotonin receptor-2 (MMSERO),
rat-derived a .alpha.-1A-adrenergic receptor (RATADRA1A),
rat-derived histamine H2 receptor (S57565), etc. was made. In
particular, the degenerate DNA primer having a nucleotide sequence
(T2A in FIG. 7, SEQ ID NO: 10) with highly common bases (highly
homologous nucleotides) was synthesized to enhance base agreement
of sequences with as many receptor cDNAs as possible even in other
regions on the basis of nucleotide sequence regions that were in
agreement with a large number of receptor cDNAs. Nucleotide
synthesis was carried out by a DNA synthesizer.
[1213] The nucleotide sequence represented by SEQ ID NO: 10 is:
[1214] 5' -GYCACCAACN.sub.2WSTTCATCCTSWN.sub.2HCTG-3'
[1215] wherein S represents G or C; Y represents C or T; W
represents A or T; H represents A, C or T and N.sub.2 represents
I.
[1216] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis, provided that I
denotes inosine.
[1217] Furthermore, a comparison of nucleotide sequences coding for
regions corresponding to or near the seventh membrane-spanning
domain among known G protein coupled receptors, i.e., human-derived
galanin receptor (HUMGALAREC), rat-derived Al adenosine receptor
(RAT1ADREC), porcine-derived angiotensin receptor (PIGA2R),
rat-derived serotonin receptor (RAT5HTRTC), human-derived dopamine
receptor (S58541), human-derived gastrin releasing peptide receptor
(HUMGRPR), mouse-derived GRP/bombesin receptor (MUSGRPBOM),
rat-derived vascular type 1 angiotensin receptor (RRVT1AIIR),
human-derived muscarinic acetylcholine receptor (HSHM4),
human-derived .beta.-1 adrenergic receptor (HUMDRB1), human-derived
gastrin receptor (HUMGARE), rat-derived cholecystokinin receptor
(RATCCKAR), rat-derived ligand unknown receptor (S59748),
human-derived somatostatin receptor (HUMSST28A), rat-derived ligand
unknown receptor (RNGPROCR), mouse-derived somatostatin receptor 1
(MUSSRI1A), human-derived .alpha.-A 1-adrenergic receptor
(HUMA1AADR), mouse-derived delta-opioid receptor (S66181),
human-derived somatostatin receptor-3 (HUMSSTR3Y), etc. was made.
In particular, the degenerate DNA primer having a nucleotide
sequence (T7A in FIG. 8, SEQ ID NO: 11) with highly common bases
(highly homologous nucleotides) was synthesized to enhance base
agreement of sequences with as many receptor cDNAs as possible even
in other regions on the basis of nucleotide sequence regions that
were in agreement with a large number of receptor cDNAs. Nucleotide
synthesis was carried out by a DNA synthesizer.
[1218] The nucleotide sequence represented by SEQ ID NO: 11 is:
[1219]
5'-ASN.sub.2SAN.sub.2RAAGSARTAGAN.sub.2GAN.sub.2RGGRTT-3'
[1220] wherein R represents A or G; S represents G or C and N.sub.2
represents I.
[1221] The parentheses indicate the incorporation of a plurality of
bases, leading to multiple oligonucleotides in the primer
preparation. In other words, nucleotide residues in parentheses of
the aforementioned DNAs were incorporated in the presence of a
mixture of plural bases at the time of synthesis, provided that I
denotes inosine.
[1222] The aforementioned abbreviations in parentheses are
reference numbers indicated when the GenBank/EMBL data base is
retrieved and are usually called "Accession Numbers".
Example 16
Cloning of Rabbit Gastropyrolic Part Smooth Muscle-Derived G
Protein Coupled Receptor Protein cDNA
[1223] (1) Preparation of Poly(A).sup.+ RNA Fraction from Rabbit
Gastropyrolic Part Smooth Muscle and Synthesis of cDNA
[1224] A total RNA was prepared from rabbit gastropyrolic part
smooth muscles by the guanidine thiocyanate method (Kaplan B. B. et
al., Biochem. J. 183, 181-184 (1979)) and, then, poly(A).sup.+ RNA
fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
Next, to 5 .mu.g of the poly(A).sup.+ RNA fraction was added a
random DNA hexamer (BRL Co.) as a primer, and the resulting mixture
was subjected to reaction with MMLV reverse transcriptase (BRL Co.)
in the buffer attached to the MMLV reverse transcriptase kit to
synthesize complementary DNAs. The reaction product was extracted
with phenol/chloroform (1:1), precipitated in ethanol, and was then
dissolved in 30 .mu.l of TE.
[1225] (2) Amplification of Receptor cDNA by PCR Using Rabbit
Gastropyrolic Part Smooth Muscle-Derived cDNA and Sequencing
[1226] By using, as a template, 1 .mu.l of cDNA prepared from the
rabbit gastropyrolic part smooth muscle in the above step (1), PCR
amplification using the DNA primer having a nucleotide sequence
represented by SEQ ID NO: 10 and the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 11 synthesized in Example 15 was
carried out.
[1227] A reaction solution was composed of the synthetic DNA
primers (SEQ: 5' primer sequence and 3' primer sequence) each in an
amount of 100 pM, 0.25 mM dNTPs, 1 .mu.l of Taq DNA polymerase and
10 .mu.l of buffer attached to the enzyme kit, and the total amount
of the reaction solution was made to be 100 .mu.l. The cycle for
amplification including 96.degree. C. for 30 sec., 45.degree. C.
for 1 min. and 60.degree. C. for 3 min. was repeated 25 times with
a Thermal Cycler (Perkin-Elmer Co.). The amplified products were
confirmed relying upon 1.2% agarose gel electrophoresis and
ethidium bromide staining.
[1228] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1229] The PCR products obtained in the above step (2) were
separated with a 1.4% low-melting temperature agarose gel, the band
parts were excised from the gel with a razor blade, and were eluted
electrophoretically, extracted with phenol and precipitated in
ethanol to recover DNAs. According to the protocol attached to a TA
Cloning Kit (Invitrogen Co.), the recovered DNAs were subcloned to
the plasmid vector, pCR.TM.II. The recombinant vectors were
introduced into E. coli JM 109 competent cells (Takara Shuzo Co.,
Japan) to produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized toothstick to
obtain 100 transformant clones.
[1230] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with the automatic
plasmid extracting machine PI-100 (Kurabo Co., Japan) to prepare
plasmid DNAs. An aliquot of the DNA thus prepared was cut by EcoRI
to confirm the size of the cDNA fragment that was inserted. An
aliquot of the remaining DNA was further processed with RNase,
extracted with phenol/chloroform, and precipitated in ethanol so as
to be condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer.
[1231] Homology retrieval was carried out based upon the determined
nucleotide sequence by using DNASIS (Hitachi System Engineering
Co., Japan). As a result, it was learned that a novel G protein
coupled receptor protein was encoded by the cDNA fragment insert in
the plasmid possessed by the transformant Escherichia coli JM
109/pMN7. FIG. 56 and FIG. 56 show the nucleotide sequences of the
cDNA fragments. To further confirm this fact, by using DNASIS
(Hitachi System Engineering Co., Japan), the nucleotide sequences
were converted into amino acid sequences [FIG. 55] and [FIG. 56],
and hydrophobicity plotting was carried out [FIG. 57]. As a result,
the presence of hydrophobic domains which prove that it is a G
protein coupled receptor protein were confirmed. Furthermore,
homology retrieval was carried out at the amino acid sequence level
to find that the DNAs were novel receptor proteins having 27%
homology relative to rat-derived .beta..sub.3-adrenaline receptor
protein (A41679), 29% homology relative to rat-derived serotonin
(5-HT6) receptor protein (JN0591), 27% homology relative to
dog-derived histamine H.sub.2 receptor protein (A39008), 27%
homology relative to human-derived somatostatin receptor (type 4)
protein (JN0605), 24% homology relative to human-derived dopamine
D.sub.1 receptor protein (S11377), 23% homology relative to
rat-derived neurotensin receptor protein (JH0164), 31% homology
relative to human-derived cholecystokinin B receptor protein
(JC1352), and 30% homology relative to rat-derived gastrin receptor
protein (JQ 1614). The aforementioned abbreviations in parentheses
are reference numbers assigned when they are registered as data to
NBRF-PIR and are usually called "Accession Numbers".
Example 17
Amplification of Receptor cDNA by PCR Using MIN6-Derived cDNA and
Sequencing
[1232] By using, as a template, 5 .mu.l of cDNA prepared from the
mouse pancreatic .beta.-cell strain, MIN6 in Working Example 4 (1),
PCR amplification using the DNA primers synthesized in Example 4
(2) as disclosed in Libert F. et al., "Science, 244:569-572, 1989",
i.e., a synthetic primer represented by the following sequence:
7 5'-CTGTG (C or T) G (C or T) (G or C) AT (C or T) GCIIT (G or T)
GA (C or T) (A or C) (SEQ ID NO: 60) G (G or C) TAC-3'
[1233] wherein I is inosine; and
[1234] a synthetic primer represented by the following
sequence:
8 5'-A (G or T) G (A or T) AG (A or T) AGGGCAGCCAGCAGAI (G or C) (A
or G) (C or T) (SEQ ID NO: 61) GAA-3'
[1235] wherein I is inosine, was carried out under the same
conditions as in Example 3 (1). The resulting PCR product was
subcloned to the plasmid vector, pCR.TM.II, in the same manner as
in Example 3 (2) to obtain a plasmid, p5S38. The plasmid p5S38 was
transfected into E. coli JM 109 to obtain transformant Escherichia
coli JM 109/p5S38.
[1236] The reaction for determining the nucleotide sequence
(sequencing) was carried out with a DyeDeoxy terminator cycle
sequencing kit (ABI Co.), the DNA was decoded with the fluorescent
automatic sequencer (ABI Co.), and the data of the nucleotide
sequence obtained were read with DNASIS (Hitachi System Engineering
Co., Japan).
[1237] FIG. 62 shows a mouse pancreatic .beta.-cell strain
MIN6-derived G protein coupled receptor protein-encoding DNA (SEQ
ID NO: 33) and an amino acid sequence (SEQ ID NO: 28) encoded by
the isolated DNA based upon the nucleotide sequence of plasmid,
p5S38. The underlined portions represent regions corresponding to
the synthetic primers.
[1238] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 62]. As a result, it was learned that a
novel G protein coupled receptor protein was encoded by the cDNA
fragment obtained. To further confirm this fact, by using DNASIS
(Hitachi System Engineering Co., Japan), the nucleotide sequence
was converted into an amino acid sequence [FIG. 62], and
hydrophobicity plotting was carried out to confirm the presence of
four hydrophobic regions [FIG. 64]. Upon comparing the amino acid
sequence with those encoded by p19P2 obtained in Example 3 (2) and
encoded by pG3-2 obtained in Example 4 (2), furthermore, a high
degree of homology was found as shown in FIG. 63. As a result, it
is strongly suggested that the mouse pancreatic .beta.-cell strain,
MIN6-derived G protein coupled receptor protein encoded by p5S38
recognizes the same ligand as the human pituitary gland-derived G
protein coupled receptor protein encoded by p19P2 does while the
animal species from which the receptor protein encoded by p5S38 is
derived is different from that from which the receptor protein
encoded by p19P2 is. It is also strongly suggested that the mouse
pancreatic .beta.-cell strain, MIN6-derived G protein coupled
receptor protein encoded by p5S38 recognizes the same ligand as the
mouse pancreatic .beta.-cell strain, MIN6-derived G protein coupled
receptor proteins encoded by pG3-2 and pG1-10 do and they are
analogous receptor proteins one another (so-called "subtype").
Example 18
Northern Hybridization with cDNA Fragment Included in MINE-Derived
Receptor Protein-Encoding p3H2-17
[1239] Mouse cell line, MIN6, Neuro-2a, poly(A).sup.+ RNA (2.5
.mu.g) and mouse brain, spleen, thymus and pancreas poly(A).sup.+
RNAs (2.5 .mu.g) were used as poly(A) RNAs. The DNA fragment
inserted into the plasmid, p3H2-17, obtained in Example 7 (3) was
recovered as a DNA fragment with about 400 bp by cutting the
plasmid with EcoRI and the resulting DNA fragment was labeled by
incorporation of [32P]dCTP (Dupont Co.) with a random prime DNA
labeling kit (Amasham Co.). The about 400 bp labeled DNA fragment
was used as a probe for hybridization.
[1240] Nylon membrane (PaLL Biodyne, U.S.A.) was used as a filter
for northern blotting and migration of the poly(A).sup.+ RNA and
adsorption (sucking) thereof with the blotting filter was carried
out according to the method as disclosed in Molecular Cloning, Cold
Spring Harbor Laboratory Press, 1989.
[1241] The hybridization was carried out by incubating the
above-mentioned filter and probe in a buffer containing 50%
formamide, 5.times.SSPE (20.times.SSPE (pH 7.4) is 3 M NaCl, 0.2 M
NaH.sub.2PO.sub.4.H.sub.2O, 25 mM EDTA), 5.times. Denhardt's
solution (Nippon Gene, Japan), 0.1% SDS and 100 .mu.g/ml of salmon
sperm DNA overnight at 42.degree. C. The filter was washed with
0.1.times.SSC (20.times.SSC is 3 M NaCl, 0.3 M sodium citrate),
0.1% SDS at 50.degree. C. and, after drying with an air, was
exposed to an X-ray film (XAR5, Kodak) for 15 days at -80.degree.
C. The results were as shown in FIG. 65.
[1242] It is considered from FIG. 65 that mRNA for the the receptor
gene encoded by the cDNA fragment included in p3H2-17 is expressed
in the cell line, MIN6, Neuro-2a, and the mouse brain, pancreas,
spleen and thymus and especially expressed in the mouse spleen and
thymus intensely. The MIN6 signal position hybridizable in the
northern hybridization plotting is different from those of other
organ cells.
Example 19
PCR Cloning of cDNA Comprising Whole Coding Regions of Receptor
Proteins from Mouse Spleen, Thymus-Derived Poly(A) RNA and
Sequencing
[1243] (1) PCR Cloning of cDNA Comprising Whole Coding Region of
Receptor Protein
[1244] In order to obtain a full-length open reading frame (coding
region) of the receptor protein encoded by the cDNA fragment
included in p3H2-17, PCR amplification was carried out by 5'RACE
and 3'RACE wherein poly(A).sup.+ RNA derived from mouse spleen and
thymus was used.
[1245] Based on the nucleotide sequence of 3H2-17 which was
disclosed, the following 4 primers were synthesized:
[1246] (Nucleotide sequence of synthesized primer)
9 (SEQ ID NO: 20) .quadrature. 5'-TAGTGTGTGGAGTCGTGTGGCTG- GCTG-3'
(SEQ ID NO: 21) .quadrature. 5'-AGTCTTTGCTGCCACAGGCATCCAGCG-3' (SEQ
ID NO: 22) .quadrature. 5'-CAAGCCAGTAAGGCTATGAAGGGCAGCAAG-3' (SEQ
ID NO: 23) .quadrature. 5'-ACAGGACCTGCTGGGCCATCCTGGCGACACA- -3'
[1247] The 5'RACE was carried out according to the protocol of
5'Ampli Finder RACE kit from ClonTech Co. (ClonTech Co.).
[1248] In an embodiment, cDNA was prepared from 2 .mu.g each of
poly(A).sup.+ RNAs derived from mouse spleen and thymus by using
the aforementioned primer .quadrature. and ligated with an anchor
attached to the 5'RACE kit. A mixture of a {fraction (1/200)}
amount of the cDNA thus prepared, the anchor and the aforementioned
primer .quadrature. was subjected to PCR using 4 polymerases, Taq
(Takara, Japan; ExTaq (Takara, Japan), Vent (New England Biolabs)
and Pfu (Stratagene) under the following conditions: 96.degree. C.
for 30 sec., 60.degree. C. for 60 sec., 72.degree. C. for 90 sec.
and 35 cycles. A 1/5 amount of the PCR product was subjected to
agarose electrophoresis and stained with ethidium bromide (EtBr).
The results are shown in FIG. 66. The amplified DNA band appeared
at an about 1 kbp position and the isolated about 1 kbp DNA band
which was synthesized from poly(A).sup.+ RNAs derived from mouse
spleen and thymus by the 5'RACE using Ex Taq polymerase was treated
with SUPREC.TM.-01 (Takara, Japan) to recover cDNA.
[1249] The isolated DNA was subcloned into pCR.TM.II vector by
using a TA Cloning Kit (Invitrogen Co.) and the vector was
transfected into E. coli JM 109 to obtain 3 transformant clones,
N26, N64 and N75. The clone, N26, holds the thymus-derived cDNA
which is amplified by the 5'RACE and the clone, N75, holds the
spleen-derived cDNA which is amplified by the 5'RACE (FIG. 68).
[1250] The 3'RACE was carried out according to the protocol of 3'
RACE kit (GIBCO BRL Co.).
[1251] In an embodiment, cDNA was prepared from 1 .mu.g each of
poly(A).sup.+ RNAs derived from mouse spleen and thymus by using an
adaptor primer attached to the 3' RACE kit. A mixture of the
adaptor primer thus prepared and a {fraction (1/10)} amount of cDNA
which was prepared by using the aforementioned primer El was
subjected to 1st PCR using 4 polymerases, Taq (Takara, Japan),
ExTaq (Takara, Japan), Vent (NEB) and Pfu (Stratagene) under the
following conditions: 96.degree. C. for 30 sec., 55.degree. C. for
60 sec., 72.degree. C for 120 sec. and 30 cycles. A mixture of a
{fraction (1/50)} amount of the 1st PCR product, the aforementioned
primer .quadrature. and the adaptor primer was subjected to 2nd PCR
using the aforementioned polymerases under the same conditions as
aforementioned herein in the 5'RACE process. A 1/5 amount of the
2nd PCR product was subjected to agarose electrophoresis and
stained with ethidium bromide. The results are shown in FIG.
67.
[1252] The amplified DNA band appeared at an about 1 kbp position
(which was synthesized from poly(A).sup.+ RNAs derived from mouse
thymus by the 3'RACE using Vent polymerase) and the amplified DNA
band appeared at an about 1 kbp position (which was synthesized
from poly(A).sup.+ RNAs derived from mouse thymus by the 3'RACE
using Pfu polymerase) were treated with SUPREC.TM.-01 (Takara,
Japan) to recover cDNA, respectively.
[1253] The isolated DNAs were treated with T4
[1254] polynucleotide kinase (Wako Pure Chemical Co., Japan) to add
phosphate to the end thereof and the phosphorylated DNAs were
ligated with pUC18 SmaI BA (Pharmacia) by using DNA Ligation Kit
(Takara, Japan) followed by transformation of E. coli JM109 to
obtain 3 transformant clones, C2, C13 and C15. The clones, C13 and
C15, hold the thymus-derived cDNA which is amplified by the 3'RACE
and the clone, C2, holds the thymus-derived cDNA which is amplified
by the 3'RACE (FIG. 68).
[1255] Based on the nucleotide sequences of clones, N26, N64 and
N75, which are considered to hold the N-terminal region of the open
reading frame (ORF) of the cDNA fragment included in p3H2-17 and
the nucleotide sequences of clones, C2, C13 and C15, which are
considered to hold the C-terminal region of the open reading flame
(ORF) of the cDNA fragment included in p3H2-17, the entire
nucleotide sequence coding for the open reading flame and
neighboring region of the receptor protein encoded by the cDNA
included in p3H2-17 was determined. To be more specific, sequencing
was carried out with the primers used in the 5'RACE and 3'RACE or
synthetic primers for sequencing by using a DyeDeoxy Terminator
Cycle Sequencing Kit (ABI Co.), the nucleotide sequences were
decoded by using a fluorescent automatic sequencer. The obtained
data of the DNA were analyzed by DNASIS (Hitachi System Engineering
Co., Japan).
[1256] PCR errors which presumably happen to occur upon PCR have
been corrected by a way of thinking that, when nucleotides between
two clones which are independently produced by PCR are identical
(e.g. those between clones, N75 and N64, are identical) each other,
the identical base is considered as correct. The determined
nucleotide sequence is shown in FIG. 69. The amino acid sequence is
deduced based on the determined nucleotide sequence (FIG. 69).
Hydrophobicity plotting was carried out based on the deduced amino
acid sequence (FIG. 70). As a result, it was learned that it was a
seven transmembrane G protein coupled receptor, as it is suggested
from the cDNA fragment included in p3H2-17.
[1257] Homology retrieval at the amino acid level indicates that it
is homologous to mouse P.sub.2Upurinoceptor and chicken
P.sub.2ypurinoceptor.
[1258] Further, the clone which are free of an error in the open
reading flame (ORF) was selected and used to construct plasmids
carrying the full-length ORF of the receptor protein encoded by
p3H2-17. In an embodiment, the cDNA fragment held by the clone,
N75, was digested with restriction enzymes, DraIII and EcoRI, to
obtain cDNA fragments which are the N-terminal region of the
receptor protein held by p3H2-17. The C-terminal cDNA fragment
encoded by C13 was digested with restriction enzymes, DraIII and
EcoRI, to delete 5'-side regions from the DraIII site of the
C-terminal and the long fragment was obtained by the digestion of
C13 with restriction enzymes, DraIII and EcoRI. The N75-derived
N-terminal cDNA DraIII-EcoRI fragment was ligated with the long
C13-derived DraIII-EcoRI fragment by using a DNA Ligation Kit
(Takara, Japan) and transfected into Escherichia coli JM 109 to
obtain transformant Escherichia coli JM 109/pMAH2-17.
[1259] (2) Electrophysiological Measurement of Receptor Encoded by
pMAH2-17
[1260] The receptor encoded by pMAH2-17 was examined
electrophsiologicallyin Xenopus oocytes. The ORF of the receptor
encoded by pMAH2-17 was inserted into the XhoI-XbaI sites of
pBluescript.TM.II SK(+) (Stratagene) with directing the sequence
thereof downstream from T7 promoter. The resulting plasmid as a
template was treated with a mCAP.TM.RNA Capping kit (Stratagene) to
produce cRNA of this receptor gene.
[1261] The cRNA was injected into Xenopus oocytes (50ng cRNA/50 nl
/oocyte), previously prepared according to the method disclosed in
Nathan Dascal et al., Proc. Natl. Acad. Sci. USA, Vol.90,
pp.6596-6600 (1993). The cRNA-injected oocytes were incubated at
20.degree. C. for 2 to 3 days and subjected to electrophysiological
measurements. The measurement was carried out with a
microelectrode-applicable high input resistance amplifier
(MEz-8300, Nippon Koden, Co., Japan), and a voltage clamping
amplifier (CEz -/200, Nippon Koden, Co., Japan). The initial
membrane potential of oocytes was set to -60 mV and responses
(current changes of the membrane) evoked by addition of ligands
were recorded with a recorder (Thermal Array recorder, Nippon
Koden, Co., Japan) (Nathan Dascal et al., Proc. Natl. Acad. Sci.
USA, Vol.90, pp.6596-6600 (1993)).
[1262] Typical inward currents elicited upon activation of
phospholipase C-coupled receptors were observed in oocytes injected
with pMAH2-17 cRNA via stimulation by 10 .mu.M ATP (FIG. 75). In
contrast, such a current was not observed in oocytes injected with
H.sub.2O, instead of pMAH2-17 cRNA, by the ATP stimulation.
[1263] In conclusion, it is considered that the receptor encoded by
pMAH2-17 cRNA is classified into a subtype within the ATP receptor,
P.sub.2 purinoceptor.
Example 20
Cloning of Rabbit Gastropyrolic,Part Smooth Muscle-Derived G
Protein Coupled Receptor Protein cDNA
[1264] (1) Preparation of Poly(A).sup.+ RNA Fraction from Rabbit
Gastropyrolic Part Smooth Muscle and Synthesis of cDNA
[1265] A total RNA was prepared from rabbit gastropyrolic part
smooth muscles by the guanidine thiocyanate method (Kaplan B. B. et
al., Biochem. J. 183, 181-184 (1979)) and, then, poly(A).sup.+ RNA
fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
Next, to 5 .mu.g of the poly(A).sup.+ RNA fraction was added a
random DNA hexamer (BRL Co.) as a primer, and the resulting mixture
was subjected to reaction with MMLV reverse transcriptase (BRL Co.)
in the buffer attached to the MMLV reverse transcriptase kit to
synthesize complementary DNAs. The reaction product was extracted
with phenol/chloroform (1: 1), precipitated in ethanol, and was
then dissolved in 30 .mu.l of TE.
[1266] (2) Amplification of Receptor cDNA by PCR Using Rabbit
Gastropyrolic Part Smooth Muscle-Derived cDNA and Sequencing
[1267] By using, as a template, 1 .mu.l of cDNA prepared from the
rabbit gastropyrolic part smooth muscle in the above step (1), PCR
amplification using the DNA primer having a nucleotide sequence
represented by SEQ ID NO: 10 and the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 4 synthesized in Example 15 was
carried out.
[1268] A reaction solution was composed of the synthetic DNA
primers (SEQ: 5' primer sequence and 3' primer sequence) each in an
amount of 100 pM, 0.25 mm dNTPs, 1 .mu.l of Taq DNA polymerase and
10 .mu.l of buffer attached to the enzyme kit, and the total amount
of the reaction solution was made to be 100 .mu.l.
[1269] The cycle for amplification including 96.degree. C. for 30
sec., 45.degree. C. for 1 min. and 60.degree. C. for 3 min. was
repeated 25 times by using a Thermal Cycler (Perkin-Elmer Co.). The
amplified products were confirmed relying upon 1.2% agarose gel
electrophoresis and ethidium bromide staining.
[1270] (3) Subcloning of PCR Product into Plasmid Vector and
Selection of Novel Receptor Candidate Clone via Decoding Nucleotide
Sequence of Inserted cDNA Region
[1271] The PCR products obtained in the above step (2) were
separated by using a 1.0% low-melting temperature agarose gel, the
band parts were excised from the gel with a razor blade, and were
electro-eluted, extracted with phenol and precipitated in ethanol
to recover DNAs. According to the protocol attached to a TA Cloning
Kit (Invitrogen Co.), the recovered DNAs were subcloned to the
plasmid vector, pCR.TM.II. The recombinant vectors were introduced
into E. coli JM 109 competent cells (Takara Shuzo Co., Japan) to
produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB agar culture medium
containing ampicillin, IPTG and X-gal. Only transformant clones
exhibiting white color were picked with a sterilized tooth stick to
obtain 100 transformant clones.
[1272] The individual clones were cultured overnight in an LB
culture medium containing ampicillin and treated with the automatic
plasmid extracting machine PI-100 (Kurabo Co., Japan) to prepare
plasmid DNAs. An aliquot of the DNAs thus prepared was cut by EcoRI
to confirm the size of the cDNA fragment that was inserted. An
aliquot of the remaining DNAs was further processed with RNase,
extracted with phenol/chloroform, and precipitated in ethanol so as
to be condensed. Sequencing was carried out by using a DyeDeoxy
terminator cycle sequencing kit (ABI Co.), the DNAs were decoded by
using a fluorescent automatic sequencer.
[1273] Homology retrieval was carried out based upon the determined
nucleotide sequence. As a result, it was learned that a novel G
protein coupled receptor protein was been encoded by the cDNA
fragment insert in the plasmid possessed by the transformant
Escherichia coli JM 109/pMN 128. The nucleotide sequences of the
cDNA fragments are shown in FIGS. 71 and 72. To further confirm
this fact, by using DNASIS (Hitachi System Engineering Co., Japan)
the nucleotide sequences were converted into amino acid sequences
[FIG. 71 and FIG. 72], and homology retrieval was carried out in
view of hydrophobicity plotting [FIG. 73] and at the amino acid
sequence level to find a novel receptor protein which has 27%
homology relative to hamster-derived .beta..sub.2-adrenaline
receptor protein (A03159), 20% homology relative to rat-derived
bradykinin receptor (type B.sub.2) protein (A41283), 24% homology
relative to human-derived dopamine D.sub.1 receptor protein
(S11377) and 23% homology relative to human-derived blue sensitive
opsin receptor protein (A03156). The aforementioned abbreviations
in parentheses are reference numbers assigned when they are
registered as data to NBRF-PIR and are usually called "Accession
Numbers".
Example 21
Cloning of cDNA Comprising Whole Coding Regions for Receptor
Protein from Human-Derived DNA Library
[1274] The DNA library constructed by Clontech wherein .lambda.
gt11 phage vector is used (CLONTECH Laboratories, Inc.; CLH L1008b)
was employed as a human placenta-derived cDNA library. The human
placenta cDNA library (1.times.10.sup.5 pfu (plaque forming units))
was thermally denatured. By using the human placenta-derived cDNA
library, PCR amplification using the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 20 and the DNA primer having a
nucleotide sequence represented by SEQ ID NO: 23 synthesized in
Example 19 was carried out.
[1275] (Nucleotide sequence of synthesized primer)
10 (SEQ ID NO: 20) .quadrature. 5'-TAGTGTGTGGAGTCGTGTGGCT- GGCTG-3'
(SEQ ID NO: 23) .quadrature.
5'-ACAGGACCTGCTGGGCCATCCTGGCGACACA-3'
[1276] The isolated DNA was subcloned using a TA Cloning Kit
(Invitrogen Co.) and sequencing was carried out. FIG. 76 shows a
nucleotide sequence of obtained cDNA fragment, ph3H2-17. As a
result, it was learned that ph3H2-17 is highly homologous to the
mouse purinoceptor cDNA fragment, p3H2-17. It is strongly suggested
that the human-derived cDNA fragment is a partial nucleotide
sequence of human purinoceptor.
[1277] Based on the nucleotide sequence of ph3H2-17 which was
sequenced, the following 2 primers were synthesized:
[1278] (Nucleotide sequence of synthesized primer)
11 .quadrature. 5'-ACAGCCATCTTCGCTGCCACAGGCAT-3' (SEQ ID NO:58)
.quadrature. 5'-AGACAGTAGCAGGCCAGCAGGGCAGCA- AA-3' (SEQ ID
NO:59)
[1279] The above synthetic 2 primers were each used in combination
with X gt 11 primers (Takara, Japan; catalogue 3864) for obtaining
full-length human prinoceptor cDNA. Thus, using thermally
denatured, human placenta-derived .lambda. gt 11 cDNA libraries
(CLONTECH; CLHL 1008b), first RCR amplification using a combination
of the DNA primer having a nucleotide sequence represented by SEQ
ID NO: 20 with .lambda. gt 11 Forward primer, of the DNA primer
having a nucleotide sequence represented by SEQ ID NO: 20 with
.lambda. gt 11 Reverse primer, of the DNA primer having a
nucleotide sequence represented by SEQ ID NO: 23 with .lambda. gt
11 Forward primer, and of the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 23 with .lambda. gt 11 Reverse
primer was carried out with ExTaq polymerase (Takara, Japan) (30
cycles; 95.degree. C./30 seconds, 55.degree. C./60 seconds, and
72.degree. C./60 seconds), respectively.
[1280] Next, by using a {fraction (1/50)} of the 1st PCR product,
second RCR amplification was carried in the same manner as in the
first PCR except for using the DNA primer having a nucleotide
sequence represented by SEQ ID NO: 58 in place of SEQ ID NO: 20 and
the DNA primer having a nucleotide sequence represented by SEQ ID
NO: 59 in place of SEQ ID NO: 23 (30 cycles; 95.degree. C./30
seconds, 65.degree. C./60 seconds and 72.degree. C/60 seconds). The
amplified product DNA was subcloned using a TA Cloning Kit
(Invitrogen Co.) and sequencing was carried out for three clones
each of 5' and 3' sides (FIG. 77).
[1281] Based on the amino acid sequence (FIG. 77) deduced from the
determined nucleotide sequence of human purinoceptor cDNA as shown
in FIG. 77, hydrophobicity plotting was carried out (FIG. 78). As a
result, it was learned that the human-derived receptor is a novel
seven transmembrane G protein coupled receptor, similarly to the
mouse type. It was also learned that the deduced amino acid
sequence of human receptor has 87% homology relative to the amino
acid sequence of mouse purinoceptor and its amino acid residues are
well conserved (FIG. 79).
[1282] Clones free of PCR errors which often occur in a PCR
amplification were selected and restriction enzyme regions
comprising overlapping areas were obtained therefrom. The
restriction enzyme regions thus obtained were subjected to
construction of plasmid phAH2-17 having a full-length open reading
frame of human purinoceptor cDNA. The plasmid phAH2-17 is possessed
by transformant Escherichia coli JM109/phAH2-17.
[1283] The DNA primers of the present invention allow efficient
amplification of DNAs that encode G protein coupled receptor
proteins. This makes it possible to efficiently screen for the DNAs
coding for G protein coupled receptor proteins and to accomplish
the cloning.
[1284] The G protein coupled receptor protein of the present
invention and their G protein coupled receptor protein-encoding DNA
are advantageously useful in:
[1285] determining ligands,
[1286] obtaining antibodies and an antisera,
[1287] constructing systems for expressing recombinant receptor
proteins,
[1288] investigating or developing receptor-binding assay systems
and screening for pharmaceutical candidate compounds, by using the
above expression system
[1289] designing drugs based upon comparisons with ligands and
receptors having a structure similar or analogous thereto,
[1290] preparing probes and/or PCR primers in gene diagnosis,
and
[1291] gene manipulating therapy.
[1292] In particular, discovering the structure and properties of
the G protein coupled receptor will lead to the development of
unique pharmaceuticals acting upon these systems.
[1293] The practice of the present invention will employ, otherwise
indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, pharmacology, immunology,
bioscience, and medical technology, which are within the skill of
the art. AU patents, patent applications, and publications
mentioned herein, both supra and infra, are hereby incorporated
herein by reference.
Sequence CWU 1
1
* * * * *