U.S. patent application number 10/090569 was filed with the patent office on 2003-01-09 for galanin receptor protein, production and use thereof.
This patent application is currently assigned to Takeda Chemical Industries, Ltd.. Invention is credited to Fujii, Ryo, Fukusumi, Shoji, Hinuma, Shuji, Hosoya, Masaki, Ohgi, Kazuhiro, Ohtaki, Tetsuya, Onda, Haruo.
Application Number | 20030008329 10/090569 |
Document ID | / |
Family ID | 27316890 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008329 |
Kind Code |
A1 |
Hinuma, Shuji ; et
al. |
January 9, 2003 |
Galanin receptor protein, production and use thereof
Abstract
Galanin receptor proteins, production and use thereof including
screening of galanin receptor agonists and antagonists are
provided. Galanin receptor proteins, etc. or salts thereof, partial
peptides thereof, DNAs coding for the above galanin receptor
protein, processes for producing the above receptor protein,
methods of screening for a galanin receptor agonist and/or
antagonist or screening kits therefor, agonist and/or antagonist
compounds or salts thereof obtained by the above screening method
or the screening kit, pharmaceutical compositions containing the
above compound or its salt, and antibodies against the above
receptor protein are provided. It is allowable to efficiently
screen a galanin receptor agonist or antagonist by using the
galanin receptor protein, the partial peptide thereof, the galanin
receptor protein-encoding DNA, the receptor protein-containing cell
or its membrane fraction. The pharmaceuticals thus screened or
characterized permits various applications including prophylactic
and/or therapeutic treatments against a variety of diseases, e.g.,
stomach ulcer, diabetes, Alzheimer's disease, dementia, etc. and a
sedative.
Inventors: |
Hinuma, Shuji; (Ibaraki,
JP) ; Fujii, Ryo; (Ibaraki, JP) ; Fukusumi,
Shoji; (Ibaraki, JP) ; Ohtaki, Tetsuya;
(Ibaraki, JP) ; Hosoya, Masaki; (Ibaraki, JP)
; Ohgi, Kazuhiro; (Ibaraki, JP) ; Onda, Haruo;
(Ibaraki, JP) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP.
P.O. BOX 9169
BOSTON
MA
02209
US
|
Assignee: |
Takeda Chemical Industries,
Ltd.
|
Family ID: |
27316890 |
Appl. No.: |
10/090569 |
Filed: |
March 4, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10090569 |
Mar 4, 2002 |
|
|
|
08540650 |
Oct 11, 1995 |
|
|
|
6399325 |
|
|
|
|
Current U.S.
Class: |
435/7.21 ;
435/320.1; 435/325; 435/358; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/723
20130101 |
Class at
Publication: |
435/7.21 ;
435/69.1; 435/320.1; 435/325; 530/350; 536/23.5; 435/358 |
International
Class: |
G01N 033/567; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 1994 |
JP |
6-247599 |
Dec 28, 1994 |
JP |
6-326610 |
May 31, 1995 |
JP |
7-134412 |
Claims
What is claimed is:
1. A galanin receptor protein comprising an amino acid sequence
selected from the group consisting of an amino acid sequence
represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5 or its
substantial equivalent thereto, or a salt thereof.
2. The receptor protein according to claim 1, which is produced by
a transformant CHO cell.
3. An isolated DNA which comprises a nucleotide sequence coding for
a galanin receptor protein of claim 1.
4. A vector comprising the DNA according to claim 3.
5. A transformant carrying the vector according to claim 4.
6. The transformant according to claim 5, wherein the host cell is
a CHO cell.
7. A process for producing a galanin receptor protein according to
claim 1, which comprises culturing a transformant of claim 5 under
conditions suitable to express said galanin receptor protein.
8. A screening method for an agonist or antagonist of a galanin
receptor protein according to claim 1, which comprises carrying out
a comparison between: (i) at least one case where galanin is
contacted with at least one component selected from the group
consisting of a galanin receptor protein according to claim 1, a
partial peptide thereof and a mixture thereof, and (ii) at least
one case where galanin together with a compound to be tested is
contacted with at least one component selected from the group
consisting of a galanin receptor protein according to claim 1, a
partial peptide thereof and a mixture thereof.
9. A kit for the screening of one or more agonists or antagonists
to a galanin receptor protein according to claim 1, which comprises
at least one component selected from the group consisting of a
galanin receptor protein according to claim 1, a partial peptide
thereof and a mixture thereof.
10. An agonist or antagonist of a galanin receptor, which is
obtained by the screening method according to claim 8 or the kit
according to claim 9.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel galanin receptor
proteins and partial peptides thereof; novel DNAs containing a
galanin receptor protein or partial peptide-encoding DNA; processes
for producing said galanin receptor protein (or partial peptide);
use of said receptor protein (or partial peptide) and said protein
(or partial peptide)-encoding DNA; a method of measuring the
physiological actions of galanin using a galanin receptor
protein-expressing cell or the galanin receptor protein; a method
of screening galanin receptor agonists/antagonists using the
galanin receptor protein-expressing cell or galanin receptor
protein; a kit for said screening; an agonist or antagonist
obtained by said screening method; and a pharmaceutical composition
containing said agonist or antagonist.
[0002] The present invention also relates to a novel mouse
pancreatic 3 cell line MIN6-derived galanin receptor protein and a
partial peptide thereof; a novel DNA coding for said mouse galanin
receptor protein or its partial peptide; processes for producing
said mouse galanin receptor protein or its partial peptide; use of
said mouse galanin receptor protein and said protein or
peptide-encoding DNA; a method of measuring the physiological
actions of galanin using a mouse-derived cell line MIN6 or the
mouse galanin receptor protein; and a method of screening a galanin
receptor agonist/antagonist using said mouse-derived cell line MIN6
or the receptor protein.
[0003] The present invention also relates to a novel human galanin
receptor protein; a partial peptide of the human galanin receptor
protein; a novel DNA which codes for the galanin receptor protein
or partial peptide; a vector carrying said DNA; a transformant
harboring said vector; a process for producing the human galanin
receptor protein (or its partial peptide); a method of screening a
galanin receptor agonist/antagonist using the human galanin
receptor protein or a human galanin receptor protein-expressing
cell (including the transformant); a kit for said screening; an
agonist or an antagonist, obtained by said screening method; and a
pharmaceutical composition containing said agonist or
antagonist.
BACKGROUND OF THE INVENTION
[0004] 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 a guanine nucleotide-binding protein (hereinafter,
sometimes referred to as "G protein") with which the receptor is
coupled and possess the common (homologous) structure, i.e. seven
transmembranes (membrane-spanning regions (domains)). Therefore,
such a receptor is generically referred to as "G protein coupled
receptor" or "seven transmembrane (membrane-spanning)
receptor".
[0005] G protein coupled receptor proteins which are widely
distributed in the functional cellular surface of cells and organs
in the living bodies 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.
[0006] 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 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. As for the
galanin and amylin, however, there has not yet been reported any
discovery concerning the structure of their receptor protein cDNA.
It is not known whether there exist any unknown receptor proteins
or receptor protein subtypes.
[0007] It is a very important means in investigating development of
new pharmaceuticals to clarify the relation between substances
controlling the complicated functions of pancreas and specific
receptors thereto. In order to develop new pharmaceuticals by
conducting an effective screening of agonists and antagonists to
the receptor proteins for controlling the functions of pancreas, it
was necessary to investigate the function of receptor protein genes
and also to express them in a suitable expression system.
[0008] 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.
[0009] Galanin is a peptide existing in central and peripheral
areas and, in central area, it shows an action of inhibition of
liberation of neurotransmitter (acetylcholine) (European Journal of
Pharmacology, vol.164, 355-360, 1989) and an action of antagonizing
foreign acetylcholine (Proceedings of National Academy of Sciences,
U.S.A., vol.85, 9841-9845, 1988) while, in pancreas, it shows a
pharmacological action such as inhibition of insulin secretion
(Diabates, vol. 34, 192-196, 1985). It has been also confirmed that
galanin has an effect of inhibiting the behavior of learning
(Neuroscience Letters, vol.88, 331-335, 1988) and of inhibiting the
feeling of fullness after a meal. Such findings suggest a
possibility that, if pharmaceuticals which inhibit the action of
galanin are developed, they may be used as intelligence tropic
agents and as remedies for obesity and for diabetes.
[0010] All of the pharmacological actions of galanin take place via
a specific galanin receptor existing in target tissues.
Accordingly, the simplest means for inhibiting the action of
galanin is to develop pharmaceuticals which specifically inhibit
the reaction of galanin with the receptor, i.e. galanin receptor
antagonists. In the development of galanin receptor antagonists, it
is usually necessary to conduct a receptor binding experiment. In
the case of galanin, experiments on galanin receptor binding using
membrane fractions of brain hippocampal formation (European Journal
of Biochemistry, vol. 181, 269-276, 1989) and of stomach and
duodenum (Peptides, vol. 11, 333-338, 1990) have been reported
already.
[0011] It has been also reported that there is a specific galanin
receptor in Rin-m-5F cells obtained from rat pancreas
(Endocrinology, vol. 124, 2635-2641, 1989). According to the
above-mentioned reports, it is already possible to conduct a
galanin receptor binding experiment. However, the amount of the
galanin receptor in those membrane fractions is as low as around 50
fmol/mg and, therefore, it was necessary to use a large amount of
cell fractions for one measurement.
[0012] Galanin exhibits the above-mentioned pharmacological actions
in living body and, if the actions can be easily measured in vitro,
that will be meaningful for the process of developing the receptor
antagonists. It has been reported already that the action of
inhibiting the insulin secretion by galanin can be substituted with
an in vitro measurement using Langerhans islet isolated from
pancreas (European Journal of Pharmacology, Vol. 203, 111-114,
1991). However, Langerhans island is required to be isolated upon
each experiment and, therefore, this method is not easily
accomplished.
[0013] As easier means, several methods using pancreatic
.beta.-cell strains (Rin-m-5F cells) have reported. They are, for
example, a method in which an effect of galanin receptor to a
second messenger system (i.e. an activity of inhibiting the
adenylate cyclase) is measured (European Journal of Biochemistry,
Vol. 177, 147-152, 1988) and a method in which an activity of
opening the potassium channel is measured (Proceedings of National
Academy of Sciences, U.S.A., Vol. 85, 1312-1316, 1988). A method in
which the activity of inhibiting the insulin secretion of galanin
using said cell strain has been reported too. However, those
methods are applicable only for insulin secretion which is
dependent upon forskolin (an adenylate cyclase activator) and the
measurement for secretion of glucose-dependent insulin is not
possible. Further, the secretion amount of insulin is small and the
sensitivity is low.
[0014] After those, a method of preparing the .beta.-cell strains
using pancreas of transgenic mice (Proceedings of National Academy
of Sciences, U.S.A., Vol. 85, 9037-9041, 1988) has been developed
and establishments of cell strains such as .beta. TC-1 cells
(Proceedings of National Academy of Sciences, U.S. A., Vol. 85,
9037-9041, 1988), IgSV 195 cells (Diabates, Vol. 38, 1056-1062,
1989) and MIN6 cells (Endocrinology, Vol. 127, 126-132, 1990) have
been reported. Among those, MIN6 cells hold the ability of insulin
secretion depending upon the glucose concentration (which is a
differentiating function inherent to .beta.-cells) in the best
manner and, in addition, they secret insulin in a high amount.
However, it has not been known yet that galanin receptor protein is
expressed in said MIN6 cells. In addition, there has been no
proposal yet for an evaluating system for the biological activity
of galanin and also for an effective method for screening the
galanin receptor agonist or antagonist using the MIN6 cells.
[0015] Recently, cDNA which codes for human galanin receptor
protein was cloned and its nucleotide sequence and also its amino
acid sequence encoded by said cDNA have been disclosed (Proceedings
of National Academy of Sciences, U.S.A., Vol. 91, 9780-9783, Oct.
11, 1994). However, there is no disclosure at all for a specific
means for screening the galanin receptor agonist/antagonist using
said receptor. Under such circumstances, a method for screening and
assessing galanin receptor agonist/antagonist in an efficient
manner is still desired.
[0016] Galanin is a polypeptide comprising 29 amino acid residues
separated from porcine small intestine [Tatemoto, K. et al., FEBS
Letter, 164, 124-128(1983)] and its primary structure is hardly
similar to those of other brain and intestinal hormones. Galanin
immunoactivity is widely distributed in central nervous system and
peripheral nervous system together with its receptor [Scofitsch, G.
and Jacobowitz, D. M., Peptides, 6, 509-546(1985); Melander, T. et
al., Journal of Comparative Neurology, 248, 475-517(1986); Rokaeus,
A., Trends in Neuroscience, 10,158-164(1987)] and, since its
distribution pattern is identical with the region containing the
traditional neurotransmitters such as 5-HT, noradrenaline and
acetylcholine, it is likely that galanin is present together with
such neurotransmitters and controls the prenervous and postnervous
actions by those neurotransmitters.
[0017] Galanin has many physiological actions and, in central
nervous system, it strongly inhibits the single synaptic reflection
in spinal nerve [Yanagisawa, M. et al., Neuroscience Letter, 70,
278-282(1986)] and its action is known to be far stronger than
somatostatin. In addition, the physiological importance of galanin
in nerve center has been greatly suggested because of stimulation
of action for taking food [Kyrokouli, S. E. et al., European
Journal of Pharmacology, 122, 159-160(1986)], participation in
formation of memory [Crawley, J. N. and Wenk, G. L., Trends in
Neuroscience, 12,278-282(1989)], inhibition of dopamine in median
elevation [Nordstrom, O. et al., Neuroscience Letter, 73,
21-26(1987)], inhibition of release of acetylcholine in hippocampal
double sides [Fisone, G. et al., Proceedings of the National
Academy of Sciences of U. S. A., 84, 7339-7343(1987)], a decrease
in metabolic circulation of 5-HT [Fuxe K., et al., Acta. Physiol.
Scand., 133, 579-581(1988)] and a decrease in a glutamic acid
release by activation of ATP-sensitive K.sup.+ channel [Ben-Ari,
Y., European Journal of Neuroscience, 2, 62-68(1990)] as a result
of administration of galanin to paraventricular nucleus of
rats.
[0018] Especially, galanin is an only neuropeptide in which choline
acetyltransferase is coexisting in the medial septal nucleus,
nucleus of diagonal band and basal nucleus [Melender, T. et al.,
Brain Research, 360, 130-138(1985); Melender, T. et al.,
Neuroscience Letter, 19, 223-240(1986); Chen-Palay, V., Brain
Research Bulletin, 21, 465-472(1988)] and is known to act on
cholinergic nerves in an inhibiting manner while, on the other
hand, it is expected that, since denaturation in cholinergic nerves
is noted in those sites in Alzheimer's disease, galanin antagonist
may prevent the denaturation of the cholinergic nerves in
Alzheimer's disease or the like [Whitehouse, P. J., et al.,
Science, 215, 1237-1239(1982); Chen-Palay, V., Journal of
Comparative Neurology, 273-543-557 (1988)]. In hypophysis, action
of stimulating the secretion of growth hormones and prolactin has
been noted [Tanoh, T., et al., Neuroendocrinology, 54, 83-88(1991);
Koshiyama, H., et al., Neuroscience Letter, 75, 49-54(1987)].
Particularly in the secretion of growth hormones, participation of
cholinergic neuron via adjustment of secretion of hypothalamic
somatostatin is noted.
[0019] On the other hand, in peripheral systems, galanin inhibits
the basal secretion of insulin both in vivo and in vitro [McDnald,
T. J. et al., Diabates, 34, 192-196(1985); Takeda, Y. et al.,
Biomedical Research, 8 (Suppl.), 117-125 (1987); Lindskog, S. et
al., Acta. Physiol. Scand., 129, 305-309(1987)] and, in addition,
it inhibits the release of insulin by stimulation of glucose
[Dunning, B. E. and Taborsky, G. J., Jr., Diabates, 37,
1157-1162(1988)]. When further immunohistological observation that
nerve fiber net containing a dense galanin immunoactivity is noted
around Langerhans islet of .beta. cells is taken into
consideration, it has been strongly suggested that galanin is one
of the nerve controlling factors for secretion of pancreatic
hormones, especially insulin. It is also noted that, in stomach,
galanin inhibits the basal secretion of somatostatin on a
dose-depending manner or it inhibits the secretion of somatostatin
or gastrin by stimulation of GRP and that nerve fiber net
containing galanin immunoactivity is observed in stomach and,
accordingly, it is suggested that, even in stomach, galanin acts as
one of the important nerve controlling factors for adjusting the
secretion in stomach [Yanaihara, N. et al., in "Galanin" (ed. by
Hokfelt, T. et al.), Macmillan Press, 185-196(1991)].
[0020] From the above descriptions, it is understood that galanin
agonist is useful as a pharmaceutical agent such as a stimulant for
secretion of growth hormones and an inhibitor for secretion of
insulin and that galanin antagonist is useful as another
pharmaceutical agent such as an inhibitor for secretion of growth
hormones and a stimulant for secretion of insulin.
[0021] Usually, in developing agonists and antagonists for
physiologically-active substances, investigations are made on the
compounds which have high affinity with the receptors to which said
substance is specifically bonded. At present, bovine hippocampal
membrane fraction is used as a galanin receptor but, because of the
difference in the animal species used, there is no guarantee that
the compound exhibiting a high affinity to said membrane fraction
has a high affinity to human galanin receptor as well. Human
galanin receptor cDNA has been cloned and reported to exhibit an
expression in COS cells [Habert-Ortoll, E. et al., Proceedings of
the National Academy of Sciences, U. S. A., 91, 9780-9783(1994)]
but, since the expressed amount is small and the expression is mere
transient, it is thought to be unsuitable for screening.
SUMMARY OF THE INVENTION
[0022] One object of the present invention is to provide novel
galanin receptor proteins and partial peptides thereof or salts
thereof; DNAs comprising a DNA coding for said galanin receptor
protein or its partial peptide; vectors carrying said DNA;
transformants harboring said vector; cell membrane fractions
obtained from said transformant; processes for producing said
receptor protein or its partial peptide, or a salt thereof; methods
for measuring the physiological actions of galanin using the
galanin receptor protein (including a cell membrane fraction
containing the receptor protein) or a galanin receptor
protein-expressing cell (including the transformant); screening
methods for a galanin receptor agonist/antagonist using the galanin
receptor protein or a galanin receptor protein-expressing cell
(including the transformant); kits for said screening; agonists or
antagonists, obtained by said screening method; pharmaceutical
compositions containing said agonist or antagonist; antibodies
against said receptor protein; immunoassays using said receptor
protein or said antibody; and use of said receptor protein and
encoding DNA.
[0023] Another object of the present invention is to provide novel
mouse pancreatic .beta. cell line MIN6-derived galanin receptor
proteins or partial peptides thereof; DNAs comprising a DNA coding
for said galanin receptor protein or partial peptide; processes for
producing said receptor protein or its partial peptide; methods of
measuring the physiological actions of galanin using a
mouse-derived cell line MIN6 or the galanin receptor protein;
screening methods for a galanin receptor agonist/antagonist using
said mouse-derived cell line MIN6 or the receptor protein;
antibodies against said receptor protein; immunoassays using said
receptor protein or said antibody; and use of said galanin receptor
protein or said receptor protein peptide-encoding DNA.
[0024] Human galanin receptor proteins manufactured by the
conventional method and the COS cells which express said human
galanin receptor protein are insufficient as receptor samples for
conducting a screening for galanin receptor agonist/antagonist.
Consequently, there has been a demand for developing a more
practical method for manufacturing human galanin receptor
proteins.
[0025] If it is possible to screen the agonist/antagonist of
galanin receptor using human galanin receptor protein, it is now
possible to overcome the disadvantage by the use of experimental
animals (for example, the possibility that, due to a difference in
species, compounds which do not achieve an effect to human being
may be obtained) whereby it is expected to conduct a development of
pharmaceutical agents effective to human being in an efficient
manner.
[0026] Yet another object of the present invention is to provide
novel human galanin receptor proteins; partial peptides of the
human galanin receptor protein; novel DNAs which code for the
galanin receptor protein or partial peptide; vectors carrying said
DNA; transformants harboring said vector; cell membrane fractions
obtained from said transformant; processes for producing the human
galanin receptor protein (or its partial peptide); methods for
measuring the physiological actions of galanin using a human
galanin receptor protein-expressing cell, said human galanin
receptor protein; screening methods for a galanin receptor
agonist/antagonist using a human galanin receptor
protein-expressing cell (including the transformant); kits for said
screening; agonists or antagonists, obtained by said screening
method; pharmaceutical compositions containing said agonist or
antagonist; antibodies against said human galanin receptor protein;
immunoassays using said receptor protein or said antibody; and use
of said human galanin receptor protein and encoding DNA.
[0027] In order to achieve the above-mentioned aims, the present
inventors have made extensive investigations. As a result, the
present inventors have succeeded in synthesizing DNA primers
effective in efficiently isolating DNAs (DNA fragments) coding for
G protein coupled receptor proteins by PCR techniques. The present
inventors have succeeded in amplifying cDNA derived from various
cells with said synthetic DNA primer, and have forwarded the
analysis. Thus, the present inventors have succeeded in isolating
novel G protein coupled receptor protein-encoding cDNAs, in
determining the partial structure thereof, and have considered that
the isolated cDNAs are homologous to known G protein coupled
receptors at the nucleotide sequence level and at the amino acid
sequence level and are each coding for a novel galanin receptor
protein. 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
succeeded in sequencing an entire amino acid sequence and entire
nucleotide sequence of said galanin receptor protein.
[0028] The present inventors have found that, when said receptor
protein expressed by a suitable means is used, an agonist or an
antagonist to said receptor protein can be screened in vivo or from
natural or nonnatural compounds by a receptor protein binding
experiment or by a measurement of intracellular second messenger as
an index. The present inventors have further found that said
agonist and antagonist can be developed as preventive and
therapeutic agents for the diseases or symptoms related to or
caused by galanin.
[0029] The present inventors have furthermore found that the
glucose or forskolin-dependent insulin secretion in cells
expressing said galanin receptor protein is inhibited by galanin.
That has been a finding for the first time. Depending upon said
finding, the present inventors have found an easy and simple method
for measuring the activity of galanin and galanin antagonist. At
the same time, the present inventors have also found that the cell
membrane fractions of cells expressing said galanin receptor
protein contain large amount of galanin receptors and succeeded in
establishing a screening for galanin receptor agonist/antagonist
using the cell membrane fractions thereof.
[0030] For example, the present inventors have amplified G protein
coupled receptor protein-encoding cDNA derived from mouse
pancreatic .beta.-cell strain MIN6 using a synthetic DNA primer for
more effective isolation thereof, whereby its analysis has been
carried out.
[0031] As a result thereof, the present inventors have succeeded in
isolating the mouse-derived cDNA fragment which codes for a novel G
protein coupled receptor protein and in elucidating its partial
structure. In said mouse-derived G protein coupled receptor
protein, there are similarities (homologies) at DNA and amino acid
levels to the known G protein coupled receptor and, therefore, it
is believed that it codes for a novel receptor protein exhibiting
an expressing function in mouse pancreas.
[0032] The present inventors further continued their studies and
have succeeded in cloning cDNA having a full-length translation
unit and in analyzing an entire amino acid sequence and an entire
nucleotide sequence of said receptor protein. Since said
mouse-derived G protein coupled receptor protein has a high
homology at DNA and amino acid levels to the human-derived galanin
receptor protein (Proceedings of National Academy of Sciences,
U.S.A., 91, 9780-9783, 1994), it has been found that said
mouse-derived G protein coupled receptor protein is identical with
a mouse-derived galanin receptor protein.
[0033] Furthermore, the present inventors have newly found that the
glucose or forskolin-dependent insulin secretion of MIN6 cells is
inhibited by galanin. Based upon said finding, the present
inventors have found an easy and simple method for measuring the
activity of galanin and galanin antagonist. At the same time, the
present inventors have also found that the cell membrane fractions
of MIN6 cells contain large amount (0.5-1.0 pmol/mg) of galanin
receptor and succeeded in establishing a method of screening
galanin receptor agonist/antagonist using the cell membrane
fractions of MIN6 cells.
[0034] To be more specific, the present inventors have amplified
and cloned novel cDNA fragments derived from mouse pancreatic
.beta. cell strain MIN6 as shown in FIG. 1 by PCR and, from the
result of analysis of their sequence, have clarified that they code
for a novel receptor protein. When said sequence was translated
into amino acid sequences, third, fourth, fifth and sixth
transmembrane domains were confirmed on hyrophobic plots (FIG. 2).
The size of the amplified DNA was about 400 bp which was almost
same as that of the known G protein coupled receptor protein.
[0035] The inventors have retrieved the data base based on, as a
template, the nucleotide sequence of the isolated DNA and observed
36% homology to human-derived somatostatin receptor subtype 4
(JN0605), 30% homology to human-derived somatostatin receptor
subtype 2 (B41795), and 30% homology to rat-derived ligand unknown
receptor (A39297), respectively (FIG. 3), 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" or "Entry Name".
[0036] Moreover, the present inventors have prepared cDNA from the
poly(A).sup.+ RNA fractions extracted from MIN6 cells and have
inserted said cDNA into lambda gt22 phage to prepare a cDNA
library. Further, the present inventors have screened the cDNA
library using, as a probe, the G protein coupled receptor protein
cDNA fragment p3H2-34 obtained by PCR and succeeded in cloning cDNA
which completely codes for the G protein coupled receptor protein
of the present invention. A nucleotide sequence of said cDNA and an
amino acid sequence encoded thereby are given in FIG. 4. A
hydrophobic plotting was conducted based upon said amino acid
sequence and the first, second, third, fourth, fifth, sixth and
seventh transmembrane domains were confirmed (FIG. 5). The G
protein coupled receptor protein of the present invention has 92%
homology at the amino acid level to the known human galanin
receptor protein.
[0037] In another aspect, the present inventors have succeeded, for
example, in cloning a DNA which codes for novel human galanin
receptor protein having an amino acid sequence which differs from
that of known human galanin receptor protein. In the known human
galanin receptor protein, the fifteenth amino acid in its amino
acid sequence is Cys while, in the human galanin receptor protein
of the present invention, the fifteenth amino acid in its amino
acid sequence (SEQ ID NO: 5 and FIGS. 12 & 13) is Trp. In
addition, in the nucleotide sequence of DNA which codes for the
known human galanin receptor protein, the base sequence which codes
for the fifteenth amino acid of said human galanin receptor protein
is .sup.15Cys (TGT) while, in the base sequence of DNA which codes
for the human galanin receptor protein of the present invention,
the base sequence which codes for the fifteenth amino acid in said
human galanin receptor protein is .sup.15Trp (TGG).
[0038] The present inventors have further succeeded in
manufacturing a CHO cell strain which expresses far more amount of
the human galanin receptor protein of the present invention than
the COS cells which express the known human galanin receptor
protein [Habert-Ortoll, E. et al., Proceedings of the National
Academy of Sciences of the U. S. A., 91, 9780-9783 (1994)]. It has
been furthermore found that, when said CHO cell strain of the human
galanin receptor protein of the present invention or partial
peptide thereof is used, it is now possible to screen the human
galanin receptor agonist/antagonist in an effective and reliable
manner. Based upon those findings, the present inventors have
continued various investigations and, as a result, they have
achieved the present invention.
[0039] Accordingly, one aspect of the present invention is
[0040] (1) a galanin receptor protein comprising an amino acid
sequence selected from the group consisting of an amino acid
sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5
and substantial equivalents thereto, or a salt thereof;
[0041] (2) the receptor protein according to the above (1), which
is produced by a transformant CHO cell;
[0042] (3) a DNA which comprises a nucleotide sequence coding for a
galanin receptor protein of the above (1);
[0043] (4) a vector comprising the DNA according to the above
(3);
[0044] (5) a transformant carrying the vector according to the
above (4);
[0045] (6) the transformant according to the above (5), wherein the
host cell is a CHO cell;
[0046] (7) a process for producing a galanin receptor protein
according to the above (1), which comprises culturing a
transformant of the above (5) under conditions suitable to express
said galanin receptor protein;
[0047] (8) a screening method for an agonist or antagonist of a
galanin receptor protein according to the above (1), which
comprises carrying out a comparison between:
[0048] (i) at least one case where galanin is contacted with at
least one component selected from the group consisting of a galanin
receptor protein according to the above (1), a partial peptide
thereof and a mixture thereof, and
[0049] (ii) at least one case where galanin together with a
compound to be tested is contacted with at least one component
selected from the group consisting of a galanin receptor protein
according to the above (1), a partial peptide thereof and a mixture
thereof;
[0050] (9) a kit for the screening of one or more agonists or
antagonists to a galanin receptor protein according to the above
(1), which comprises at least one component selected from the group
consisting of a galanin receptor protein according to the above
(1), a partial peptide thereof and a mixture thereof; and
[0051] (10) an agonist or antagonist of a galanin receptor, which
is obtained by the screening method according to the above (8) or
by the kit according to the above (9).
[0052] Another aspect of the present invention is
[0053] (11) a mouse-derived galanin receptor protein comprising an
amino acid sequence selected from the group consisting of an amino
acid sequence represented by SEQ ID NO: 1 and substantial
equivalents thereto; or a salt thereof;
[0054] (12) a mouse-derived galanin receptor protein according to
the above (11), which comprises an amino acid sequence selected
from the group consisting of an amino acid sequence represented by
SEQ ID NO: 2 and substantial equivalents thereto; or a salt
thereof;
[0055] (13) a human galanin receptor protein comprising an amino
acid sequence selected from the group consisting of an amino acid
sequence represented by SEQ ID NO: 5 and substantial equivalents
thereto; or a salt thereof;
[0056] (14) a partial peptide of a galanin receptor protein
according to the above (1), or a salt thereof;
[0057] (15) a partial peptide of a mouse-derived galanin receptor
protein according to the above (11) or (12), or a salt thereof;
[0058] (16) a partial peptide of a human galanin receptor protein
according to the above (13), or a salt thereof;
[0059] (17) a DNA which comprises a nucleotide sequence coding for
a mouse-derived galanin receptor protein of the above (11) or
(12);
[0060] (18) a DNA which comprises a nucleotide sequence coding for
a human galanin receptor protein of the above (13);
[0061] (19) a DNA of the above (17) comprising a nucleotide
sequence represented by SEQ ID NO: 3; 1
[0062] (20) a DNA of the above (17) comprising a nucleotide
sequence represented by SEQ ID NO: 4;
[0063] (21) a DNA of the above (18) comprising a nucleotide
sequence represented by SEQ ID NO: 6;
[0064] (22) a vector comprising a DNA according to the above
(17);
[0065] (23) a vector comprising a DNA according to the above
(18);
[0066] (24) a transformant (including a transfectant) carrying a
vector of the above (22);
[0067] (25) a transformant (including a transfectant) carrying a
vector of the above (23);
[0068] (26) a process for producing a mouse-derived galanin
receptor protein or a salt thereof according to the above (11),
which comprises culturing a transformant of the above (24) to
produce said galanin receptor on the membrane of the
transformant;
[0069] (27) a process for producing a human galanin receptor
protein or a salt thereof according to the above (13), which
comprises culturing a transformant of the above (25) under
conditions to express said galanin receptor;
[0070] (28) a cell or membrane fraction containing a galanin
receptor protein according to the above (1);
[0071] (29) a cell or membrane fraction containing a mouse-derived
galanin receptor protein according to the above (11) or (12);
[0072] (30) a cell or membrane fraction containing a human galanin
receptor protein according to the above (13);
[0073] (31) a screening method for a galanin receptor agonist
and/or antagonist, which comprises using a galanin receptor protein
according to the above (1), a partial peptide according to the
above (14) or a cell or membrane fraction according to the above
(28);
[0074] (32) a screening method for a mouse-derived galanin receptor
agonist and/or antagonist, which comprises using a mouse-derived
galanin receptor protein according to the above (11) or (12), a
partial peptide according to the above (15) or a cell or membrane
fraction according to the above (29);
[0075] (33) a screening method for a human galanin receptor agonist
and/or antagonist, which comprises using a human galanin receptor
protein according to the above (13), a partial peptide according to
the above (16) or a cell or membrane fraction according to the
above (30);
[0076] (34) a screening method for a galanin receptor agonist
and/or antagonist, which comprises carrying out a comparison
between:
[0077] (i) at least one case where galanin is contacted with at
least one component selected from the group consisting of a galanin
receptor protein or a salt thereof according to the above (1), a
partial peptide or a salt thereof according to the above (14), a
cell or membrane fraction according to the above (28), and a
mixture thereof, and
[0078] (ii) at least one case where galanin together with a sample
(including a compound) to be tested is contacted with at least one
component selected from the group consisting of a galanin receptor
protein or a salt thereof according to the above (1), a partial
peptide or a salt thereof according to the above (14), a cell or
membrane fraction according to the above (28), and a mixture
thereof;
[0079] (35) a screening method for a mouse-derived galanin receptor
agonist and/or antagonist, which comprises carrying out a
comparison between:
[0080] (i) at least one case where galanin is contacted with at
least one component selected from the group consisting of a
mouse-derived galanin receptor protein or a salt thereof according
to the above (11), a partial peptide or a salt thereof according to
the above (15), and a mixture thereof, and
[0081] (ii) at least one case where galanin together with a sample
(including a compound) to be tested is contacted with at least one
component selected from the group consisting of a mouse-derived
galanin receptor protein or a salt thereof according to the above
(11), a partial peptide or a salt thereof according to the above
(15), and a mixture thereof;
[0082] (36) a screening method for a human galanin receptor agonist
and/or antagonist, which comprises carrying out a comparison
between:
[0083] (i) at least one case where galanin is contacted with at
least one component selected from the group consisting of a human
galanin receptor protein or a salt thereof according to the above
(13), a partial peptide or a salt thereof according to the above
(16), a cell or membrane fraction according to the above (30), and
a mixture thereof, and
[0084] (ii) at least one case where galanin together with a sample
(including a compound) to be tested is contacted with at least one
component selected from the group consisting of a human galanin
receptor protein or a salt thereof according to the above (13), a
partial peptide or a salt thereof according to the above (16), a
cell or membrane fraction according to the above (30), and a
mixture thereof;
[0085] (37) a kit for the screening of a galanin receptor agonist
and/or antagonist, which comprises at least one component selected
from the group consisting of a galanin receptor protein or a salt
thereof according to the above (1), a partial peptide or a salt
thereof according to the above (14), a cell or membrane fraction
according to the above (28), and a mixture thereof;
[0086] (38) a kit for the screening of a mouse-derived galanin
receptor agonist and/or antagonist, which comprises at least one
component selected from the group consisting of a mouse-derived
galanin receptor protein or a salt thereof according to the above
(11) or (12), a partial peptide or a salt thereof according to the
above (15), a cell or membrane fraction according to the above
(29), and a mixture thereof;
[0087] (39) a kit for the screening of a human galanin receptor
agonist and/or antagonist, which comprises at least one component
selected from the group consisting of a galanin receptor protein or
a salt thereof according to the above (13), a partial peptide or a
salt thereof according to the above (16), a cell or membrane
fraction according to the above (30), and a mixture thereof;
[0088] (40) a galanin receptor agonist and/or antagonist, obtained
by a method according to any of the above (31) to (36) or a kit
according to any of the above (37) to (39);
[0089] (41) a galanin receptor agonist and/or antagonist, obtained
by a method according to the above (32) or (35) or a kit according
to the above (38);
[0090] (42) a galanin receptor agonist and/or antagonist, obtained
by a method according to the above (33) or (36) or a kit according
to the above (39);
[0091] (43) a pharmaceutical composition comprising an effective
amount of the galanin receptor agonist according to (40);
[0092] (44) a pharmaceutical composition comprising an effective
amount of the galanin receptor agonist according to (41);
[0093] (45) a pharmaceutical composition comprising an effective
amount of the galanin receptor agonist according to (42);
[0094] (46) a pharmaceutical composition comprising an effective
amount of the galanin receptor antagonist according to (40);
[0095] (47) a pharmaceutical composition comprising an effective
amount of the galanin receptor antagonist according to (41);
[0096] (48) a pharmaceutical composition comprising an effective
amount of the galanin receptor antagonist according to (42);
[0097] (49) a pharmaceutical composition according to (43) which is
an inhibitor for acetylcholine liberation, an inhibitor for insulin
secretion, a stimulant for growth hormone secretion, an inhibitor
for learning behavior or satiety;
[0098] (50) a pharmaceutical composition according to (46) which is
an agent for promoting the acetylcholine liberation, an agent for
inhibiting the growth hormone secretion, an agent for promoting the
insulin secretion, an agent for promoting the learning behavior or
an agent for promoting satiety;
[0099] (51) an antibody against at least one component selected
from the group consisting of a galanin receptor protein or a salt
thereof according to the above (1) and a partial peptide or a salt
thereof according to the above (14);
[0100] (52) an antibody against at least one component selected
from the group consisting of a mouse-derived galanin receptor
protein or a salt thereof according to the above (11) or (12) and a
partial peptide or a salt thereof according to the above (15);
and
[0101] (53) an antibody against at least one component selected
from the group consisting of a human galanin receptor protein or a
salt thereof according to the above (13) and a partial peptide or a
salt thereof according to the above (16).
[0102] To be more specific, the present invention relates to the
following:
[0103] (54) a method of screening a galanin receptor agonist or
antagonist, characterized in that, the binding amount of the
labeled galanin with the galanin receptor protein or its salt
according to (1) (e.g., the mouse-derived galanin receptor protein
or its salt according to (11), etc.) or with the partial peptide or
its salt according to (14) (e.g., the partial peptide of the
mouse-derived galanin receptor protein or its salt according to
(15), etc.) is measured in the case where the labeled galanin is
contacted with the galanin receptor protein or its salt according
to (1) (e.g., the mouse-derived galanin receptor protein or its
salt according to (11), etc.) or with the partial peptide or its
salt according to (14) (e.g., the partial peptide of the
mouse-derived galanin receptor protein or its salt according to
(15), etc.) and also in the case where the labeled galanin and the
test compound are contacted with the galanin receptor protein or
its salt according to (1) (e.g., the mouse-derived galanin receptor
protein or its salt according to (11), etc.) or with the partial
peptide or its salt according to (14) (e.g., the partial peptide of
the mouse-derived galanin receptor protein or its salt according to
(15), etc.) and the comparison is made between them;
[0104] (55) a method of screening a galanin receptor agonist or
antagonist, characterized in that, the labeled galanin is contacted
with the cells (except mouse-derived MIN6 cells [FERM BP-4954])
containing the galanin receptor protein according to (1) (e.g., the
mouse-derived galanin receptor protein according to (11), etc.) and
the labeled galanin and the test compound are contacted with the
cells (except mouse-derived MIN6 cells [FERM BP-4954]) containing
the galanin receptor protein according to (1) (e.g., the
mouse-derived galanin receptor protein according to (11), etc.) and
the binding amounts of the labeled galanin with said cells in both
cases are measured and compared;
[0105] (56) a method of screening a galanin receptor agonist or
antagonist, characterized in that, the labeled galanin is contacted
with the cell membrane fractions of cells (except mouse-derived
MIN6 cells [FERM BP-4954]) containing the galanin receptor protein
according to (1) (e.g., the mouse-derived galanin receptor protein
according to (11), etc.) and the labeled galanin and the test
compound are contacted with the cell membrane fraction of cells
(except mouse-derived MIN6 cells [FERM BP-4954]) containing the
galanin receptor protein according to (1) (e.g., the mouse-derived
galanin receptor protein according to (11), etc.) and the binding
amounts of the labeled galanin with the membrane fractions of said
cells in both cases are measured and compared;
[0106] (57) a method of screening a galanin receptor agonist or
antagonist, characterized in that, the labeled galanin is contacted
with the galanin receptor protein according to (1) (e.g., the
mouse-derived galanin receptor protein according to (11), etc.)
expressed in cell membranes of the transformant according to (5)
(e.g., the mouse-derived galanin receptor protein-expressible
transformant according to (24), etc.) by culturing said
transformant and the labeled galanin and the test compound are
contacted with the galanin receptor protein according to (1) (e.g.,
the mouse-derived galanin receptor protein according to (11), etc.)
expressed in cell membranes of the transformant according to (5)
(e.g., the mouse-derived galanin receptor protein-expressible
transformant according to (24), etc.) by culturing said
transformant and the binding amounts of the labeled galanin with
said galanin receptor in both cases are measured and compared;
[0107] (58) a method of screening a galanin receptor agonist or
antagonist, characterized in that, galanin is contacted with the
cells (except the mouse-derived MIN6 cells [FERM BP-4954])
containing the galanin receptor protein according to (1) (e.g., the
mouse-derived galanin receptor protein according to (11), etc.) and
galanin and the test compound are contacted with the cells (except
the mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin
receptor protein according to (1) (e.g., the mouse-derived galanin
receptor protein according to (11), etc.) and the resulting
cell-stimulating activities via the galanin receptor protein in
both cases are measured and compared;
[0108] (59) a method of screening a galanin receptor agonist or
antagonist, characterized in that, galanin is contacted with the
galanin receptor protein according to (1) (e.g., the mouse-derived
galanin receptor protein according to (11), etc.) expressed in cell
membranes of the transformant according to (5) (e.g., the
mouse-derived galanin receptor protein-expressible transformant
according to (24), etc.) by culturing said transformant and galanin
and the test compound are contacted with the galanin receptor
protein according to (1) (e.g., the mouse-derived galanin receptor
protein according to (11), etc.) expressed in cell membranes of the
transformant according to (5) (e.g., the mouse-derived galanin
receptor protein-expressible transformant according to (24), etc.)
by culturing said transformant and the resulting cell stimulating
activities via the galanin receptor protein are measured and
compared;
[0109] (60) a method of screening according to (58) or (59) in
which the cell-stimulating activity is an activity which
accelerates or inhibits arachidonic acid liberation, acetylcholine
liberation, intracellular Ca.sup.2+ liberation, intracellular cAMP
production, intracellular cGMP production, inositol phosphate
production, cell membrane potential variation, phosphorylation of
intracellular protein, activation of c-fos, a decrease in pH,
insulin secretion, etc. (especially the activity which accelerates
or inhibits the intracellular cAMP production or insulin
secretion);
[0110] (61) a galanin receptor agonist or antagonist obtained by a
screening methods according to any of (31), (34) (e.g., (32), (35),
etc.) and (54) to (60);
[0111] (62) an agent for inhibiting acetylcholine liberation,
insulin secretion, learning behavior or feeling of satiety after a
meal characterized in containing the galanin receptor agonist
according to (61);
[0112] (63) an agent for accelerating acetylcholine liberation,
insulin secretion, behavior of learning or feeling of fulfillment
after a meal characterized in containing the galanin receptor
antagonist according to (61);
[0113] (64) an intelligence tropic agent or a remedy for obesity or
for diabetes characterized in containing the galanin receptor
antagonist according to (40) (e.g., (41), etc.) or (61);
[0114] (65) a kit for screening according to (37) (e.g., (38),
etc.), characterized in comprising a cell containing the galanin
receptor protein according to (1) (e.g., the mouse-derived galanin
receptor protein according to (11), etc.);
[0115] (66) a kit for screening according to (37) (e.g., (38),
etc.), characterized in containing the membrane fractions of the
cells which contain the galanin receptor protein according to (1)
(e.g., the mouse-derived galanin receptor protein according to
(11), etc.);
[0116] (67) a galanin receptor agonist or antagonist obtained by
the use of the kit for screening according to (37) (e.g., (38),
etc.), (65) or (66);
[0117] (68) an agent for inhibiting acetylcholine liberation,
insulin secretion, learning behavior or feeling of fulfillment
after a meal characterized in containing the galanin receptor
agonist according to (67);
[0118] (69) an agent for accelerating acetylcholine liberation,
insulin secretion, behavior of learning or feeling of fulfillment
after a meal characterized in containing the galanin receptor
antagonist according to (67);
[0119] (70) an intelligence tropic agent or a remedy for obesity or
for diabetes characterized in containing the galanin receptor
antagonist according to (40) (e.g., (41), etc.) or (69); and
[0120] (71) a method of quantitative determination of the galanin
receptor protein or its salt according to (1) (e.g., the
mouse-derived galanin receptor protein or its salt according to
(11), etc.) or the partial peptide or its salt according to (14)
(e.g., the partial peptide of the mouse-derived galanin receptor
protein or its salt according to (15), etc.), characterized in
that, the antibody according to (51) (e.g., the antibody according
to (52), etc.) is contacted with the galanin receptor protein or
its salt according to (1) (e.g., the mouse-derived galanin receptor
protein or its salt according to (11), etc.) or the partial peptide
or its salt according to (14) (e.g., the partial peptide of the
mouse-derived galanin receptor protein or its salt according to
(15), etc.).
[0121] The present invention furthermore provides the
following:
[0122] (72) a method of measuring the physiological activity of
galanin, characterized in that, the biological activity of the
mouse-derived MIN6 cells when the mouse-derived MIN cells (FERM
BP-4954) or the cell membrane fractions thereof are contacted with
galanin;
[0123] (73) a method of screening a galanin receptor agonist or
antagonist, characterized in that, a comparison is made between the
cases where (i) galanin is contacted with the mouse-derived MIN6
cells (FERM BP-4954) or cell membrane fractions thereof and (ii)
galanin and the test compound are contacted with the mouse-derived
MIN6 cells (FERM BP-4954) or cell membrane fractions thereof;
[0124] (74) a kit for screening for a galanin receptor agonist or
antagonist characterized in containing the mouse-derived MIN6 cells
(FERM BP-4954) or cell membrane fractions thereof;
[0125] (75) a galanin receptor agonist or antagonist obtained by
the method for screening according to (73) or by the kit for
screening according to (74);
[0126] (76) an inhibitor for liberation of acetylcholine, an
inhibitor for secretion of insulin, an inhibitor for the behavior
of learning or an inhibitor for feeling satiety after a meal
characterized in containing the galanin receptor agonist according
to (75);
[0127] (77) an accelerator for liberation of acetylcholine, an
accelerator for secretion of insulin, an accelerator for the
behavior of learning or an accelerator for feeling satiety after a
meal characterized in containing the galanin receptor antagonist
according to (75);
[0128] (78) a method for screening a galanin receptor agonist or
antagonist, characterized in that, the labeled galanin is contacted
with the mouse-derived MIN6 cells (FERM BP-4954) and the labeled
galanin and the test compound are contacted with the mouse-derived
MIN6 cells (FERM BP-4954) and the binding amounts of the labeled
galanin with said mouse-derived galanin MIN6 cells in both cases
are measured and compared;
[0129] (79) a method of screening a galanin receptor agonist or
antagonist, characterized in that, the labeled galanin is contacted
with the cell membrane fractions of the mouse-derived MIN6 cells
(FERM BP-4954) and the labeled galanin and the test compound are
contacted with the cell membrane fractions of the mouse-derived
MIN6 cells (FERM BP-4954) and the binding amounts of the labeled
galanin with said membrane fractions of the mouse-derived MIN6
cells in both cases are measured and compared;
[0130] (80) a method of screening a galanin receptor agonist or
antagonist, characterized in that, galanin is contacted with the
mouse-derived MIN6 cells (FERM BP-4954) and galanin and the test
compound are contacted with the mouse-derived MIN6 cells (FERM
BP-4954) and the resulting cell-stimulating activities via the
mouse-derived galanin receptor (especially the activity of
secretion of insulin from MIN6 cells or the activity of inhibiting
or accelerating the CAMP production in the MIN6 cells) in both
cases are measured and compared;
[0131] (81) a method of screening according to the above (80) in
which the cell-stimulating activity is an activity for accelerating
or inhibiting the arachidonic acid liberation, acetylcholine
liberation, intracellular Ca.sup.2+ liberation, intracellular cAMP
production, intracellular cGMP production, inositol phosphate
production, cell membrane potential variation, phosphorylation of
intracellular protein, activation of c-fos, a decrease in pH,
secretion of insulin, etc. (especially the activity which
accelerates or inhibits the intracellular cAMP production or the
insulin secretion);
[0132] (82) a galanin receptor agonist or antagonist obtained by a
method of screening according to any of (73) and (78) to (81);
[0133] (83) an inhibitor for liberation of acetylcholine, an
inhibitor for secretion of insulin, an inhibitor for the behavior
of learning and an inhibitor for feeling fulfillment after a meal
characterized in containing the galanin receptor agonist according
to (75) or (82);
[0134] (84) an accelerator for liberation of acetylcholine, an
accelerator for secretion of insulin, an accelerator for the
behavior of learning and an accelerator for feeling satiety after a
meal characterized in containing the galanin receptor antagonist
according to (75) or (82); and
[0135] (85) an intelligence tropic agent or a remedy for obesity or
for diabetes characterized in containing the galanin receptor
antagonist according to (75) or (82).
[0136] Yet another aspect of the present invention is:
[0137] (86) a partial peptide according to (16) in which the
partial peptide is a region exposed outside the cell membrane of
the human galanin receptor protein molecule according to (13);
[0138] (87) a vector according to (23) in which the vector is an
expression vector for the human galanin receptor protein as
indicated by pTS863;
[0139] (88) a transformant according to (25) in which the host cell
is a CHO cell;
[0140] (89) a CHO cell according to (88) in which the CHO cell is
CHO/pTS863-5 or CHO/pTS863-7; `
[0141] (90) a cell or cell membrane fraction thereof according to
(30) in which the cell is CHO/pTS863-5 or CHO/pTS863-7;
[0142] (91) a method of screening the galanin receptor agonist or
antagonist according to (33), which comprises carrying out a
comparison between the cases where (i) galanin is contacted with
the human galanin receptor protein or salt thereof according to
(13) or with the partial peptide or salt thereof according to (16)
and (ii) galanin and the test compound are contacted with the human
galanin receptor protein or salt thereof according to (13) or with
the partial peptide or salt thereof according to (16);
[0143] (92) a method of screening the galanin receptor agonist or
antagonist according to (33), which comprises measuring and
comparing the binding amounts of the labeled galanin to said human
galanin receptor protein, partial peptide thereof or salt thereof
in the cases where (i) the labeled galanin is contacted with the
human receptor protein or salt thereof according to (13) or with
the partial peptide or salt thereof according to (16) and (ii) the
labeled galanin and the test compound are contacted with the human
galanin receptor protein or salt thereof according to (13) or with
the partial peptide or salt thereof according to (16);
[0144] (93) a method of screening the galanin receptor agonist or
antagonist according to (33), which comprises carrying out a
comparison between the cases where (i) the labeled galanin is
contacted with the cell or cell membrane fraction thereof according
to (30) and (ii) the labeled galanin and the test compound are
contacted with the cell or the cell membrane fraction thereof
according to (30);
[0145] (94) a method of screening the galanin receptor agonist or
antagonist according to (33), which comprises measuring and
comparing the the binding amounts of the labeled galanin with said
cell or cell membrane fraction thereof in the cases where (i) the
labeled galanin is contacted with the cell or the cell membrane
fraction thereof according to (30) and (ii) the labeled galanin and
the test compound are contact with the cell or the cell membrane
fraction thereof according to (30);
[0146] (95) a method of screening the galanin receptor agonist or
antagonist according to (33), which comprises measuring and
comparing cell stimulating activities via the recombinant human
galanin receptor (for example, activities which promote or inhibit
the opening of K.sup.2+ channel, closing of N type Ca.sup.+
channel, liberation of arachidonic acid, liberation of
acetylcholine, variations in intracellular Ca.sup.2+ concentration,
inhibition of intracellular cAMP production, production of inositol
phosphate, cell membrane potential changes, phosphorylation of
intracellular protein, activation of c-fos, decrease in pH, cell
migration activity, secretion of hormones, activation of G protein
and cell promulgation, etc.) in the cases where (i) galanin is
contacted with the cell or the cell membrane fraction thereof
according to (30) and (ii) galanin and the test compound are
contacted with the cell or the cell membrane fraction thereof
according to (30);
[0147] (96) a pharmaceutical composition according to (45) for
inhibiting liberation of acetylcholine, inhibiting secretion of
insulin, stimulating secretion of growth hormones, inhibiting
learning behavior or inhibiting satiety;
[0148] (97) a pharmaceutical composition according to (45) which is
a prophylactic or therapeutic agent for schizophrenic illness or
stomach ulcer or is a sedative;
[0149] (98) a pharmaceutical composition according to (48) for
promoting the acetylcholine liberation, inhibiting the growth
hormone secretion, promoting the insulin secretion, promoting the
learning behavior or promoting satiety;
[0150] (99) a pharmaceutical composition according to (48) which is
a prophylactic and therapeutic agent for diabetes, Alzheimer's
disease or dementia;
[0151] (100) a preventive and therapeutic agent containing the DNA
according to (18) for a galanin receptor protein-deficient disease;
and
[0152] (101) a preventive and therapeutic agent according to (100)
in which the galanin receptor protein-deficient disease is
diabetes, Alzheimer's disease or dementia.
[0153] Yet another aspect of the present invention is:
[0154] (102) a galanin receptor protein according to the above (1)
which comprises
[0155] an amino acid sequence selected from the group consisting of
an amino acid sequence represented by SEQ ID NO: 1, amino acid
sequences wherein one or more amino acid residues (preferably from
1 to 30 amino acid residues, more preferably from 1 to 10 amino
acid residues) are deleted from the amino acid sequence of SEQ ID
NO: 1, amino acid sequences wherein one or more amino acid residues
(preferably from 1 to 30 amino acid residues, more preferably from
1 to 10 amino acid residues) are added to the amino acid sequence
of SEQ ID NO: 1, and amino acid sequences wherein one or more amino
acid residues (preferably from 1 to 30 amino acid residues, more
preferably from 1 to 10 amino acid residues) in the amino acid
sequence of SEQ ID NO: 1 are substituted with one or more other
amino acid residues, or a salt thereof;
[0156] (103) a galanin receptor protein according to the above (1)
which comprises
[0157] an amino acid sequence selected from the group consisting of
an amino acid sequence represented by SEQ ID NO: 2, amino acid
sequences wherein one or more amino acid residues (preferably from
1 to 30 amino acid residues, more preferably from 1 to 10 amino
acid residues) are deleted from the amino acid sequence of SEQ ID
NO: 2, amino acid sequences wherein one or more amino acid residues
(preferably from 1 to 30 amino acid residues, more preferably from
1 to 10 amino acid residues) are added to the amino acid sequence
of SEQ ID NO: 2, and amino acid sequences wherein one or more amino
acid residues (preferably from 1 to 30 amino acid residues, more
preferably from 1 to 10 amino acid residues) in the amino acid
sequence of SEQ ID NO: 2 are substituted with one or more other
amino acid residues, or a salt thereof; and
[0158] (104) a galanin receptor protein according to the above (1)
which comprises
[0159] an amino acid sequence selected from the group consisting of
an amino acid sequence represented by SEQ ID NO: 5, amino acid
sequences wherein one or more amino acid residues (preferably from
1 to 30 amino acid residues, more preferably from 1 to 10 amino
acid residues) are deleted from the amino acid sequence of SEQ ID
NO: 5, amino acid sequences wherein one or more amino acid residues
(preferably from 1 to 30 amino acid residues, more preferably from
1 to 10 amino acid residues) are added to the amino acid sequence
of SEQ ID NO: 5, and amino acid sequences wherein one or more amino
acid residues (preferably from 1 to 30 amino acid residues, more
preferably from 1 to 10 amino acid residues) in the amino acid
sequence of SEQ ID NO: 5 are substituted with one or more other
amino acid residues, or a salt thereof.
[0160] Yet another aspect of the present invention is:
[0161] (105) a process according to the above (27), wherein said
transformant is produced by transforming a host cell, CHO cell,
with a vector comprising a nucleotide sequence coding for a
human-derived galanin receptor protein;
[0162] (106) a pharmaceutical composition comprising an effective
amount of an agonist according to the above (40) or a salt thereof
in admixture with a pharmaceutically acceptable diluent, carrier or
excipient;
[0163] (107) a pharmaceutical composition according to the above
(106), which inhibits liberation of acetylcholine, secretion
insulin, learning action, or satiety;
[0164] (108) a pharmaceutical composition comprising an effective
amount of an antagonist according to the above (40) or a salt
thereof in admixture with a pharmaceutically acceptable diluent,
carrier or excipient;
[0165] (109) a pharmaceutical composition according to the above
(108), which promotes liberation of acetylcholine, secretion
insulin, learning action, or satiety; and
[0166] (110) a transformant CHO cell capable of expressing
human-derived galanin receptor proteins.
[0167] 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
[0168] FIG. 1 is the partial nucleotide sequence of the novel
receptor protein cDNA clone, p3H2-34, obtained from mouse
pancreatic .beta.-cell line, 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
[0169] FIG. 2 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 1, 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.
[0170] FIG. 3 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.
[0171] FIG. 4 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
[0172] FIG. 5 is the hydrophobicity plotting profile, prepared
based upon the amino acid sequence shown in FIG. 4, 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.
[0173] FIG. 6 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.
[0174] FIG. 7 is the plotting profile of the binding amounts (PMB)
of labeled galanin to MIN 6 cells against the concentrations of
standard porcine galanin, rat galanin, galanin (1-16) partial
peptide or galanin antagonist (galantide).
[0175] FIG. 8 is the plotting profile of the amount of insulin
secretion from MIN 6 cells against the amount of galanin.
[0176] FIG. 9 is the plotting profile of the amount of insulin
secretion from MIN 6 cells against the amount of galanin.
[0177] FIG. 10 is the slotting profile of the amount of
intracellular cAMP in MIN 6 cells against the amount of
galanin.
[0178] FIG. 11 is the nucleotide sequence and deduced amino acid
sequence (1st to 135th) of the human galanin receptor protein
obtained in Example 11.
[0179] FIG. 12 is the nucleotide sequence and deduced amino acid
sequence (136th to 349th) of the human galanin receptor protein
obtained in Example 11.
[0180] FIG. 13 is the construction of expression plasmid, pTS863,
containing the human galanin receptor protein cDNA obtained in
Example 12. The striped region of expression plasmid, pTS863,
indicates the human galanin receptor protein cDNA. DHFR; dhfr gene
and Amp.sup.r; ampicillin resistant gene.
[0181] FIG. 14 depicts a profile of northern blot analysis of
poly(A).sup.+ RNA from mouse tissues, Neuro-2a, and MIN6 cells with
p3H2-34. Poly(A).sup.+ RNA (5 .mu.g/lane) was denatured by
treatment with glyoxal and then electrophoresed on a 1.2% agarose
gel. The RNAs were transferred onto a nitrocellulose filter and
hybridized with a .sup.32P-labeled cDNA insert of p3H2-34 as a
probe. Lanes: 1, Neuro-2a; 2, MIN6; 3, intestinal smooth muscle; 4,
testis; 5, pancreas; 6, kidney; 7, liver; 8, heart; 9, lung; 10,
spleen; 11, thymus; 12, brain. Arrowheads indicate the sizes of the
molecular weight markers.
[0182] FIG. 15 illustrates a comparison of porcine
[.sup.125I]galanin binding to CHO cells transformed with or without
a mouse galanin receptor cDNA. CHO-MGR20 cells transformed with a
full-length translation unit or mock transformed CHO cells were
incubated with [.sup.125I]galanin (100 pM at final concentration)
at 37.degree. C. for 1 hr in the absence (open column) or presence
(closed column) of unlabeled porcine galanin (1 .mu.M at final
concentration). The amounts of [.sup.125I]galanin bound are
represented as a percentages of the radioactivity remaining on the
cells after washing. Values indicated are mean.+-.S.E.M. in
triplicate.
[0183] FIG. 16 is a primary structure comparison of mouse and human
galanin receptors. Identical residues are indicated by the vertical
line. Putative membrane spanning domains I-VII are boxed.
[0184] FIG. 17 depicts a profile of Scatchard analysis of
[.sup.125I] galanin binding to the membranes from CHO cells
transformed with the mouse galanin receptor cDNA. Membrane
fractions (1 .mu.g of protein) were incubated with increasing
concentrations of porcine [.sup.125I] galanin for 75 min at
25.degree. C. The results shown are from one representative
experiment performed in triplicate assays. Each symbol represents
the mean value.+-.S.E.M. The values for K.sub.d and B.sub.MAX were
45 pM and 5 pmol/mg protein, respectively. B, [.sup.125I] galanin
bound (pmol/mg protein), B/F, bound to free ratio (pmol/mg
protein.cndot.nM).
[0185] FIG. 18 illustrates a result of competitive experiments on
the binding of porcine [.sup.125I] galanin to mouse galanin
receptor. Competitions to the porcine [.sup.125I] galanin (100 pM
at final concentrations) bindings were examined with unlabeled
porcine (.DELTA.), rat (.circle-solid.), human (.box-solid.)
galanins, galanin (1-16) (.largecircle.), and M15
(.tangle-solidup.). Membrane fractions (1 .mu.g of protein) were
incubated with the ligands for 75 min at 25.degree. C. The amounts
of [.sup.125I] galanin bound were expressed as percentages against
the control. Each symbol represents the mean value.+-.S.E.M. of the
triplicate assays. IC.sub.50 values were 0.25.+-.0.03 nM (porcine
galanin), 0.25.+-.0.01 nM (rat galanin), 0.43.+-.0.03 nM (human
galanin), 0.83.+-.0.01 nM (M15), and 3.6.+-.0.04 nM
[galanin-(1-16)], respectively.
[0186] FIG. 19 shows a galanin receptor-mediated inhibition of
forskolin-stimulated cAMP production. CHO-MGR20 or mock transformed
CHO cells were incubated with forskolin (10 .mu.M) and porcine
galanin (0.1 .mu.M) at 37.degree. C. for 30 min. The reaction was
terminated by extracting the cells with ice-cold ethanol. The
amounts of intracellular cAMP were quantitated by EIA. Values
indicated are mean.+-.S.E.M. in triplicate assays.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0187] According to the present invention, galanin receptor
proteins and partial peptides thereof or salts thereof; DNAs
comprising a DNA coding for said galanin receptor protein or its
partial peptide; vectors carrying said DNA; transformants harboring
said vector; cell membrane fractions obtained from said
transformant; processes for producing said receptor protein or its
partial peptide, or a salt thereof; methods for measuring the
physiological actions of galanin using the galanin receptor protein
(including a cell membrane fraction containing the receptor
protein) or a galanin receptor protein-expressing cell (including
the transformant); screening methods for a galanin receptor
agonist/antagonist using the galanin receptor protein or a galanin
receptor protein-expressing cell (including the transformant); kits
for said screening; agonists or antagonists, obtained by said
screening method; pharmaceutical compositions containing said
agonist or antagonist; antibodies against said receptor protein;
immunoassays using said receptor protein or said antibody; use of
said receptor protein and encoding DNA; etc. may be successfully
provided. For example, template DNAs coding for part or all of the
polypeptide sequence of galanin receptor protein, can be
successfully obtained and various DNA sequences encoding part or
all of the polypeptide sequence of galanin receptor protein can be
isolated and characterized. Further, galanin receptor proteins,
partial peptides derived from the galanin receptor protein,
modified derivatives or analogues thereof, and salts thereof are
recognized, predicted, deduced, produced, expressed, isolated and
characterized. More specifically, DNA sequences comprising each a
nucleotide sequence indicated by a SEQ ID NO selected from the
group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6
have been isolated and characterized. Galanin receptor proteins
comprising each part or all of an amino acid sequence selected from
the group consisting of an amino acid sequence represented by SEQ
ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 and its substantial
equivalents thereto, or a salt thereof.
[0188] These galanin receptor proteins are those derived from all
cells and tissues (e.g. amygdaloid nucleus, pituitary gland,
pancreas, brain (including whole brain, midbrain nigra and other
regions), kidney, liver, gonad, thyroid gland, cholecyst, bone
marrow, adrenal, skin, muscle, lung, digestive duct, stomach, blood
vessel, heart, thymus, spleen, leukocyte, etc.) of warm-blooded
animals (e.g. guinea,pig, rat, mouse, swine, sheep, cattle, horse,
monkey, human being, rabbit, cat, dog, etc.), and any of galanin
receptor 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: 1, an amino acid sequence represented by
SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 5,
and substantial equivalents to the amino acid sequence represented
by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5. These galanin
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: 1, an amino acid sequence
represented by SEQ ID NO: 2, and an amino acid sequence represented
by SEQ ID NO: 5, proteins wherein the amino acid sequence thereof
is about 90% to 99.9% homologous to an amino acid sequence
represented by SEQ ID NO: 1, an amino acid sequence represented by
SEQ ID NO: 2 or an amino acid sequence represented by SEQ ID NO: 5
and the activity thereof is substantially equivalent to the protein
having an amino acid sequence represented by SEQ ID NO: 1, an amino
acid sequence represented by SEQ ID NO: 2 or an amino acid sequence
represented by SEQ ID NO: 5 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.
[0189] In one embodiment of the present invention, mouse-derived
galanin receptor proteins are those derived from all mouse-derived
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.), and any of proteins as long as they
comprise an amino acid sequence represented by SEQ ID NO: 1, and
substantial equivalents thereto. The mouse-derived galanin receptor
proteins may include proteins having an amino acid sequence
represented by SEQ ID NO: 1, proteins wherein the amino acid
sequence thereof is about 90% to 99.9% homologous to an amino acid
sequence represented by SEQ ID NO: 1 and the activity thereof is
substantially equivalent to the protein having an amino acid
sequence represented by SEQ ID NO: 1 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.
[0190] In another embodiment of the present invention,
mouse-derived galanin receptor proteins include mouse pancreatic
.beta.-cell line, MIN6 (FERM BP-4954)-derived galanin receptor
proteins comprising an amino acid sequence represented by SEQ ID
NO: 1, etc. Examples of the mouse-derived galanin receptor protein
are mouse-derived galanin receptor proteins having an amino acid
sequence represented by SEQ ID NO: 1, proteins wherein one or more
amino acid residues (preferably from 1 to 30 amino acid residues,
more preferably from 1 to 10 amino acid residues) are deleted from
the amino acid sequence of SEQ ID NO: 1, proteins wherein one or
more amino acid residues (preferably from 1 to 30 amino acid
residues, more preferably from 1 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 1, proteins wherein
one or more amino acid residues (preferably from 1 to 30 amino acid
residues, more preferably from 1 to 10 amino acid residues) in the
amino acid sequence of SEQ ID NO: 1, are substituted with one or
more amino acid residues, etc.
[0191] More specific examples of the mouse-derived galanin receptor
protein are mouse pancreatic .beta.-cell line, MIN6-derived galanin
receptor proteins having an amino acid sequence represented by SEQ
ID NO: 2, proteins having a substantial amino acid sequence thereto
(for example, the amino acid sequence thereof is about 90% to 99.9%
homologous to an amino acid sequence represented by SEQ ID NO: 2),
proteins wherein one or more amino acid residues (preferably from 1
to 30 amino acid residues, more preferably from 1 to 10 amino acid
residues) are deleted from the amino acid sequence of SEQ ID NO: 2,
proteins wherein one or more amino acid residues (preferably from 1
to 30 amino acid residues, more preferably from 1 to 10 amino acid
residues) are added to the amino acid sequence of SEQ ID NO: 2,
proteins wherein one or more amino acid residues (preferably from 1
to 30 amino acid residues, more preferably from 1 to 10 amino acid
residues) in the amino acid sequence of SEQ ID NO: 2, are
substituted with one or more amino acid residues, etc.
[0192] In yet another embodiment of the present invention, human
galanin receptor proteins are those derived from all human-derived
cells and tissues (e.g. stomach, pituitary gland, pancreas, brain
(including whole brain, midbrain nigra and other regions), kidney,
liver, gonad, thyroid gland, cholecyst, bone marrow, adrenal, skin,
muscle, lung, digestive duct, blood vessel, heart, etc.), and any
of proteins as long as they comprise an amino acid sequence
represented by SEQ ID NO: 5, and substantial equivalents thereto.
The human galanin receptor proteins may include proteins having an
amino acid sequence represented by SEQ ID NO: 5, proteins wherein
the amino acid sequence thereof is about 90% to 99.9% homologous to
an amino acid sequence represented by SEQ ID NO: 5 and the activity
thereof is substantially equivalent to the protein having an amino
acid sequence represented by SEQ ID NO: 5 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.
[0193] In another embodiment of the present invention, human
galanin receptor proteins include human-derived galanin receptor
proteins comprising an amino acid sequence represented by SEQ ID
NO: 5, etc. Examples of the human galanin receptor protein are
human-derived galanin receptor proteins having an amino acid
sequence represented by SEQ ID NO: 5, proteins wherein one or more
amino acid residues (preferably from 1 to 20 amino acid residues,
more preferably from 1 to 10 amino acid residues) are deleted from
the amino acid sequence of SEQ ID NO: 5, proteins wherein one or
more amino acid residues (preferably from 1 to 20 amino acid
residues, more preferably from 1 to 10 amino acid residues) are
added to the amino acid sequence of SEQ ID NO: 5, proteins wherein
one or more amino acid residues (preferably from 1 to 20 amino acid
residues, more preferably from 1 to 10 amino acid residues) in the
amino acid sequence of SEQ ID NO: 5, are substituted with one or
more amino acid residues, etc.
[0194] A portion of the amino acid sequence may be modified (e.g.
addition, deletion, substitution with other amino acids, etc.) in
the galanin receptor proteins of the present invention.
[0195] Furthermore, the galanin 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.
[0196] However, the known human galanin receptor protein having an
amino acid sequence in which the fifteenth Trp in the amino acid
sequence represented by SEQ ID NO: 5 is substituted with Cys is
excluded from the coverage of the human galanin receptor protein of
the present invention.
[0197] The salt of said galanin 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.
[0198] The galanin 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 galanin receptor protein
encoding DNA. The protein or its salt of the present invention may
be manufactured by the peptide synthesis as described herein
below.
[0199] The galanin receptor protein fragment (the partial peptide
of said galanin receptor protein) may include, for example, the
site which is exposed outside cell membranes, among the galanin
receptor protein molecule. Examples of the partial peptide are
peptides containing a region which is analyzed as an extracellular
area (hydrophilic region or site) in a hydrophobic plotting
analysis on the galanin receptor protein. 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 (or peptide fragment) which contains
plural domains at the same time will be used as well.
[0200] In an embodiment of the present invention, the partial
peptide of said mouse-derived galanin receptor protein may include,
for example, the site which is exposed outside cell membranes,
among the galanin receptor protein molecule. Examples of the
mouse-derived galanin receptor partial peptide are peptides
containing a region which is analyzed as an extracellular area
(hydrophilic region or site) in a hydrophobic plotting analysis on
the galanin receptor protein, represented by FIG. 2.
[0201] The salt of said galanin receptor partial peptide 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.
[0202] The partial peptide of the galanin 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) galanin 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 {circle
over (1)} to {circle over (5)}:
[0203] {circle over (1)} M. Bodanszky and M. A. Ondetti: Peptide
Synthesis, Interscience Publishers, New York (1966).
[0204] {circle over (2)} Schroeder and Luebke: The Peptide,
Academic Press, New York, (1965).
[0205] {circle over (3)} Nobuo Izumiya et al.: Fundamentals and
Experiments of the Peptide Synthesis, Maruzen K K, Japan
(1975).
[0206] {circle over (4)} 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.
[0207] {circle over (5)} Haruaki Yajima (ed): Development of
Pharmaceuticals (Second Series), Vol. 14, Peptide Synthesis,
Hirokawa Shoten, Japan.
[0208] 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.
[0209] Furthermore, the product may be manufactured by culturing
the transformant (transfectant) containing the DNA coding for said
partial peptide.
[0210] The galanin receptor protein-encoding DNA of the present
invention may be any coding DNA as long as it contains a nucleotide
sequence coding for a galanin receptor protein which contains an
amino acid sequence substantially equivalent to the amino acid
sequence having SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5,
provided that the known galanin receptor protein wherein 15th Trp
in the the amino acid sequence of SEQ ID NO: 5 is replaced with Cys
is excluded.
[0211] The DNA of the present invention may be any one of a genome
DNA, a genome DNA library, a tissue and cell-derived cDNA, a 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.
[0212] In an embodiment, the DNA coding for the mouse-derived
galanin receptor protein may be any coding DNA as long as it
contains a nucleotide sequence coding for a mouse-derived galanin
receptor protein which contains an amino acid sequence
substantially equivalent to the amino acid sequence having SEQ ID
NO: 1 and/or which has an activity substantially equivalent to the
amino acid sequence having SEQ ID NO: 1. Examples of the DNA coding
for the mouse-derived galanin receptor protein comprising the amino
acid sequence of SEQ ID NO: 1 includes DNA having a nucleotide
sequence represented by SEQ ID NO: 3, etc. The DNA coding for the
mouse-derived galanin receptor protein comprising the amino acid
sequence of SEQ ID NO: 2 includes DNA having a nucleotide sequence
represented by SEQ ID NO: 4, etc.
[0213] In another embodiment, the DNA coding for the human galanin
receptor protein may be any coding DNA as long as it contains a
nucleotide sequence coding for a human- derived galanin receptor
protein which contains an amino acid sequence substantially
equivalent to the amino acid sequence having SEQ ID NO: 5 and/or
which has an activity substantially equivalent to the amino acid
sequence having SEQ ID NO: 5, provided that the known human galanin
receptor protein wherein 15th Trp in the the amino acid sequence of
SEQ ID NO: 5 is replaced with Cys is excluded. Examples of the DNA
coding for the human galanin receptor protein comprising the amino
acid sequence of SEQ ID NO: 5 includes DNA having a nucleotide
sequence represented by SEQ ID NO: 6, etc.
[0214] The DNA completely coding for the galanin receptor protein
of the present invention can be cloned by
[0215] (1) carrying out the PCR amplification using a synthetic DNA
primer having a partial nucleotide sequence (nucleotide fragment)
of the galanin receptor protein; or
[0216] (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 galanin
receptor protein (e.g., human galanin receptor protein, etc.) 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 or manuals attached thereto.
[0217] For example, the DNA completely coding for the mouse-derived
galanin receptor protein of the present invention is cloned by (1)
carrying out the PCR amplification using a synthetic DNA primer
having a partial nucleotide sequence (nucleotide fragment) of the
mouse-derived galanin 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 or mouse-derived galanin receptor protein
or a labeled synthetic DNA having part or all of the coding region
thereof.
[0218] The cloned galanin receptor protein-encoding 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.
[0219] A vector containing the galanin receptor protein-encoding
DNA (for example, an expression vector for the galanin receptor
protein; specifically, an expression plasmid comprising the human
galanin receptor protein-encoding DNA, etc.) can be produced by,
for example, (a) cutting out a target DNA fragment from the galanin
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 (for example, an
expression plasmid compatible with the human galanin receptor
protein-encoding DNA, etc.).
[0220] The vector may include plasmids derived from Escherichia
coli (e.g., pBR322, pBR325, pUC12, pUC13, etc.), plasmids derived
from Bacillus subtilis (e.g., pUB110, 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.
[0221] 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, SPO2 promoters, penP promoters, etc.
When the host is an yeast, the promoters are preferably PHO5
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 (CMV) promoters, SR.alpha.
promoters, etc. An enhancer can be effectively utilized for the
expression.
[0222] As required, furthermore, a host-compatible signal sequence
is added to the N-terminal side of the galanin 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
.alpha.-amylase signal sequences, subtilisin signal sequences, etc.
When the host is an yeast, they may include mating factor .alpha.
signal sequences, invertase signal sequences, etc. When the host is
an animal cell, they may include insulin signal sequences,
.alpha.-interferon signal sequences, antibody molecule signal
sequences, etc.
[0223] Further, a preferred method of constructing an expression
plasmid among the vectors containing the human galanin receptor
protein DNA of the present invention will be concretely given as
hereunder.
[0224] Examples of the plasmid used are pAKKO-111 (sometimes
referred to as pA1-11), pRc/CMV, pRc/RSV, etc. and, among them, the
use of pAKKO-111 (pA1-11) is preferred. With respect to a promoter,
anything may be used so far as it functions in an effective manner
in host cells and its examples are SV40 early gene promoter, CMV
promoter, HSV-TK promoter, SR.alpha. promoter, RSV promoter, etc.
Among them, CMV promoter and SR.alpha. promoter are preferred and
the use of SR.alpha. promoter is particularly preferred.
[0225] With respect to an expression plasmid, the use of the agent
containing an enhancer, a splicing signal, a poly A adding signal,
a selective marker, etc. besides the above-mentioned ones is
preferred. Examples of the selective marker are dihydrofolate
reductase (hereinafter, sometimes referred to as "dhfr") gene and
neomycin phosphate transferase (hereinafter, sometimes referred to
as "neo" gene). The dhfr gene gives a resistance to
methotrexate,(MTX) while the neo gene gives a resistance to G-418.
Especially when a dhfr gene-deficient CHO cell is used and a dhfr
gene is utilized as a selective marker, it is possible to select
even by a medium free from thymidine.
[0226] Specific and preferred examples of the expression plasmid
carrying the human galanin receptor protein encoding DNA of the
present invention are those in which the above-mentioned promoters
(e.g., particularly, SR.alpha.-promoter, etc.) are inserted in the
upstream of the human galanin receptor protein DNA and, preferably,
an SV early gene poly A addition signal is inserted to the
downstream of the human galanin receptor protein DNA followed by
inserting dhfr gene, ampicillin-resisting gene, etc. into the
downstream of the poly A addition signal.
[0227] More specific and preferred example is an expression plasmid
designated pTS863 (FIG. 13) in which SR.alpha. promoter is inserted
in an upstream of the human galanin receptor protein DNA, an SV
early gene poly A addition signal is inserted in a downstream of
the human galanin receptor protein DNA, a dhfr gene is inserted in
a downstream thereof and then an ampicillin-resisting gene is
inserted in the downstream thereof, etc.
[0228] When the expression plasmid containing the human galanin
receptor protein DNA prepared as such is introduced into a host
cell, it is possible to produce a cell which is able to highly
express the DNA which codes for the human galanin receptor
protein.
[0229] A transformant or transfectant is produced by using the
vector thus constructed, which carries the galanin 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 MI114 [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.sup.-, 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.sup.- CHO cell), CHO K-1, human FL cell, 293 cell, L cell,
myeloma cell, C127 cell, Balb/c3T3 cell, Sp-2/0 cell, etc.
[0230] 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 harboring the expression vector
carrying a galanin receptor protein-encoding DNA are produced
according to the aforementioned techniques.
[0231] Among the above-mentioned host cells, animal cells are
particularly preferred as the host cell for an expression plasmid
containing the human galanin receptor protein DNA of the present
invention. The examples thereof are 293 cell, CHO cell, Vero cell,
L cell, myeloma cell, C127 cell, Balb/c3T3 cell and Sp-2/O cell,
etc. Among them, CHO cell and 293 cell are preferred and
particularly CHO cell [Journal of Experiment of Medicine, 108,
945(1958)] is more preferred. Among said CHO cell, the preferred
ones are dhfr gene-deficient CHO cell (hereinafter, sometimes
referred to as CHO (dhfr.sup.-) cell) [Proceedings of the National
Academy of Sciences of the U. S. A., 77, 4216-4220(1980)], CHO K-1
cell [Proceedings of the National Academy of Sciences of the U. S.
A., 60, 1275(1968)], etc. When dhfr gene is inserted in an
expression plasmid as a selective marker, CHO(dhfr.sup.-) and the
like are suitable.
[0232] With respect to the combination of the expression plasmid
with the host cell, the preferred one can be suitably selected and,
for example, CHO(dhfr.sup.-) cell and the like are suitable as the
host cell of the expression plasmid (FIG. 13) indicated by pTS863.
In introducing the expression plasmid into animal cells, known
methods such as a calcium phosphate method [Graham, F. L. and van
der Eb, A. J.: Virology, 52, 456-467(1973)], an electroporation
[Neumann, E. et al., EMBO Journal, 1,841-845(1982)], etc. may be
used.
[0233] As such, a transformant in which a transformation is carried
out using a vector containing a human galanin receptor protein DNA
is produced. In addition, the transformant prepared by a
transformation using an expression plasmid containing the human
galanin receptor protein DNA may be used for the manufacture of
human galanin receptor protein.
[0234] Cells which are able to highly express the human galanin
receptor protein can be obtained by selecting the cells wherein the
above-mentioned expression plasmid is incorporated in the
chromosome by means of clone selection. Briefly, the transformant
is first selected using the above-mentioned selective marker as an
index for selection. Then the transformant produced as such using
the selective marker is repeatedly subjected to a clone selection
to give a cell strain which stably exhibits a high ability of
expressing the human galanin receptor protein. When a dhfr gene is
used as a selective marker, the resisting cells are selected by a
culture with a sequential increase in the methotrexate (MTX)
concentration to amplify the introduced gene in the cells whereby a
cell strain exhibiting far higher expression can be obtained.
[0235] Even when CHO (dhfr.sup.-) cell is used as a host, the CHO
containing an expression plasmid indicated by pTS863 also has a
dhfr gene as a result because a dhfr gene is introduced, for
example, into an expression plasmid indicated by pTS863 (FIG. 13).
In this specification, the CHO cell obtained by giving an
expression plasmid (e.g. pTS 863, etc.) containing dhfr gene may be
sometimes referred to as "CHO(dhfr.sup.+) cell".
[0236] An example of the transformant which is able to highly
express the human galanin receptor protein DNA in the present
invention is a CHO (dhfr.sup.+) cell obtained by giving an
expression plasmid indicated by pTS863 obtained in Example 11
(mentioned herein later) to a CHO (dhfr.sup.-) cell, etc. More
specific examples are CHO (dhfr.sup.+) cell indicated by
CHO/pTS863-5, CHO (dhfr.sup.+) cell indicated by CHO/pTS863-7, etc.
As compared with the known human galanin receptor
protein-expressing COS cells, the above-mentioned CHO (dhfr.sup.+)
cells are able to express more amount of human galanin receptor
protein and, further, there are some which exhibit a receptor
activity (e.g., ligand binding activity, etc.) of about 10 to
100-fold (preferably about 100-fold) as compared with natural
tissues containing human galanin receptor proteins (e.g. human
melanoma Bowes cells, etc.). Accordingly, those CHO (dhfr.sup.+)
cells are effective in conducting a method of screening for the
human galanin receptor agonist/antagonist which will be mentioned
herein later.
[0237] The cells which contain the human galanin receptor protein
of the present invention can be also manufactured by culturing the
transformant containing the vector (particularly, the expression
plasmid) carrying the human galanin receptor protein DNA of the
present invention under a condition where the human galanin
receptor protein DNA can be expressed.
[0238] 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, vitamins,
growth-promoting factors, etc. It is desired that the culture
medium is pH from about 5 to about 8.
[0239] 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.beta.-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 for the Biological Medicine, Vol. 73, 1 (1950)],
.alpha.-MEM medium, etc. which are containing, for example, about 5
to 20% of fetal calf serum. Especially when CHO (dhfr.sup.-) cells
and dhfr selective marker gene are used, it is preferred to use a
DMEM medium containing a dialyzed fetal bovine serum which rarely
contains thymidine. 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 72 hours. As required, medium
exchange, aeration and stirring may be applied.
[0240] As such, cells containing the human galanin receptor protein
can be manufactured from the transformant retaining the vector
(particularly, expression plasmid) containing the human galanin
receptor protein-encoding DNA. Examples of the cell containing the
human galanin receptor protein are CHO cells containing the human
galanin receptor protein and the like. The cells containing the
human galanin receptor protein can be obtained by culturing CHO
(dhfr.sup.+) cell indicated by CHO/pTS863-5, CHO (dhfr.sup.+) cell
indicated by CHO/pTS863-7, etc.
[0241] Separation and purification of the galanin receptor protein
(for example, human galanin receptor protein, etc.) from the
above-mentioned cultures can be carried out according to methods
described herein below.
[0242] To extract galanin 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
galanin 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").
[0243] In the case where galanin 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 galanin
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.
[0244] In case where the galanin 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 galanin 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.
[0245] The galanin 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 galanin receptor
protein thus formed can be measured by experimenting the coupling
(or binding) with a ligand including galanin or by enzyme
immunoassays (enzyme linked immunoassays) using specific
antibodies.
[0246] When the DNA which codes for the known human galanin
receptor protein is used in the above-mentioned means instead of
that which codes for the human galanin receptor protein of the
present invention, it is also possible to isolate the cells which
highly express recombinant human galanin receptor protein or to
isolate recombinant human galanin receptor protein.
[0247] Although the known human galanin receptor protein is
expressed by COS cells, the expressed amount in the case of the COS
cells are usually small. However, in accordance with a method of
constructing the expression plasmid of the present invention, it is
possible to manufacture not only the cells (particularly CHO cells)
which highly express the human galanin receptor protein of the
present invention but also the cells (particularly CHO cells) which
highly express the known human galanin receptor protein.
[0248] The cell membrane fraction of a cell containing said galanin
receptor protein (for example, cell membrane fraction of a cell
containing human galanin receptor protein, cell membrane fraction
of a cell containing mouse-derived galanin receptor protein, etc.)
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 containing the galanin receptor protein
(for example, the human galanin receptor protein, the mouse-derived
galanin receptor protein, etc.). 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 (15,000 rpm to 30,000 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 galanin
receptor protein (for example, the human galanin receptor protein,
the mouse-derived galanin receptor protein, etc.) and a lot of
membrane components such as phospholipids and membrane proteins
derived from the cells.
[0249] The amount of the galanin receptor protein in the membrane
fraction cell containing said galanin receptor protein is
preferably 10.sup.3 to 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 a large amount of samples within the
same lot.
[0250] The galanin receptor protein, the partial peptide thereof
and the galanin receptor protein-encoding DNA of the present
invention can be used for:
[0251] {circle over (1)} obtaining an antibody and an
antiserum,
[0252] {circle over (2)} constructing a system for expressing a
recombinant receptor protein,
[0253] {circle over (3)} developing a receptor-binding assay system
using the above developing system and screening pharmaceutical
candidate compounds,
[0254] {circle over (4)} designing drugs based upon the comparison
with ligands and receptors which have a similar or analogous
structure,
[0255] {circle over (5)} preparing a probe in the analysis of genes
and preparing a PCR primer,
[0256] {circle over (6)} gene manipulating therapy,
[0257] {circle over (7)} producing a transgenic animal (for
example, transgenic mouse, etc.),
[0258] {circle over (8)} producing a model animal suffering from
diseases caused by gene deficiency, etc.
[0259] In particular, it is allowable to screen a galanin receptor
agonist or antagonist specific to a warm-blooded animal such as
human being by a receptor-binding assay system which uses a system
for expressing a recombinant galanin 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.
[0260] For example, the mouse-derived galanin receptor protein, the
partial peptide of the mouse-derived galanin receptor protein and
the DNA which codes for the mouse-derived galanin receptor protein
can be used for {circle over (1)} obtaining antibody and antiserum;
{circle over (2)} construction of an expression system of the
recombinant receptor protein; {circle over (3)} development of the
receptor-binding assay system using said expression system and
screening of the candidate compounds as pharmaceuticals; {circle
over (4)} conducting a drug design based upon a comparison with
ligands and receptors which have a similar or analogous structure;
{circle over (5)} preparing probes and designing PCR primers in
gene diagnosis; {circle over (6)} gene therapy, etc.
[0261] The human galanin receptor protein, the partial peptide of
the human galanin receptor protein and the DNA which codes for the
human galanin receptor protein can be used for {circle over (1)}
obtaining antibody and antiserum; {circle over (2)} construction of
an expression system of the recombinant receptor protein; {circle
over (3)} development of the receptor-binding assay system using
said expression system and screening of the candidate compounds as
pharmaceuticals; {circle over (4)} conducting a drug design based
upon a comparison with ligands and receptors which have a similar
or analogous structure; {circle over (5)} preparing probes and
designing PCR primers in gene diagnosis; {circle over (6)} gene
therapy, etc. Especially when the receptor binding assay system
utilizing the expression system for the human galanin receptor
protein of the present invention is used, it is possible to screen
the galanin receptor agonist or antagonist which is specific to
warm-blooded animals (especially, human being) whereupon said
agonist or antagonist can be used as a preventive and therapeutic
agent for various diseases.
[0262] Concretely described below are uses of galanin receptor
proteins, partial peptides thereof (peptide fragments thereof),
galanin receptor protein-encoding DNAs and antibodies against the
galanin receptor protein according to the present invention.
[0263] (1) Quantitative Measurement of Galanin
[0264] The galanin receptor protein, a partial peptide thereof or a
salt thereof has a binding property to galanin and, therefore, it
is capable of determining quantitatively an amount of galanin in
vivo with good sensitivity.
[0265] This quantitative measurement may be carried out by, for
example, combining with a competitive method. Thus, samples to be
measured is contacted with galanin receptor proteins or partial
peptide thereof so that the galanin concentration in said sample
can be measured. In one embodiment of the quantitative measurement,
the protocols described in the following {circle over (1)} and
{circle over (2)} or the methods similar thereto may be used:
[0266] {circle over (1)} Hiroshi Irie (ed): "Radioimmunoassay"
(Kodansha, Japan, 1974); and
[0267] {circle over (2)} Hiroshi Irie (ed): "Radioimmunoassay,
Second Series" (Kodansha, Japan, 1979).
[0268] Further, the quantitative determination method of galanin
according to the present invention can be used as a diagnostic
method for the diseases caused by increase/decrease in galanin
concentrations such as stomach ulcer, diabetes and Alzheimer's
disease.
[0269] (2) Screening of Galanin Receptor Agonist and/or
Antagonist
[0270] Galanin receptor proteins or partial peptides thereof are
used. Alternatively, expression systems for recombinant type
galanin receptor proteins or partial peptides 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 galanin with the galanin receptor
protein. Such a compound includes a compound exhibiting a galanin
receptor-mediated cell stimulating activity (e.g. activity of
promoting or activity of inhibiting physiological reactions
including liberation of arachidonic 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,
secretion of insulin, etc.; in particular, activity of promoting or
activity of inhibiting endocellular cAMP production and secretion
of insulin) (so-called "galanin receptor agonist"), a compound free
of such a cell stimulating activity (so-called "galanin receptor
antagonist"), etc.
[0271] Thus, the present invention provides a method of screening a
galanin receptor agonist and/or galanin receptor antagonist with a
galanin receptor protein or a salt thereof, characterized in
comparing the following cases:
[0272] (i) the case wherein galanin is contacted with the galanin
receptor protein or salt thereof, or a partial peptide thereof or a
salt thereof; and
[0273] (ii) the case wherein galanin is contacted with a mixture of
the galanin receptor protein or salt thereof or the partial peptide
or salt thereof and said test sample (including a test
compound).
[0274] In said screening method, one characteristic feature of the
present invention resides in that the amount of galanin bonded with
said galanin receptor protein (for example, human or mouse-derived
galanin receptor protein) or partial peptide thereof, the cell
stimulating activity of galanin, etc. are measured in the case
where (i) galanin is contacted with said galanin receptor protein
(for example, human or mouse-derived galanin receptor protein) or
its partial peptide and in the case where (ii) galanin and a test
sample (including a test compound) are contacted with the galanin
receptor protein or its partial peptide, respectively and then
compared therebetween.
[0275] In the screening of the galanin receptor agonist or
antagonist, it may be considered to use human hippocampus as a
human galanin receptor protein source. However, tissues derived
from human being are hardly available and, therefore, they are not
suitable for use in screening whereupon recombinant human galanin
receptor proteins which are abundantly expressed in cells
(particularly, animal cells such as CHO cells) are suitable in
practice. More preferably, the cell strain in which human galanin
receptor proteins are continuously and stably expressed is
advantageously used.
[0276] Accordingly, the human galanin receptor protein of the
present invention or the salt thereof, the human galanin receptor
protein-partial peptide of the present invention or the salt
thereof and the cell or cell fraction thereof containing the human
galanin receptor protein of the present invention are remarkably
useful as a reagent for screening the galanin receptor agonist or
antagonist.
[0277] Briefly, the present invention offers a method of screening
galanin receptor agonist or antagonist, characterized in that, the
human galanin receptor protein of the present invention or the salt
thereof, the partial peptide of the human galanin receptor protein
of the present invention or the salt thereof or the cells or cell
fraction thereof containing the human galanin receptor protein of
the present invention is used.
[0278] More specifically, the present invention offers:
[0279] (I) a method of screening the human galanin receptor agonist
or antagonist, characterized in that, a comparison is conducted
between the cases where (i) the human galanin receptor protein of
the present invention or the partial peptide or the salt thereof is
contacted with galanin and where (ii) the human galanin receptor
protein of the present invention or the partial peptide or the salt
thereof is contacted with galanin and a test compound; and
[0280] (II) a method of screening the galanin receptor agonist or
antagonist, characterized in that, a comparison is conducted
between the cases where (i) cells containing the human galanin
receptor protein of the present invention or cell membrane fraction
thereof are contacted with galanin and where (ii) cells containing
the human galanin receptor protein of the present invention or cell
membrane fraction thereof are contacted with galanin and a test
compound.
[0281] To be more specific, in the screening methods (I) and (II)
of the present invention, the cell-stimulating activity and/or
binding amount of galanin to said human galanin receptor protein or
partial peptide or salt thereof or cells containing the human
galanin receptor protein or cell membrane fraction thereof in (i)
and (ii) are measured and compared.
[0282] In one more specific embodiment of the present
invention,
[0283] {circle over (1)} a method of screening a galanin receptor
agonist and/or galanin receptor antagonist or a salt thereof,
characterized in that, when a labeled galanin is contacted with a
galanin receptor protein (e.g., human-derived galanin receptor
protein, etc.) or a partial peptide thereof and when a labeled
galanin and a test compound are contacted with a galanin receptor
protein (e.g., human-derived galanin receptor protein, etc.) or a
partial peptide thereof, the amounts of the labeled galanin bonded
with said protein or partial peptide thereof or salt thereof are
measured and compared;
[0284] {circle over (2)} (a) (i) a method of screening a galanin
receptor agonist and/or galanin receptor antagonist or a salt
thereof, characterized in that, when a labeled galanin is contacted
with galanin receptor protein (e.g., human or mouse galanin
receptor protein, etc.)-containing cells (e.g., mouse MIN 6 cell
(FERM BP-4954), etc.) or a membrane fraction of said cells and when
a labeled galanin and a test compound are contacted with galanin
receptor protein (e.g., human or mouse galanin receptor protein,
etc.)-containing cells (e.g., mouse MIN 6 cell (FERM BP-4954),
etc.) or a membrane fraction of said cells, the amounts of the
labeled galanin binding with said protein or partial peptide
thereof or a salt thereof are measured and compared;
[0285] (b) (ii) a method of screening a galanin receptor agonist
and/or galanin receptor antagonist or a salt thereof, characterized
in that, when a galanin receptor protein-activating compound (e.g.
galanin) is contacted with galanin receptor protein (e.g., human or
mouse galanin receptor protein, etc.)-containing cells (e.g., mouse
MIN 6 cell (FERM BP-4954), etc.) or a membrane fraction of said
cells and when the galanin receptor protein-activating compound and
a test compound are contacted with galanin receptor protein (e.g.,
human or mouse galanin receptor protein, etc.)-containing cells
(e.g., mouse MIN 6 cell (FERM BP-4954), etc.) or a membrane
fraction of said cells, the resulting galanin receptor
protein-mediated cell stimulating activities (e.g. activities of
promoting or activities of inhibiting physiological responses
including the opening of K.sup.+ channel, closing of N type
Ca.sup.+ channel liberation of arachidonic acid, liberation of
acetylcholine, endocellular Ca.sup.2+ changes, endocellular cAMP
production (or its depression), endocellular cGMP production,
insulin secretion, production of inositol phosphate, cell membrane
potential changes, phosphorylation of endocellular proteins,
activation of c-fos, decrease of pH, cell migration activity,
secretion of hormones, activation of G protein, cell promulgation,
etc.) are measured and compared; and
[0286] {circle over (3)} a method of screening a galanin receptor
agonist and/or galanin receptor antagonist or a salt thereof,
characterized in that, when a labeled galanin is contacted with
galanin receptor proteins (e.g., human or mouse galanin receptor
proteins, etc.) expressed on the cell membrane by culturing a
transformant containing a galanin receptor protein encoding DNA and
when a labeled galanin and a test compound are contacted with
galanin receptor proteins (e.g., human or mouse galanin receptor
proteins, etc.) expressed on the cell membrane by culturing a
transformant containing a galanin receptor protein encoding DNA,
the amounts of the labeled galanin binding with said galanin
receptor protein are measured and compared;
[0287] {circle over (4)} a method of screening a galanin receptor
agonist and/or galanin receptor antagonist or a salt thereof,
characterized in that, when a galanin receptor protein-activating
compound (e.g. galanin) is contacted with galanin receptor proteins
(e.g., human or mouse galanin receptor proteins, etc.) expressed on
cell membranes by culturing transformants containing galanin
receptor protein-encoding DNA and when a galanin receptor
protein-activating compound and a test compound are contacted with
the galanin receptor protein expressed on the cell membrane by
culturing the transformant containing the galanin receptor
protein-encoding DNA, the resulting galanin receptor
protein-mediated cell stimulating activities (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,
endocellular cGMP production, production of inositol phosphate,
changes in cell membrane potential, phosphorylation of endocellular
proteins, activation of c-fos, lowering of pH, insulin secretion,
etc.) are measured and compared: are provided.
[0288] In the above-mentioned screening methods {circle over (1)}
or {circle over (2)} (a), a compound which binds with a galanin
receptor protein (e.g., human galanin receptor protein, etc.) and
inhibits the binding of galanin with the galanin receptor protein
can be selected as a galanin receptor agonist or antagonist.
[0289] Further, in the above-mentioned screening method {circle
over (2)} (b), a compound which exhibits a cell-stimulating
activity (for example, activities of promoting or inhibiting the
opening of K.sup.+ channel, closing of N type Ca.sup.+ channel,
liberation of arachidonic acid, liberation of acetylcholine,
variations in intracellular Ca.sup.2+ concentration, inhibition of
intracellular cAMP production, production of inositol phosphate,
variations in cell membrane potential, phosphorylation of
intracellular protein, activation of c-fos, decrease in pH, cell
migration activity, secretion of hormones, activation of G protein,
cell promulgation, etc.) via the galanin receptor (e.g., human
galanin receptor, etc.) upon said galanin receptor binding can be
selected as a galanin receptor agonist.
[0290] On the other hand, in the above-mentioned screening methods
{circle over (1)} and {circle over (2)} (a), a compound having no
stimulating activity to said cells among the test compounds which
exhibit an activity of inhibiting the binding of galanin with the
galanin protein receptor (e.g., human galanin receptor protein,
etc.) can be selected as a galanin receptor antagonist.
[0291] Before the cells containing the galanin receptor protein
(e.g., human galanin receptor protein, etc.) of the present
invention were developed, there was no cell which highly expressed
the galanin receptor protein (e.g., human galanin receptor protein,
etc.) and, therefore, it has not been possible to conduct an
effective screening of galanin receptor agonists or
antagonists.
[0292] Specific explanations of the screening method will be given
as hereunder.
[0293] First, with respect to the galanin receptor protein such as
the mouse-derived galanin receptor protein used for the screening
method of the present invention, any product may be used so far as
it contains galanin receptor proteins or partial peptide thereof
such as mouse-derived galanin receptor proteins or partial peptide
thereof although the use of a membrane fraction derived from
mammalian organs, tissues, cells, including mouse, is suitable.
Galanin receptor proteins which are expressed in a large amount
using a recombinant are suitable for the screening.
[0294] In the manufacture of the galanin receptor protein (for
example, mouse-derived galanin receptor protein, etc.), 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.
[0295] In order to introduce the galanin receptor protein-encoding
DNA fragment (for example, mouse-derived galanin receptor
protein-encoding DNA fragment, etc.) 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).
[0296] Accordingly, in the screening method, the substance
containing a galanin receptor protein or a partial peptide thereof
(for example, mouse-derived galanin receptor protein or its partial
peptide, etc.) may be a galanin receptor protein or its partial
peptide (for example, mouse-derived galanin receptor protein or its
partial peptide, etc.) 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.
[0297] When the galanin 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.
[0298] For example, the mouse-derived galanin receptor
protein-containing cells are host cells expressing the
mouse-derived galanin receptor, naturally occurring cells
containing the mouse-derived galanin receptor protein, etc.
Examples of said host cells may include Escherichia coli, Bacillus
subtilis, yeasts, insect cells, animal cells such as CHO cell and
COS cell, etc. The host cell expressing the mouse-derived galanin
receptor can be produced by the method according to the
above-mentioned transformant production.
[0299] In conducting the above-mentioned methods {circle over (1)},
{circle over (2)} (a) and {circle over (3)} for screening the
galanin receptor agonist and/or galanin receptor antagonist, a
suitable galanin receptor fraction and a labeled galanin are
necessary. With respect to the galanin receptor fraction, it is
preferred to use naturally occurring galanin receptors (natural
type galanin receptors), recombinant type galanin receptor
fractions with the activity equivalent to that of the natural type
galanin receptor, cells expressing the recombinant type
mouse-derived galanin receptor, naturally occurring cells
containing the mouse-derived galanin receptor, etc. Here the term
"activity equivalent to" means the same galanin binding activity,
or the substantially equivalent galanin binding activity.
[0300] With respect to the labeled galanin, it is possible to use
labeled galanin, labeled galanin analogized compounds, etc. For
example, galanin labeled with [.sup.3H], [.sup.125I], [.sup.14C],
[.sup.35S], etc. and other labeled substances may be utilized.
Known galanin agonists and antagonists labeled with [.sup.3H],
[.sup.125I], [.sup.14C], etc. may be utilized. Preferred examples
of the labeled galanin are galanin labeled with [.sup.125I]
(Dupont/NEN), etc.
[0301] Specifically, galanin receptor protein-containing cells or
cell membrane fractions (for example, human or mouse galanin
receptor protein-containing cells or cell membrane fractions of the
present invention) or the galanin receptor proteins or partial
peptides thereof are first suspended in a buffer which is suitable
for the measuring method to prepare the receptor sample in
conducting the screening for a galanin receptor agonist and/or
galanin receptor antagonist. 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 galanin with the
receptor may be used.
[0302] 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), gelatin, etc.
may be added to the buffer with an object of decreasing the
nonspecific binding. Further, a protease inhibitor such as PMSF,
leupeptin, bacitracin, aprotinin, E-64 (manufactured by Peptide
Laboratory, Japan), pepstatin, etc. may be added with an object of
inhibiting the decomposition of the receptor and galanin by
protease. A labeled galanin in a certain amount (for example, about
10,000 cpm to 1,000,000 cpm in case of 2000Ci/mmol; 5,000 cpm to
500,000 cpm in other cases) 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 coexists. 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.
[0303] 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 (for
example, the amount of [.sup.125I], etc.) retained in the glass
fiber filter, etc. is measured by means of a liquid scintillation
counter or a .gamma.-counter. Although a manifold or a cell
harvester may be used for the filtration, the use of cell harvester
is recommended for improving the efficiency. 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 inhibitory candidate substance, i.e., agonist and/or
antagonist candidate compound.
[0304] In an embodiment of the screening using human galanin
receptor proteins, the operation is carried out in accordance with
the following procedures:
[0305] (i) A reaction buffer (pH: 7.4) comprising 20 mM of
Tris-HCl, 1 mM of EDTA, 0.1% of BSA, 0.05% of CHAPS, 0.5mM of PMSF,
40 .mu.g/ml of leupeptin, 20 .mu.g/ml of E-64 and 10 .mu.g/ml of
pepstatin is prepared.
[0306] (ii) A test compound solution (2 .mu.l) in which the test
compound is suspended in the reaction buffer is placed in a
reaction tube on an ice bath. The final concentration of the test
compound is adjusted to 100 .mu.M.
[0307] (iii) The cell membrane fraction containing human galanin
receptor protein freeze-dried at -80.degree. C. is returned to a
room temperature and then vortex is gently generated and diluted to
a suitable concentration to prepare a cell membrane fraction
solution (for example, 0.5 mg protein/ml (bovine hippocampal
membrane fraction, CHO cell membrane fraction) etc.). This cell
membrane fraction solution is passed through a cell strainer and
each 200 .mu.l of it is placed in each reaction tube using a
separator.
[0308] (iv) Each 2 .mu.l of [.sup.125I] galanin diluted in a
reaction buffer is placed in a reaction tube on an ice bath.
[0309] (v) The reaction is carried out at 25.degree. C. for 60 or
75 minutes.
[0310] (vi) A B/F separation is conducted using a manifold. The
filter (GF/F, Whatman) which is used therefor is previously dipped
in a PEI solution (20 mM Tris-HCl and 0.3% polyethyleneimide; pH:
7.4) for more than one hour.
[0311] (vii) The filter is counted using a gamma-counter. The
compound which inhibits the specific binding to an extent of 40-50%
or more and of 50% or more is evaluated as.+-.and +,
respectively.
[0312] In conducting the above-mentioned methods {circle over (2)}
(b) and {circle over (4)} for screening the galanin receptor
agonist and/or galanin receptor antagonist, the galanin receptor
protein (e.g., mouse-derived galanin 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, endocellular
cGMP production, production of inositol phosphate, changes in the
cell membrane potential, phosphorylation of endocellular proteins,
activation of c-fos, lowering of pH, secretion of insulin, etc.)
may be measured by known methods or by the use of commercially
available measuring kits.
[0313] In conducting a screening method of the above-mentioned
{circle over (2)} (b), it is possible to measure the cell
stimulating activity via the galanin receptor protein (e.g., human
galanin receptor protein, etc.) (for example, activities of
promoting or inhibiting the opening of K.sup.+ channel, closing of
N type Ca.sup.+ channel, liberation of arachidonic acid, liberation
of acetylcholine, variations in intracellular Ca.sup.2+
concentration, inhibition of intracellular cAMP production,
production of inositol phosphate, variations in cell membrane
potential, phosphorylation of intracellular protein, activation of
c-fos, decrease in pH, cell migration activity, secretion of
hormones, activation of G protein, cell promulgation, etc.) by
known method or by commercially-available measuring kits. To be
more specific, galanin receptor protein (e.g., human or
mouse-derived galanin receptor protein, etc.)-containing cells are
at first cultured in a multiwell plate or the like.
[0314] 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.
[0315] In conducting a screening by measuring the cell stimulating
activity, cells in which a suitable galanin receptor protein (e.g.,
mouse-derived galanin receptor protein) is expressed are necessary.
Preferred galanin receptor protein (e.g., mouse-derived galanin
receptor protein)-expressing cells are naturally occurring
mouse-derived galanin receptor protein (natural type mouse-derived
galanin receptor protein)-containing cell lines or strains (e.g.,
mouse MIN6 (FERM BP-4954), etc.), the above-mentioned recombinant
type mouse-derived galanin receptor protein-expressing cell lines
or strains, etc. Among them, the natural type mouse-derived galanin
receptor protein-containing cell line, mouse pancreas-derived MIN6
cell, is capable of secreting insulin from intracellular regions
when galanin binds with galanin receptors on the cell membrane of
said cell. In case where the insulin secretion is used as an index
for the cell stimulating activity, mouse pancreas-derived MIN6 cell
is particularly preferred.
[0316] 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.
[0317] A kit for screening the galanin receptor agonist and/or
galanin receptor antagonist comprises a galanin receptor protein or
a salt thereof according to the present invention (e.g., human or
mouse-derived galanin receptor protein or its salt) or a partial
peptide thereof according to the present invention (e.g., human or
mouse-derived galanin receptor partial peptide or its salt), a
galanin receptor protein (e.g., human or mouse-derived galanin
receptor protein)-containing cell or its cell membrane fraction
according to the present invention, etc.
[0318] Examples of the screening kit include as follows:
[0319] 1. Reagent for Determining Ligand.
[0320] {circle over (1)} Buffer for Measurement and Buffer for
Washing.
[0321] The product wherein 0.05% of bovine serum albumin
(manufactured by Sigma) is added to Hanks' Balanced Salt Solution
(manufactured by Gibco).
[0322] 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.
[0323] {circle over (2)} Sample of Mouse-Derived Galanin Receptor
Protein.
[0324] CHO cells in which a mouse-derived galanin 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 atmosphere for two days to prepare the
sample.
[0325] {circle over (3)} Labeled Galanin.
[0326] The galanin which is labeled with commercially available
[.sup.3H], [.sup.125I], [.sup.14C], [.sup.35S], etc.
[0327] 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 5 .mu.M
with a buffer for the measurement.
[0328] {circle over (4)} Standard Galanin Solution.
[0329] Galanin is dissolved in PBS containing 0.1% of bovine serum
albumin (manufactured by Sigma) to make 100 .mu.M and stored at
-20.degree. C.
[0330] 2. Method of the Measurement.
[0331] {circle over (1)} CHO cells are cultured in a 12-well tissue
culture plate to express mouse-derived galanin receptor proteins.
The receptor protein-expressing CHO cells are washed with 1 ml of
buffer for the measurement twice. Then 490 .mu.l of buffer for the
measurement is added to each well.
[0332] {circle over (2)} Five .mu.l of a test compound solution of
10.sup.-3 to 10.sup.-10 M is added, then 5 .mu.l of a labeled
galanin 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
galanin of 10.sup.-4 M is added instead of the test compound.
[0333] {circle over (3)} 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).
[0334] {circle over (4)} Radioactivity is measured using an
automatic .gamma.-counter or a liquid scintillation counter
(manufactured by Beckmann) and PMB (percent of maximum binding) is
calculated by the following expression (1):
PMB=[(B-NSB)/(B.sub.0-NSB)].times.100 (1)
[0335] PMB: Percent of maximum binding
[0336] B: Value when a sample is added
[0337] NSB: Nonspecific binding
[0338] B.sub.0: Maximum binding
[0339] Another example of the screening kit is as follows:
Reagents for the Screening
[0340] {circle over (1)} Buffer for Measurement and for
Washing.
[0341] Hanks buffer to which 0.01% of bovine serum albumin and
0.05% of CHPS are added is used. This is filtered through a filter
with a pore size of 0.22 .mu.m, sterilized and stored at 4.degree.
C. or may be prepared upon use.
[0342] {circle over (2)} Human Galanin Receptor Protein Sample.
[0343] Cells containing the human galanin receptor protein are
subcultured in a 12-well plate at 5.times.10.sup.5/well and
cultured at 37.degree. C. with 5% CO.sub.2 and 95% air until cells
become confluent.
[0344] {circle over (3)} Labeled Galanin.
[0345] Commercially-available galanin which is labeled with
[.sup.3H], [.sup.125I], [.sup.14C], etc. is used. That which is in
a state of solution is stored at 4.degree. C. or -20.degree. C.
and, upon use, it is diluted to 1 .mu.l with a buffer for the
measurement.
[0346] {circle over (4)} Standard Galanin Solution.
[0347] Galanin is diluted with sterilized water to make 10.sup.-4 M
and stored at -20.degree. C.
Method of Measurement
[0348] {circle over (1)} Cells containing human galanin receptor
protein cultured in a 12-well culturing plate are washed, about
twice, with 1 ml of a buffer for measurement.
[0349] {circle over (2)} After the buffer for measurement is sucked
out, 5 .mu.l of a test compound solution (10.sup.-3 to 10.sup.-10
M) cooled at 4.degree. C. is added, then 0.5 ml of a buffer for
measurement, containing 100 pM of a labeled galanin, is added and
the mixture is made to react at 37.degree. C. for one hour with 5%
CO.sub.2 and 95% air. In order to know the nonspecific binding
amount, 1 .mu.M of galanin is added together with the test
compound.
[0350] {circle over (3)} The reaction solution is removed and
washed, three times, with 1 ml of buffer for washing which is kept
at 37.degree. C. The labeled galanin bonded to the cells is removed
with 0.5 ml of 0.2N NaOH and the radioactivity is measured by a
.gamma.-counter to calculate the PMB (percent of maximum binding)
from the above formula (1).
[0351] In the above-mentioned screening methods and screening kit,
it is also possible to use recombinant human galanin receptor
protein manufactured from DNA such as known human galanin receptor
protein DNA or the like or partial peptide thereof or cells
containing said recombinant human galanin receptor protein or a
cell membrane fraction thereof instead of the human galanin
receptor protein of the present invention or the partial peptide
thereof or cells containing the human galanin receptor protein or a
cell membrane fraction thereof.
[0352] The compound or its salt obtained by the screening method or
screening kit of the present invention is a compound which inhibits
the binding of galanin with a galanin receptor protein and, more
particularly, it is a compound having a cell stimulating activity
mediated via a galanin receptor or a salt thereof (so-called
"galanin receptor agonist") or a compound having no said
stimulating activity (so-called "galanin 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. The galanin receptor agonist or
antagonist obtained by the screening method or the screening kit of
the present invention is a compound or salt thereof selected from
the test sample including the compounds (for example, peptides,
proteins, nonpeptidic compounds, synthesized compounds, fermented
products, cell extracts, plant extracts, animal tissue extracts,
cell or tissue cultures, biological fluids, etc.; said test
compounds may be either novel or known) using the screening method
or the screening kit of the present invention and is a compound
which inhibits the binding of galanin with the recombinant human
galanin receptor protein of the present invention. Among those
compounds, galanin receptor agonist is a compound which exhibits a
cell-stimulating activity via a human galanin receptor while
galanin receptor antagonist is a compound which does not exhibit
said cell-stimulating activity.
[0353] In addition, the compounds in which the structure of said
galanin receptor agonist or antagonist obtained by the screening
method or the screening kit of the present invention is chemically
modified or substituted or the compounds in which a design is
conducted based upon said structure are also included in the
galanin receptor agonist or antagonist obtained by the screening
method or the screening kit of the present invention.
[0354] With respect to the salt of said galanin receptor agonist or
antagonist, physiologically-acceptable acid addition salts thereof
are particularly preferred. Examples of such salts are those with
inorganic acids (for example, hydrochloric acid, phosphoric acid,
hydrobromic acid and sulfuric acid) or with organic acids (for
example, acetic acid, formic acid, propionic acid, fumaric acid,
maleic acid, succinic acid, tartaric acid, citric acid, malic acid,
oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic
acid).
[0355] The galanin receptor agonist exhibits all or part of the
physiological activities of galanin or its equivalent and,
therefore, it is useful as an active ingredient for a
pharmaceutical composition with safety and low toxicity depending
upon said physiological activities. On the other hand, the galanin
receptor antagonist may inhibit all or part of the physiological
activities of galanin or its equivalent and, therefore, it is
useful as an active ingredient for a pharmaceutical composition
with safety and low toxicity inhibiting said physiological
activities.
[0356] More specifically, galanin receptor agonists are useful as
an inhibitor for liberation of acetylcholine, an inhibitor for
insulin secretion, a stimulant for growth hormone secretion, an
inhibitor for learning behavior or an inhibitor for satiety, etc.
and, moreover, it is useful as a preventive and therapeutic agent
for schizophrenic disease, gastric ulcer, as a sedative, etc. On
the other hand, galanin receptor antagonists are useful as an
accelerator for liberation of acetylcholine, an accelerator for
insulin secretion, an inhibitor for growth hormone secretion, an
accelerator for learning behavior and an accelerator for satiety,
etc. and, moreover, it is useful as a preventive and therapeutic
agent for diabetes, Alzheimer's disease, dementia, etc.
[0357] When the galanin receptor agonist and/or galanin receptor
antagonist 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,
elixirs, 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 can 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] Dose levels of said galanin receptor agonist and/or galanin
receptor antagonist 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 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.
[0362] (3) Preventive and Therapeutic Agent for of Galanin Receptor
Protein Deficiency Diseases
[0363] The galanin receptor protein (e.g., human galanin receptor
protein)-encoding DNA can be used a preventive and/or therapeutic
agent for treating said galanin receptor protein deficiency
diseases.
[0364] For example, when there is a patient for whom the
physiological action of galanin cannot be expected because of a
decrease in the galanin receptor protein (e.g., human galanin
receptor protein) in vivo, the amount of the galanin receptor
protein in the cells of said patient can be increased whereby the
action of galanin can be fully achieved by:
[0365] (a) administering the galanin receptor protein (e.g., human
galanin receptor protein)-encoding DNA to the patient to express
it; or
[0366] (b) inserting the galanin receptor protein (e.g., human
galanin receptor protein)-encoding DNA into cells or the like to
express it, followed by transplanting said cells or the like to
said patient. Accordingly, the galanin receptor protein (e.g.,
human galanin receptor protein)-encoding DNA can be used as a safe
and less toxic preventive and therapeutic agent for the galanin
receptor protein (e.g., human galanin receptor protein) deficiency
diseases (e.g., diabetes, Alzheimer's disease, dementia, etc.). It
may be used in treating or remedying defects by promoting the
acetylcholine liberation, inhibiting the growth hormone secretion,
promoting the insulin secretion, promoting the learning behavior,
promoting satiety, etc.
[0367] When the DNA of the present invention 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.
[0368] 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.
[0369] 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.).
[0370] 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). For other animals, the dose calculated from the
above based upon the body weight may be administered.
[0371] Furthermore, the method of inserting the DNA of the present
invention into cells to express said galanin receptor protein
(e.g., human galanin receptor protein, etc.) followed by
transplanting said cells to the patient may be carried out by a
method which is known per se or is similar thereto.
[0372] (4) Manufacture of Antibody or Antiserum against the Galanin
Receptor Protein of the Present Invention, Its Partial Peptide or
Its Salt.
[0373] Antibodies (e.g. polyclonal antibody and monoclonal
antibody) and antisera against the galanin receptor protein or salt
thereof of the present invention or against the peptide fragment of
the galanin 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 galanin receptor
protein or its salt of the present invention or the partial peptide
(fragment) of the galanin receptor protein or its salt of the
present invention. For example, monoclonal antibodies can be
manufactured by the method as given herein below.
Preparation of Monoclonal Antibody
[0374] (a) Preparation of Monoclonal Antibody-Producing Cells.
[0375] The galanin receptor protein of the present invention or its
salt or the partial peptide of the galanin receptor protein of the
present invention or its salt 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
chickens and hamsters and the use of mice, rats and hamsters is
preferred.
[0376] 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
galanin 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.
[0377] Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1,
etc. and the use of P3U1 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.
[0378] Various methods may be applied for screening a hybridoma
which produces anti-galanin receptor antibody. For example, a
supernatant liquid of hybridoma culture is added to a solid phase
(e.g. microplate) to which the galanin 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-galanin 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 galanin receptor labeled with a radioactive substance or an
enzyme is added and anti-galanin receptor monoclonal antibodies
bonded with the solid phase is detected.
[0379] Selection and cloning of the anti-galanin 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-galanin receptor in
the antiserum.
[0380] 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.
[0381] (b) Purification of the Monoclonal Antibody.
[0382] Like in the separation/purification of conventional
polyclonal antibodies, the separation/purification of the
anti-galanin 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.
[0383] The galanin receptor antibody of the present invention which
is manufactured by the aforementioned method (a) or (b) is capable
of specifically recognizing galanin receptors and, accordingly, it
can be used for a quantitative determination of the galanin
receptor in test liquid samples and particularly for a quantitative
determination by sandwich immunoassays.
[0384] Thus, the present invention provides, for example, the
following methods:
[0385] (i) a quantitative determination of a galanin receptor in a
test liquid sample, which comprises
[0386] (a) competitively reacting the test liquid sample and a
labeled galanin receptor with an antibody which reacts with the
galanin receptor of the present invention, and
[0387] (b) measuring the ratio of the labeled galanin receptor
binding with said antibody; and
[0388] (ii) a quantitative determination of a galanin receptor in a
test liquid sample, which comprises
[0389] (a) reacting the test liquid sample with an antibody
immobilized on an insoluble carrier and a labeled antibody
simultaneously or continuously, and
[0390] (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 galanin receptor while another
antibody is capable of recognizing the C-terminal region of the
galanin receptor.
[0391] When the monoclonal antibody of the present invention
recognizing a galanin receptor (hereinafter, may be referred to as
"anti-galanin receptor antibody") is used, galanin 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 galanin 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.
[0392] 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.
[0393] 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.
[0394] In a sandwich (or two-site) method, the test liquid is made
to react with an insolubilized anti-galanin receptor antibody (the
first reaction), then it is made to react with a labeled
anti-galanin receptor antibody (the second reaction) and the
activity of the labeling agent on the insoluble carrier is measured
whereupon the amount of the galanin 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.
[0395] In the method of measuring galanin receptors by the sandwich
method of the present invention, the preferred anti-galanin
receptor antibodies used for the first and the second reactions are
antibodies wherein their sites binding to the galanin 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 galanin
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.
[0396] The anti-galanin 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.
[0397] 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.
[0398] 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.
[0399] 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 galanin 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 (Immunochemical 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.
[0400] As such, the amount of galanin receptor proteins can now be
determined with a high precision using the anti-galanin receptor
antibody of the present invention.
[0401] (5) Preparation of Animals Having the Galanin Receptor
Protein-Encoding DNA of the Present Invention.
[0402] It is possible to prepare transgenic animals expressing
galanin receptors using galanin receptor protein-encoding DNA.
Examples of the animals are warm-blooded mammals such as rats,
mice, rabbit, sheep, swines, cattle, cats, dogs and monkeys.
[0403] In transferring the galanin 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 galanin
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 galanin 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 galanin receptor protein in a high amount can be prepared.
[0404] 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.
[0405] Transfer of the galanin 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 galanin 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 galanin 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 galanin receptor
protein in all of their embryonal cells and somatic cells.
[0406] The transgenic animal to which the galanin 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.
[0407] The animal to which the galanin receptor protein DNA is
transferred highly expresses the galanin receptor protein and,
accordingly, it is useful as the animal for screening for an
agonist or an antagonist to said galanin receptor protein.
[0408] 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 galanin receptor
protein can be analyzed. Cells of the galanin 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 galanin receptor
proteins therefrom.
[0409] 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.
[0410] DNA: Deoxyribonucleic acid
[0411] cDNA: Complementary deoxyribonucleic acid
[0412] A: Adenine
[0413] T: Thymine
[0414] G: Guanine
[0415] C: Cytosine
[0416] RNA: Ribonucleic acid
[0417] mRNA: Messenger ribonucleic acid
[0418] dATP: Deoxyadenosine triphosphate
[0419] dTTP: Deoxythymidine triphosphate
[0420] dGTP: Deoxyguanosine triphosphate
[0421] dCTP: Deoxycytidine triphosphate
[0422] ATP: Adenosine triphosphate
[0423] EDTA: Ethylenediamine tetraacetic acid
[0424] SDS: Sodium dodecyl sulfate
[0425] EIA: Enzyme Immunoassay
[0426] G, Gly: Glycine (or Glycyl)
[0427] A, Ala: Alanine (or Alanyl)
[0428] V, Val: Valine (or Valyl)
[0429] L, Leu: Leucine (or Leucyl)
[0430] I, Ile: Isoleucine (or Isoleucyl)
[0431] S, Ser: Serine (or Seryl)
[0432] T, Thr: Threonine (or Threonyl)
[0433] C, Cys: Cysteine (or Cysteinyl)
[0434] M, Met: Methionine (or Methionyl)
[0435] E, Glu: Glutamic acid (or Glutamyl)
[0436] D, Asp: Aspartic acid (or Aspartyl)
[0437] K, Lys: Lysine (or Lysyl)
[0438] R, Arg: Arginine (or Arginyl)
[0439] H, His: Histidine (or Histidyl)
[0440] F, Phe: Pheylalanine (or Pheylalanyl)
[0441] Y, Tyr: Tyrossine (or Tyrosyl)
[0442] W, Trp: Tryptophan (or Tryptophanyl)
[0443] P, Pro: Proline (or Prolyl)
[0444] N, Asn: Asparagine (or Asparaginyl)
[0445] Q, Gln: Glutamine (or Glutaminyl)
[0446] NVal: Norvaline (or Norvalyl)
[0447] pGlu: Pyroglutamic acid (or Pyroglutamyl)
[0448] Blc: .gamma.-Butyrolacton-.gamma.-carbonyl
[0449] Kpc: 2-Ketopiperidinyl-6-carbonyl
[0450] Otc: 3-Oxoperhydro-1,4-thiazin-5-carbonyl
[0451] Me: Methyl
[0452] Et: Ethyl
[0453] Bu: Butyl
[0454] Ph: Phenyl
[0455] TC: Thiazolidinyl-4(R)-carboxamide
[0456] The transformant Escherichia coli, designated JM109/p3H2-34,
which is obtained in the Example 3 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from Oct. 12, 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-4828. It is also on deposit from Oct. 12,
1994 with the Institute for Fermentation, Osaka, Japan (IFO) and
has been assigned the Accession Number IFO 15749.
[0457] The transformant Escherichia coli, designated JM109/pMGR20,
which is obtained in the Example 4 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 Dec. 14, 1994 with IFO and has been
assigned the Accession Number IFO 15773.
[0458] The mouse pancreatic .beta. cell line, designated MIN6, is
on deposit under the terms of the Budapest Treaty from Dec. 27,
1994, with NIBH and has been assigned the Accession Number FERM
BP-4954. It is also on deposit from Apr. 11, 1995 with IFO and has
been assigned the Accession Number IFO 50454.
[0459] The transformant Escherichia coli, designated SURE/pTS863,
which is obtained in the Example 12 mentioned herein below, is on
deposit under the terms of the Budapest Treaty from May 25, 1995,
with NIBH and has been assigned the Accession Number FERM BP-5110.
It is also on deposit from Jun. 1, 1995 with IFO and has been
assigned the Accession Number IFO 15826.
[0460] The transformant CHO cell, designated CHO/pTS863-5, which is
obtained in the Example 13 mentioned herein below, is on deposit
under the terms of the Budapest Treaty from May 25, 1995, with NIBH
and has been assigned the Accession Number FERM BP-5111. It is also
on deposit from Jun. 1, 1995 with IFO and has been assigned the
Accession Number IFO 50456.
[0461] The transformant CHO cell, designated CHO/pTS863-7, which is
obtained in the Example 13 mentioned herein below, is on deposit
under the terms of the Budapest Treaty from May 25, 1995, with NIBH
and has been assigned the Accession Number FERM BP-5112. It is also
on deposit from Jun. 1, 1995 with IFO and has been assigned the
Accession Number IFO 50457.
[0462] Each SEQ ID NO set forth in the SEQUENCE LISTING of the
specification refers to the following sequence:
[0463] [SEQ ID NO: 1] is a partial amino acid sequence encoded by
the mouse pancreatic .beta.-cell line, MIN6-derived galanin
receptor protein cDNA included in p3H2-34,
[0464] [SEQ ID NO: 2] is a full length amino acid sequence encoded
by the mouse pancreatic .beta.-cell line, MIN6-derived galanin
receptor protein cDNA included in pMGR20,
[0465] [SEQ ID NO: 3] is a nucleotide sequence of the mouse
pancreatic .beta.-cell line, MIN6-derived galanin receptor protein
cDNA fragment included in p3H2-34,
[0466] [SEQ ID NO: 4] is a nucleotide sequence of the translational
unit in the mouse pancreatic .beta.-cell line, MIN6-derived galanin
receptor protein cDNA fragment included in pMGR20,
[0467] [SEQ ID NO: 5] is a full length amino acid sequence encoded
by the human galanin receptor protein cDNA obtained in Example
11.
[0468] [SEQ ID NO: 6] is a nucleotide sequence of the translational
unit in the human galanin receptor protein cDNA obtained in Example
11.
[0469] 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. All patents, patent applications, and publications
mentioned herein, both supra and infra, are hereby incorporated
herein by reference.
EXAMPLES
[0470] 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.
Incidentally, the gene operation using Escherichia coli is carried
out by a method described in Maniatis, et al.: "Molecular Cloning"
(Cold Spring Harbor Laboratory, 1989).
Reference Example 1
Preparation of Synthetic DNA Primer for Amplifying DNA Coding for G
Protein Coupled Receptor Protein
[0471] 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.-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 .alpha..sub.2B
receptor protein (M91466, RATA2BAR) was made. As a result, highly
homologous regions or parts were found.
[0472] 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), mouse-derived TRH receptor protein
(S43387, S43387), rat-derived neuromedin K receptor protein
(J05189, RATNEURA), rat-derived adenosine A1 receptor protein
(M69045, RATALARA), human-derived neurokinin A receptor protein
(M57414, HUMNEKAR), rat-derived adenosine A3 receptor protein
(M94152, RATADENREC), human-derived 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.
[0473] 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 (CD019, 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).
[0474] 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: 7 which is complementary to the homologous nucleotide sequence
and the degenerate synthetic DNA having a nucleotide sequence
represented by SEQ ID NO: 8 which is complementary to the
homologous nucleotide sequence were produced. Nucleotide synthesis
was carried out by a DNA synthesizer.
Synthetic DNAs
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) CCTG-3' (SEQ ID NO: 7)
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) GGCAAA-3' (SEQ ID NO: 8)
[0475] 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 1
Preparation of Poly(A).sup.+ RNA Fraction from Mouse Pancreatic
.beta.-Cell Strain, MIN6 and Synthesis of cDNA
[0476] A total RNA was prepared from the mouse pancreatic
.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).
Example 2
Amplification of Receptor cDNA by PCR Using MIN6-Derived cDNA and
Sequencing
[0477] By using, as a template, 5 .mu.l of cDNA prepared from the
mouse pancreatic .beta.-cell strain, MIN6 in Example 1, PCR
amplification using the DNA primers synthesized in Reference
Example 1 was carried out. A reaction solution was composed of the
synthetic DNA primers 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.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.
Example 3
Subcloning of PCR Product into Plasmid Vector and Selection of
Novel Receptor Candidate Clone via Decoding Nucleotide Sequence of
Inserted cDNA Region
[0478] The PCR products obtained in Example 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 (Invitrogen Co.). The recombinant vectors were
introduced into E. coli JM109 competent cells (Takara Shuzo Co.,
Japan) to produce transformants. Then, transformant clones having a
cDNA-inserted fragment were selected in an LB (Luria-Bertani) agar
culture medium containing ampicillin, IPTG
(isopropylthio-.beta.-D-galactoside) and X-gal
(5-bromo-4-chloro-3-indoly- l-.beta.-D-galactoside). Only
transformant clones exhibiting white color were picked with a
sterilized toothstick to obtain transformant Escherichia coli
JM109/p3H2-34.
[0479] 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).
[0480] Homology retrieval was carried out based upon the determined
nucleotide sequence [FIG. 1]. 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/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. 1], and
homology retrieval was carried out in view of hydrophobicity
plotting [FIG. 2] and at the amino acid sequence level to find
homology relative to human somatostatin receptor subtype 4
(JN0605), human somatostatin receptor subtype 2 (B41795) and
rat-derived ligand unknown receptor (A39297) [FIG. 3].
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 4
Cloning of cDNA containing Whole Coding Region for Receptor Protein
from Mouse Pancreatic .beta.-Cell Strain, MIN6-Derived cDNA
Library
[0481] Superscript.TM. Lambda System (BRL, Cat. 8256) distributed
by BRL Co. and Gigapack 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).sup.+ RNA. The cDNA library was mixed with
E. coli Y1090r.sup.- 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 Pure Chemical Co., Japan) 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.
[0482] The filter was incubated overnight at 42.degree. C. together
with the probe mentioned herein below in a buffer containing 50%
formamide, 4.times.SSPE(20.times.SSPE (pH 7.4) is 3 M NaCl, 0.2 M
NaH.sub.2PO4.H.sub.2O, 25 mM EDTA), 5.times.Denhardt's solution,
0.1% SDS and 100 .mu.g/ml of salmon sperm DNA for
hybridization.
[0483] The probe used was obtained by cutting the DNA fragment
inserted in the plasmid, p3H2-34, obtained in Example 2, with
EcoRI, followed by recovery and labeling by incorporation of
[.sup.32P]dCTP (Dupont/NEN) with a random prime DNA labelling kit
(Amersham Co.).
[0484] 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.
[0485] 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 JM109 was
transformed with the plasmid to obtain a transformant E. coli
JM109/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 Example 2. 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 2.
Example 5
Sequencing of MIN6-Derived Receptor Protein Full-Length cDNA
[0486] Among the NotI-SalI fragments inserted in the plasmid,
pMGR20, obtained in Example 4, 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.
[0487] 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).
[0488] FIG. 4 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. 4. The amino acid
sequence of the receptor protein encoded by the DNA insert was that
as represented by SEQ ID NO: 2 (FIG. 4). Since the amino acid
sequence has 92% homology to the human-derived galanin receptor
protein at the amino acid sequence level, it was learned that the
cDNA insert in the pMGR20 is a mouse-derived galanin receptor
protein-encoding cDNA.
Example 6
Galanin Receptor Binding Experiment using MIN6 Cell Membrane
Fraction.
[0489] (1) Preparation of Membrane Fractions from MIN6 Cells.
[0490] MIN6 cells were cultured by a known method (Endocrinology,
vol. 127, pages 126-132, 1990). Thus, the culturing was carried out
in a Dulbecco modified Eagle's medium containing 15% of fetal
bovine serum, 4.5 g/liter of glucose, 5 .mu.l/liter of
mercaptoethanol, 75 mg/ml of penicillin and 50 mg/ml of
streptomycin in the presence of 5% carbon dioxide gas. The cultured
cells were washed with PBS containing EDTA and exfoliated from the
culturing device. The exfoliated cells were recovered by
centrifugation and subjected to the following method of preparing
the membrane fractions.
[0491] The recovered cells (about 2.5 ml) were suspended in a
buffer for homogenization (containing 10 mM of NaHCO.sub.3, 5 mM of
EDTA, 0.5 mM of PMSF, 10 .mu.g/ml of pepstatin, 20 .mu.g/ml of
leupeptin and 4 .mu.g/ml of E-64; pH: 7.2) and homogenized with a
polytron homogenizer (Kinematica) at 23,000 rpm for one minute. The
resulting homogenate was centrifuged in a Hitachi RP24A rotor using
a Hitachi High-Speed Centrifuging Machine (type: CR26H) at 5,000
rpm for ten minutes. The supernatant liquid after centrifugation
was recovered and subjected to an ultracentrifugation in a Hitachi
RP42 rotor using a Hitachi Ultracentrifugal Machine (type: SCP70H)
at 30,000 rpm for one hour to give pellets. The resulting pellets
were again suspended in a buffer for the homogenization and stored
at -70.degree. C. until its actual use.
[0492] (2) Receptor Binding Experiment using MIN6 Cell Membrane
Fractions.
[0493] The MIN6 cell membrane fractions prepared by the method of
the above-mentioned (1) were diluted with a buffer for the receptor
binding experiment (containing 20 mM of Tris, 1 mM of EDTA, 0.03%
of NaN , 0.1% of BSA, 0.05% of CHAPS, 0.5 mM of PMSF, 10 .mu.g/ml
of pepstatin, 20 .mu.g/ml of leupeptin and 4 .mu.g/ml of E-64; pH:
7.4) to make the membrane protein concentration 50 .mu.g/ml. Each
100 .mu.l of the diluted membrane fractions was charged in a test
tube made of polypropylene (Falcon; type 2038) and subjected to the
following receptor binding experiment. In the meanwhile, porcine
galanin (New England Nuclear) which was labeled with a commercially
available [.sup.125I] radioisotope was diluted with a buffer for
the receptor binding experiment to make its concentration 5 nM and
used in the following experiments.
[0494] Standard porcine galanin solution or galanin-related peptide
solution (3 .mu.l) with varied concentrations and 2 .mu.l of 5 nM
labeled galanin solution were mixed with 100 .mu.l of the
above-mentioned membrane fraction. The mixture was allowed to stand
in a water bath of 25.degree. C. for 75 minutes to promote the
receptor binding reaction. Thereafter, 1.5 ml of an ice-cooled
buffer for the binding experiment was added to the reaction
solution for quenching the binding reaction and filtered with a
glass fiber filter (GF/F, manufactured by Whatman) immediately
whereupon the membrane fractions were collected on the filter
paper. Then the filter paper was washed with 1.5 ml of the same
buffer and the amount of the radioisotope in the filter paper was
determined by a gamma-ray detector.
[0495] Amount of the labeled galanin bound therewith was expressed
in terms of PMB (percent of maximum binding) as calculated by the
following equation (2):
PMB=[(B-NSB)/(B.sub.0-NSB)].times.100 (2)
[0496] in which PMB: percent of maximum binding
[0497] B: the value when the sample is added
[0498] NSB: nonspecific binding amount (the binding amount of the
labeled galanin in the presence of 1 .mu.M of standard galanin)
[0499] B.sub.0: maximum binding amount (the binding amount of the
labeled galanin in the absence of the standard porcine galanin)
[0500] The result wherein the binding amounts (PMB) of the labeled
galanin as determined hereinabove were plotted against the
concentrations of standard porcine galanin, rat galanin, galanin
(1-16) partial peptide or galanin antagonist (galantide) is shown
in FIG. 7. From the result, each of the concentrations (IC.sub.50)
giving 50% of PMB was calculated and given in Table 1.
1 TABLE 1 Peptides IC.sub.50 Porcine Galanin 0.12 nM Rat Galanin
0.13 nM Galanin (1-16) 3.3 nM Galantide 0.69 nM
[0501] It is noted from Table 1 that, when the cell membrane
fractions of MIN6 cells were used, the receptor binding affinity of
the ligand to the galanin receptor was able to be determined by
means of a galanin receptor binding experiments.
Example 7
Screening of Galanin Receptor Agonist/Antagonist by Galanin
Receptor Binding Experiments
[0502] It is possible to conduct a screening of galanin receptor
angonist/antagonist by a method mentioned in Example 6. Thus, 1
.mu.l of the solution of test compounds and 2 .mu.l of a 5 nM
labeled galanin solution are mixed with 100 .mu.l of MIN6 cell
membrane fractions by the same manner as in Example 6. Thereafter,
the binding amount is determined by the same manner as in Example 6
whereby the compounds which decreased the PMB to an extent of lower
than a certain level are screened as galanin receptor
agonists/antagonists.
Example 8
Detection for Biological Activity of Galanin Using MIN6 Cells
[0503] It has been known that galanin is biologically active in
inhibiting the insulin secretion of a Langerhans islet of pancreas
by stimulation of glucose. Such an activity can be easily detected
by the following methods using MIN6 cells. Among the detecting
methods, measurement of insulin secretion by glucose stimulation
was principally conducted according to a known method
(Diabetologia, volume 36, pages 1139-1145, 1993).
[0504] Briefly, 3.times.10.sup.5 MIN6 cells were seeded on a
24-well plate and cultured in a Dulbecco modified Eagle's medium
(supplemented with 15% of fetal bovine serum, 4.5 g/liter of
glucose, 5 .mu.l/liter of mercaptoethanol, 75 mg/ml of penicillin
and 50 mg/ml of streptomycin) in the presence of 5% carbon dioxide
gas for three days.
[0505] The cells were washed for three times with a
Krebs-Ringer-HEPES buffer (containing 119 mM of NaCl, 4.74 mM of
KCl, 2.54 mM of CaCl.sub.2, 1.19 mM of MgSO.sub.4, 1.19 mM of
KH.sub.2PO.sub.4, 25 mM of NaHCO.sub.3, 10 mM of HEPES and 0.5% of
BSA) and cultured in a Krebs-Ringer-HEPES buffer to which 5 mM of
glucose was added at 37.degree. C. for 30 minutes.
[0506] Then the cells were washed with the Krebs-Ringer-HEPES
buffer twice. The cells were cultured at 37.degree. C. for 90
minutes in a Krebs-Ringer-HEPES buffer to which a varied amount of
rat galanin and 25 mM of glucose. The supernatant liquid after the
culturing was collected and the amount of insulin which was
secreted into the supernatant liquid was determined by a
commercially available radioimmunoassay kit (Amersham).
[0507] FIG. 8 shows the result in which the amount of insulin
secretion was plotted against the amount of galanin. It is apparent
from FIG. 8 that, when about 100 pM of galanin is added, the
insulin secretion which increased by a glucose stimulation is
decreased to an extent of about one half. As such, it is now clear
that the activity of galanin can be easily detected using the
above-mentioned method.
Example 9
Detection for Biological Activity of Galanin Using MIN6 Cells
[0508] When MIN6 cells were treated with forskolin, the cAMP
concentration in the cells increased whereupon secretion of insulin
increased. Such a phenomenon can be inhibited by addition of
galanin and can be used as a method of measuring the biological
activity of galanin.
[0509] The cells which were cultured and pretreated (by the
operations of washing, treating with 5 mM of glucose and washing)
by the same manner as in Example 8 were cultured at 37.degree. C.
for 90 minutes in a Krebs-Ringer-HEPES buffer to which varied
concentration of rat-type galanin, 100 .mu.M of forskolin, 200
.mu.M of isobutylmethylxantine (IBMX) and 6.25 mM of glucose were
added.
[0510] The supernatant after the culturing was recovered and the
amount of insulin which was secreted into the supernatant was
determined by a commercially available radioimmunoassay kit
(Amersham). In addition, the cells were treated with perchloric
acid to extract the cAMP in the cells and the cAMP in the extract
was determined by a commercially available radioimmunoassay kit
(Amersham). FIG. 9 shows the result in which the secreted amount of
insulin is plotted against the amount of galanin. FIG. 10 shows the
result in which the amount of intracellular cAMP was plotted. From
FIG. 9 and FIG. 10, it is noted that the activity of galanin can be
easily detected when secretion of insulin from MIN6 cells or cAMP
concentration in MIN6 cells was measured.
Example 10
Detection for Biological Activity of Galanin Agonist/Antagonist
Using MIN6 Cells
[0511] It is possible to measure the activity of galanin
agonist/antagonist by a method as mentioned in Example 8. Briefly,
the cells which are cultured and pretreated (by the operations of
washing, treating with 5 mM of glucose and washing) by the same
manner as in Example 8 are cultured at 37.degree. C. for 90 minutes
in a Krebs-Ringer-HEPES buffer to which a suitable concentration of
test compounds, 100 pM of rat galanin and 25 mM of glucose are
added.
[0512] The supernatant liquid after the culturing is recovered and
the amount of insulin secreted into the supernatant liquid is
determined using a commercially available radioimmunoassay kit
(Amersham). As a control, the amount of insulin is determined by
the same manner for the supernatant liquid obtained after culturing
in a Krebs-Ringer-HEPES buffer containing 25 mM glucose only and
also in a Krebs-Ringer-HEPES buffer containing 100 pM of rat
galanin and 25 mM of glucose. It is noted from the result that the
activity of galanin agonist/antagonist can be easily measured using
MIN6 cells.
Example 11
Cloning of cDNA Coding for Human Galanin Receptor Protein
[0513] Human melanoma Bowes cells were cultured using a DMEM medium
with high concentrations of glucose containing 10% fetal bovine
serum at 37.degree. C. under the condition of 95% air/5% CO.sub.2
and, when it became almost confluent, total RNA was prepared by a
guanidine-thiocyanate method. From the resulting total RNA was
prepared a poly A.sup.+ RNA fraction by an oligo(dT) cellulose
column. The poly A.sup.+ RNA (10 .mu.g) was treated with a random
hexamer and a reverse transcriptase to synthesize a single-stranded
DNA and then treated with Escherichia coli DNA polymerase I and
RNase H to synthesize a double-stranded DNA whereupon a
double-stranded cDNA was synthesized from poly A.sup.+ RNA. This
double-stranded cDNA was blunt ended with a T4 DNA polymerase and
then EcoRI adapters were added thereto. The resulting
double-stranded cDNA wherein both terminals were added with EcoRI
adapters was subjected to a gel filtration to remove cDNA of about
1,000 bp and less and then phosphate group was introduced into the
EcoRI adapters using a T4 polynucleotide kinase.
[0514] Then this cDNA was incorporated into a .lambda. gt11 EcoRI
arm and subjected to an in vitro packaging to prepare a cDNA
library (average chain length: about 1.6 kbp; rate of insertion:
98%) of melanoma Bowes cells having about 1.5.times.10.sup.6 pfu in
total. The .lambda. phage of this cDNA library was infected with
Escherichia coli Y109r.sup.- strain, seeded on each of soft agar
plates at about 1.8.times.10.sup.4 plaques each and incubated
overnight at 42.degree. C. to form plaques. The plaques were
transferred to a nitrocellulose filter, successively treated with a
modifying solution (0.5N sodium hydroxide and 1.5M sodium
chloride), a neutralizing solution (0.5M Tris-HCl (pH: 7.0) and
1.5M sodium chloride) and 3.times.SSC (20.times.SSC=3M sodium
chloride and 0.3M sodium citrate), air-dried and baked at
80.degree. C. for three hours whereupon the phage DNA was
immobilized on the nitrocellulose filter.
[0515] On the other hand, in order to obtain the cDNA fragments to
be used as a probe, synthetic oligonucleotides {circle over (1)}
and {circle over (2)} were synthesized based upon a base sequence
of the known human galanin receptor cDNA [Habert-Ortoll, E. et al.,
Proceedings of the National Academy of Sciences of the U.S.A., 91,
9780-9783 (1994)].
5'-TCCGTGGACCGCTACGTGGCCATCGTG-3'(SEQ ID NO: 9) {circle over
(1)}
[0516] It is a synthetic oligonucleotide containing a sense
sequence of +388 to +414 (wherein the translation initiation site
was named +1).
5'-GACTTATCACACATGAGTACAATTGGTTGATGG-3'(SEQ ID NO: 10) {circle over
(2)}
[0517] It is a synthetic oligonucleotide containing an antisense
sequence of +1024 to +1053.
[0518] An RT-PCR was carried out using those two synthetic
nucleotides as primers and 5 .mu.g of human melanoma Bowes cell
poly A.sup.+ RNA as a template whereupon cDNA fragments of 669 bp
containing C-terminals of human galanin receptor protein were
obtained. The cDNA fragments were incorporated into the HincII site
of pUC119 to give a plasmid pHGR54-7. The pHGR54-7 was subjected to
a double digestion with BamHI and HincII and the resulting cDNA
fragments containing the human galanin receptor protein C-terminal
were used as probes for screening the human melanoma Bowes cell
cDNA library.
[0519] Labeling of the probes was conducted by subjecting the
above-mentioned cDNA fragments to a random priming method using
[.alpha.-.sup.32P]dCTP. A hybridization was carried out at
85.degree. C. in a buffer for hybridization (5.times.SSPE,
5.times.Denhardt's solution, 100 .mu.g/l thermally modified salmon
sperm DNA, 0.1% SDS) containing labeled probes. The filter was
finally washed in 0.1.times.SSC, 0.1% SDS solution at 50.degree. C.
and subjected to an autoradiography to detect the plaques which
were hybridized with the probes.
[0520] After the phage DNA was extracted from the phage clone
lambda HGR2 obtained by that method, cDNA fragments were cut out by
digesting with a restriction enzyme EcoRI and inserted into the
EcoRI sites of the pUC118 plasmid to give pHGR2-3. The base
sequence of the cDNA fragments inserted thereinto was determined by
a conventional method using [.alpha.-.sup.32P]dCTP whereupon it was
found that said cDNA fragment was composed of 1,882 bp (FIG. 11 and
FIG. 12; SEQ ID NO: 6). There was one substitution with a base as
compared with the base sequence of the human galanin receptor cDNA
which was reported already [Habert-Ortoll, E. et al., Proceedings
of the National Academy of Sciences of the U.S.A., 91, 9780-9783
(1994)]. Said substitution with a base was within a translation
domain and is accompanied by an amino acid substitution, i.e.,
.sup.15Cys(TGT).fwdarw..sup.15Trp(TGG) (FIG. 11 and FIG. 12; SEQ ID
NO: 6). As such, a plasmid pHGR2-3 containing the human galanin
receptor DNA fragments was obtained.
Example 12
Construction of Expression Plasmid containing Human Galanin
Receptor Protein cDNA
[0521] pAKKO-111 (shown as pA1-11 in FIG. 13) was used as an
expression vector. The pAKKO-111 was constructed as follows:
Briefly, pTB1417 according to Japanese Unexamined Patent
Publication No. Hei-05/076385 was treated with HindIII and ClaI to
give DNA fragments of 1.4 kb containing SR.alpha. promoters and
poly A added signals. Further, pTB348 [Naruo, K. et al.,
Biochemical and Biophysical Research Communications, 128,
256-264(1985)] was treated with ClaI and SalI to give DNA fragments
of 4.5 kb containing dihydrofolate reductase (dhfr) genes. Those
DNA fragments were blunt ended with a T4 polymerase and ligated by
a T4 ligase to construct pAKKO-111 plasmid.
[0522] Human galanin receptor cDNA expression plasmid was prepared
by a method as shown in FIG. 13 from the plasmids pHGR2-3 and
pHGR54-7 containing the human galanin receptor cDNA fragments
obtained in Example 11. First, pHGR2-3 was subjected to a double
digestion using restriction enzymes BamHI and MunI and the
resulting DNA fragments of about 1,190 bp were inserted between
BamHI and MunI sites of pHGR54-7. In the meanwhile, pHGR2-3 was
digested with NcoI, the resulting fragments of 495 bp were blunt
ended with DNA polymerase I Klenow fragments, SalI linkers were
added thereto, then subjected to a double digestion with AgeI and
SacII and the resulting DNA fragments of 200 bp were inserted
between XmaI and SacII sites of the above-prepared plasmid
whereupon a plasmid pTS862 which contained the translation unit
only of the human galanin receptor cDNA was obtained.
[0523] Finally, SalI DNA fragments containing the translation
domain of the human galanin receptor protein cDNA of about 1.0 kbp
obtained by digesting the plasmid pTS862 with SalI were introduced
into the SalI site of pAKKO-111 in a regular order to give a human
galanin receptor protein cDNA expression plasmid pTS863. This
expression plasmid pTS863 was introduced into Escherichia coli to
give a transformant Escherichia coli SURE/pTS863.
Example 13
Expression of Human Galanin Receptor Protein cDNA in CHO
(dhfr.sup.-) Cells
[0524] Four kinds of CHO (dhfr.sup.-) cells (in which cell numbers
were stepwisely changed within a range of 3.times.10.sup.4 to
1.times.10.sup.6 cells) were sowed on laboratory dishes with 10 cm
diameter and cultured for 24 hours with a Ham's F12 medium
containing 10% of fetal bovine serum. The human galanin receptor
cDNA expression plasmid pTS863 (1.5 .mu.g) obtained in Example 12
was transfected to the above-prepared cells by a calcium phosphate
method. After 24 hours from the transfection, the medium was
substituted with a DMEM medium containing 10% of dialyzed fetal
bovine serum and the cells wherein the plasmid was incorporated in
chromosomes were selected. Colonies of the selected cells were
cloned to give two clones of cell strains CHO/pTS863-5 and
CHO/pTS863-7 which highly expressed the human galanin receptor in a
stable manner.
Example 14
Measurement of Human Galanin Receptor Activity of Human Galanin
Receptor Expression CHO Cells
[0525] Human galanin receptor expression CHO cells were seeded on a
12-well plate, cultured at 37.degree. C. under the condition of 95%
air/5% CO.sub.2 using a DMEM medium containing 10% of dialyzed
fetal bovine serum until a confluent was resulted and the medium
was exchanged on one day before the binding experiment was done
whereupon the human galanin receptor having an amino acid sequence
having SEQ ID NO: 5 was expressed.
[0526] Then the binding experiment to [.sup.125I] galanin (porcine)
was conducted as follows. First, the cells were washed twice with
each 1 ml of a buffer for measuring a binding (Hanks solution
containing 0.1% BSA and 0.05% of CHAPS) warmed at 37.degree. C.,
said buffer for binding measurement was sucked, then 0.5 ml of a
buffer for binding measurement containing 100 pM of [.sup.125I]
galanin (porcine) was added and a binding reaction was carried out
for one hour at 37.degree. C. under the condition of 95% air/5%
CO.sub.2. After completion of the reaction, the buffer for the
binding measurement was removed and washed thrice with each 1 ml of
a buffer for the binding measurement warmed at 37.degree. C. The
amount of [.sup.125I] galanin (porcine) bound with the cells was
measured by a gamma-counter after removing the cells with 0.2N
sodium hydroxide and was defined as a total binding amount.
Incidentally, the same operation was conducted after adding 1 .mu.M
of unlabeled galanin (porcine) at the binding reaction and the
amount of [.sup.125I] galanin bound with the cells was defined as
the nonspecific binding amount. The results are given in Table
2.
2TABLE 2 Total Binding Nonspecific Binding Cell Strain No. Amt.
(cpm) Amt (cpm) CHO/pTS863-5 50466.8 .+-. 502.9 1458 .+-. 100.1
CHO/pTS863-7 59158.6 .+-. 2095.1 1962.4 .+-. 56.3
[0527] It was confirmed from Table 2 that CHO/pTS863-5 and
CHO/pTS863-7 which are cell strains expressing the human galanin
receptor protein of the present invention in a high and stable
manner were capable of specifically binding with galanin which is a
ligand.
Example 15
Saturation Binding Experiments and Scatchard Plot Analysis with
Human [.sup.125I] Galanin in GAL5 Cell Membrane Fractions
[0528] CHO cells expressing human galanin receptor proteins (GAL5
cell, denoted by CHO/pTS863-5 in Example 13) were cultured in a
DMEM medium containing 10% dialyzed serum, 2 mM glutamine,
penicillin and streptomycin at 37.degree. C. under the condition of
95% air/5% CO.sub.2. The cells were collected with a
phosphate-buffered saline (PBS) containing 1 g/l EDTA, suspended in
a buffer for homogenization (10 mM HEPES, 5 mM EDTA, 0.03 %
NaN.sub.3, 10 .mu.g/ml of pepstatin, 0.5 mM phenylmethylsulfonyl
fluoride (PMSF), 20 .mu.g/ml of E-64, 40 .mu.g/ml of leupeptin, pH
7.3) and homogenized with a Polytron homogenizer. The resultant
homogenates were centrifuged at 2,500 rpm for 10 min under
4.degree. C. The resultant supernatant was ultracentrifuged at
30,000 rpm for 60 min under 4.degree. C. Pellets were suspended in
a buffer for homogenization to form a suspension as a GAL5 cell
membrane fraction.
[0529] The GAL5 cell membrane fraction was diluted with an assay
buffer (20 mM Tris., 1 mM EDTA, 0.08 % NaN.sub.3, 10 .mu.g/ml of
pepstatin, 0.5 mM PMSF, 20 .mu.g/ml of E-64, 40 .mu.g/ml of
leupeptin, 0.1% BSA, and 0.05% CHAPS, pH 7.4) to make the membrane
protein concentration 2 .mu.g/ml. Each 100 .mu.l of the diluted
membrane fractions was charged in a test tube.
[0530] The GAL5 cell membrane fractions were incubated with 15 pM
to 500 pM concentrations of human [.sup.125I] galanin for 75 min at
25.degree. C., then diluted with 1.5 ml of a filtration buffer (20
mM Tris., 1 mM EDTA, 0.03 % NaN.sub.3, 0.1% BSA, and 0.05% CHAPS,
pH 7.4, 4.degree. C.) and subjected to filtration through glass
fiber filters (GF/F, Whatman, Kent, UK) treated with
polyethylenimine. The filters were rinsed with 1.5 ml of the same
filtration buffer and the radiolabeled ligands remaining were
quantitated with an auto-.gamma.-counter (Beckman Instruments,
Inc., Palo Alto, Calif.). Nonspecific binding was determined in the
presence of 20 nM of unlabeled human galanin. Scatchard plot
analysis indicated its dissociation constant (K.sub.d) of 20 pM and
maximal number of binding sites (B.sub.MAX) of 9.6 pmol/mg
protein.
Example 16
Northern Blot Analysis Using Mouse Galanin Receptor Protein
Encoding cDNA
[0531] Five micrograms of poly (A).sup.+ RNAs from mouse brain,
thymus, spleen, lung, heart, liver, kidney, pancreas, testis,
intestinal smooth muscle, MIN6, and Neuro-2a were electrophoresed
on 1.2% agarose gel after denaturation with glyoxal and dimethyl
sulfoxide (Thomas, P.S., Proc. Natl. Acad. Sci. U.S.A., 77,
5201-5205, 1980). After the electrophoresis, the RNAs were
transferred onto a nitrocellulose filter (Schleicher & Schuell,
Dassel, Germany) and the filter was baked at 80.degree. C. for 2
hr. As a probe, the cDNA insert of p3H2-34 was excised by EcoRI
digestion and labeled with [.alpha..sup.32P]dCTP(222 TBq/mmol,
Dupont/NEN) by a Multiprime DNA labeling kit (Amersham
International PLC, Amersham Place, UK). Hybridization was conducted
overnight at 42.degree. C. in a buffer containing 50% formamide,
5.times.SSC, 50 mM NaHPO.sub.4, pH 6.5, 10.times.Denhardt's
solution, and 100 .mu.g/ml salmon sperm DNA. The filter was then
washed with 2.times.SSC, 0.1% SDS at 50.degree. C., and then
autoradiographed at -80.degree. C. for 12 days on an X-Omat film AR
(Eastman Kodak Company, Rochester, N.Y.) with an intensifying
screen. In Northern blot analysis using poly(A) RNAs from mouse
tissues, the faintly hybridizing signals for the mouse galanin
receptor only in the brain and small intestine (FIG. 14) were
detected . The result of the Northern blot indicated that the
expression level of galanin receptor mRNA was substantially lower
in mouse normal tissues.
Example 17
[0532] (1) Expression of Mouse Galanin Receptor cDNA in CHO
Cells
[0533] A cDNA clone with a complete translation unit, pMGR20
(obtained in Example 4), was digested with NotI, blunt ended, and
ligated with XbaI linker (Takara Shuzo Co., Ltd., Kyoto, Japan).
The cDNA fragment was excised by SalI and XbaI digestion and
inserted between SalI and SpeI sites of a mammalian cell expression
vector, pAKKO-111H (Hinuma, et al., Biochim. Biophys. Acta, 1219,
251-259 (1994)). A resultant expression plasmid with the mouse
galanin receptor cDNA downstream of the SR.alpha. promoter and with
dhfr gene as a selection marker was designated as pAKKOMGR20. The
plasmid DNA was transfected into CHO dhfr.sup.- cells with a
CellPhect Transfection Kit (Pharmacia). Transformants were selected
in .alpha.-MEM medium without deoxyribonucleoside and
ribonucleoside (GIBCO BRL) supplemented with dialyzed fetal bovine
serum (GIBCQ BRL).
[0534] (2) Binding Assay with Porcine [.sup.125I] Galanin
[0535] CHO cells transformed with pAKKOMGR20 and pAKKO-111H were
grown in a 12-well tissue culture plate at 2.0.times.10.sup.5
cells/well and cultured for one day. After two washings with Hanks'
balanced salt solution (HBSS) containing 0.1% BSA, the cells were
incubated with 100 pM porcine [.sup.125I] galanin (Dupont/NEN) at
room temperature for 1 hour in the presence or absence of unlabeled
porcine galanin (1 .mu.M at final concentration). The cells were
then washed three times with HBSS containing BSA, lysed with 0.1N
NaOH, 1% SDS, and the radiolabeled ligands remaining were
quantitated with an auto-.gamma.-counter (Beckman Instruments,
Inc., Palo Alto, Calif.). For competitive binding experiments and
Scatchard plot analysis, membrane fractions were prepared from the
transformed CHO cells. The cells grown in 225-cm.sup.2 tissue
culture flasks for three days were dispersed in a
phosphate-buffered saline (PBS) containing 5 mM EDTA and then
harvested by centrifugation. The cells were washed with the same
buffer, and then suspended in 10 mM sodium carbonate buffer (pH
7.5), including 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), 20 .mu.g/ml of leupeptin, 4 .mu.g/ml of E-64, and 0.5
.mu.g/ml of pepstatin. After the cells were homogenized with a
Polytron homogenizer, the homogenates were centrifuged at 3,000 rpm
for 10 min in a Hitachi RR2A2 rotor. The resultant supernatant was
ultracentrifuged at 30,000 rpm for 60 min in a Beckman Type 30
rotor. Pellet suspension was then done in a buffer containing 20 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.5 mM PMSF, 20 .mu.g/ml of
leupeptin, 4 .mu.g/ml of E-64, and 0.5 .mu.g/ml of pepstatin, and
used as a membrane fraction.
[0536] Competitive binding and saturation binding experiments were
performed as described by Ohtaki et al. (J. Biol. Chem., 268,
26650-26657, (1993)).
[0537] In brief, the membrane fractions were incubated in a buffer
containing 20 mM Tris-HCl (pH 7.5), 0.05% CHAPS, 0.1% BSA, 5 mM
EDTA, 0.5 mM of PMSF, 20 .mu.g/ml of leupeptin, 4 .mu.g/ml of E-64,
and 0.5 .mu.g/ml of pepstatin with porcine [.sup.125I] galanin at
25.degree. C. for 75 min. Bound and free ligands were separated by
filtration through glass fiber filters (GF/F, Whatman, Kent, UK)
treated with 0.3% of polyethylenimine. Nonspecific binding was
determined in the presence of 1 .mu.M of unlabeled porcine galanin.
In competitive binding experiments the concentrations of galanin
and galanin analogs were added to the buffer simultaneously with
porcine [.sup.125I] galanin.
[0538] CHO cells transformed with the expression plasmid for the
mouse galanin receptor cDNA bound significant amounts of
[.sup.125I] galanin as compared with control cells (FIG. 15).
[0539] Membrane fractions (1 .mu.g of protein) were incubated with
concentrations of porcine [.sup.125I] galanin for 75 min at
25.degree. C. in Scatchard plot analysis. The results shown are
from one representative experiment performed in triplicate assays.
Each symbol represents the mean value.+-.S.E.M. B, [.sup.125I]
galanin bound (pmol/mg protein), B/F, bound to free ratio (pmol/mg
protein.cndot.nM). Scatchard plot analysis indicated the presence
of a single class of high-affinity binding site with a dissociation
constant (K.sub.d) of 45 pM and maximal number of binding sites
(B.sub.MAX) of 5 pmol/mg protein (FIG. 17).
[0540] Competitive experiments on the binding of porcine
[.sup.125I] galanin to mouse galanin receptor were conducted.
Competitions to the porcine [.sup.125I] galanin (100 pM at final
concentrations) bindings were examined with unlabeled porcine
(.DELTA.), rat (.circle-solid.), human (.box-solid.) galanins,
galanin-(1-16) (.largecircle.), and M15 (.tangle-solidup.).
Membrane fractions (1 .mu.g of protein) were incubated with the
ligands for 75 min at 25.degree. C. The amounts of [.sup.125I]
galanin bound were expressed as percentages against the control.
Each symbol represents the mean value.+-.S.E.M. of the triplicate
assays. IC.sub.50 values were 0.25.+-.0.03 nM (porcine galanin),
0.25.+-.0.01 nM (rat galanin), 0.43.+-.0.03 nM (human galanin),
0.83.+-.0.01 nM (Ml5), and 3.6.+-.0.04 nM [galanin-(1-16)],
respectively.
[0541] The binding of [.sup.125I] galanin was competitively
inhibited by galanin and galanin-derived peptides. Porcine and rat
galanin exhibited almost the same high efficiency in inhibiting the
[.sup.125I] galanin binding whereas human galanin was somewhat
lower. The Ki values of porcine, rat, and human galanins were
0.072.+-.0.008, 0.069.+-.0.002, and 0.12.+-.0.008 nM, respectively.
The galanin receptor antagonists M15 and galanin-(1-16) also
effectively inhibit the [.sup.125I] galanin binding, and their Ki
values were 0.23.+-.0.003 and 1.0.+-.0.011 nM, respectively (FIG.
18). These obtained values were almost comparable to those from
MIN6 cell membranes.
[0542] (3) cAMP Assay
[0543] The CHO cells were seeded at 2.0.times.10.sup.5 cells/well
in 24-well tissue culture plates and cultured for two days. The
cells were washed two times with HBSS containing 0.1% BSA and 1 mM
IBMX, and then the same buffer with experimental agents were added
to the wells. After incubation at 37.degree. C. for 30 min, the
media were discarded and intracellular cAMP was extracted with
ice-cold ethanol. The aliquots of extracts were evaporated and the
amounts of cAMP were quantitated by a cAMP EIA system (Amersham) as
described by the manufacturer.
[0544] Galanin receptor-mediated inhibition of forskolin-stimulated
cAMP production was observed. CHO-MGR20 or mock transformed CHO
cells were incubated with forskolin (10 .mu.M) and porcine galanin
(0.1 .mu.M) at 37.degree. C. for 30 min. The reaction was
terminated by extracting the cells with ice-cold ethanol. The
amounts of intracellular cAMP were quantitated by EIA. Values
indicated are mean.+-.S.E.M. in triplicate assays.
[0545] It is examined by the assessment of galanin-induced signal
transduction to confirm further that the mouse galanin receptor
expressed in CHO cells was functional. The pancreatic galanin
receptor has been demonstrated to induce the inhibition of insulin
release through a pathway involving G proteins negatively coupled
to adenylate cyclase (Cormont, M.,et al., Diabates, 40, 1170-1176,
1991; Gillison, S., et al., Diabates, 43, 24-32, 1994). The
treatment with porcine galanin potently inhibited
forskolin-stimulated cAMP accumulation in the galanin receptor
cDNA-introduced CHO cells (FIG. 19). The CHO transformants with the
plasmid vector without cDNA insert also showed forskolin-stimulated
cAMP accumulation, but it was not inhibited by the galanin
treatment. The treatment with galanin alone did not alter the cAMP
levels in CHO transformants (FIG. 19).
[0546] The galanin receptor protein of the present invention and
the DNA coding for said protein can be used for {circle over (1)}
acquisition of antibody and antiserum; {circle over (2)}
construction of expression system for of a recombinant type
receptor protein; {circle over (3)} development of the receptor
binding assay system using said expression system and screening of
the candidate compounds for pharmaceuticals; {circle over (4)}
conducting a drug design based upon a comparison with structurally
analogous ligands and receptors; {circle over (5)} preparation of
probes and PCR primers for a gene diagnosis; {circle over (6)}
preparation of transgenic animals; and {circle over (7)}
preparation of model patient animals deficient in the receptor
protein DNA. Elucidation of the structure and property of the
mouse-derived galanin receptor is particularly related to the
development of unique pharmaceuticals which act on such a
system.
[0547] Furthermore, the human galanin receptor protein of the
present invention is a novel protein having an amino acid sequence
which is different from that of the known human galanin receptor
protein. The cells (particularly CHO cells) retaining the
expression vector containing the human galanin receptor of the
present invention are capable of expressing far more amount of
human galanin receptor protein than the known COS cells containing
the human galanin receptor protein are.
[0548] The human galanin receptor protein of the present invention
or partial peptide thereof or the cells containing the human
galanin receptor protein or a cell membrane fraction thereof is
capable of effectively screening the human galanin receptor agonist
or antagonist.
[0549] When the screening method of the present invention is used,
it is possible to advantageously select the agonist or the
antagonist whereby pharmaceutical agents can be developed in
earlier stage. The agonist is useful, for example, as an inhibitor
for acetylcholine liberation, an inhibitor for insulin secretion,
an inhibitor for a learning behavior or an inhibitor for satiety
and also as a preventive and therapeutic agent for schizophrenic
disease and as sedative while the antagonist is useful, for
example, as an accelerator for acetylcholine liberation, an
accelerator for insulin secretion, an inhibitor for growth hormone
secretion, an accelerator for a learning behavior or as an
accelerator for satiety and also as a preventive and therapeutic
agent for diabetes, Alzheimer's disease and dementia.
Sequence CWU 1
1
* * * * *