U.S. patent application number 10/027049 was filed with the patent office on 2003-01-30 for neuropeptide y receptor y5 and nucleic acid sequences.
Invention is credited to Bloomquist, Brian T., Cornfield, Linda J., Flores-Riveros, Jaime R., Hu, Yinghe, McCaleb, Michael L..
Application Number | 20030022283 10/027049 |
Document ID | / |
Family ID | 21768840 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022283 |
Kind Code |
A1 |
Hu, Yinghe ; et al. |
January 30, 2003 |
Neuropeptide Y receptor Y5 and nucleic acid sequences
Abstract
The present invention provides novel NPY/PYY receptor proteins
and the nucleic acid sequence encoding them. The invention is
directed to the isolation, characterization, and pharmacological
use of these receptors and nucleic acids. In particular, this
invention provides human and rat NPY/PYY receptors (which we call
the NPY Y5 receptor) and nucleic acids. Also provided are
recombinant expression constructs useful for transfecting cells and
expressing the protein in vitro and in vivo. The invention further
provides methods for detecting expression levels of the protein as
well as methods for screening for receptor antagonists and agonists
to be used for the treatment of obesity or anorexia,
respectively.
Inventors: |
Hu, Yinghe; (North Haven,
CT) ; McCaleb, Michael L.; (Madison, CT) ;
Bloomquist, Brian T.; (New Haven, CT) ;
Flores-Riveros, Jaime R.; (Madison, CT) ; Cornfield,
Linda J.; (Hamden, CT) |
Correspondence
Address: |
Michael S. Greenfield
McDonnell Boehnen Hulbert & Berghoff
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Family ID: |
21768840 |
Appl. No.: |
10/027049 |
Filed: |
December 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10027049 |
Dec 20, 2001 |
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09327035 |
Jun 7, 1999 |
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6368824 |
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09327035 |
Jun 7, 1999 |
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08838399 |
Apr 7, 1997 |
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5965392 |
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60014969 |
Apr 8, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/320.1; 435/325; 530/350; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61P 43/00 20180101; A61K 38/1709 20130101; G01N 2800/303 20130101;
A61P 3/10 20180101; A61P 3/00 20180101; G01N 33/6893 20130101; C07K
14/70571 20130101; A61P 3/04 20180101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/252.3; 435/320.1; 530/350; 536/23.2 |
International
Class: |
C07K 014/705; C07H
021/04; C12P 021/02; C12N 005/06; C12N 001/21 |
Claims
We claim:
1. An isolated nucleic acid encoding a neuropeptide Y receptor
comprising a molecule having a nucleotide sequence substantially
the same as SEQ ID NO 1, SEQ ID NO 3, or SEQ ID NO 5.
2. A homogenous composition of the neuropeptide Y receptor
comprising a molecule having an amino acid sequence substantially
the same as SEQ ID NO 2, SEQ ID NO 4, or SEQ ID NO 6.
3. A vector comprising the nucleic acid according to claim 1.
4. A vector according to claim 3 adapted for expression in a cell
further comprising regulatory elements necessary for expression of
the nucleic acid operatively linked to the nucleic acid to enable
expression of the nucleic acid.
5. A vector according to claim 4 wherein the cell is a mammalian
cell.
6. A vector according to claim 5 wherein the cell is a human 293
cell.
7. A vector according to claim 4 that is a plasmid.
8. A vector according to claim 7 wherein the plasmid is the
pBluescript plasmid.
9. A vector according to claim 7 wherein the plasmid is the pcDNA3
plasmid.
10. A vector according to claim 3 which is self-replicating.
11. A cell transformed with the nucleic acid according to claim 1
that expresses the nucleic acid.
12. A cell according to claim 11 that is a bacterium cell, an
insect cell, or a yeast cell.
13. A cell according to claim 11 that is a mammalian cell.
14. A cell according to claim 13 that is a human 293 cell.
15. A nucleic acid probe comprising a nucleic acid complementary to
the nucleic acid according to claim 1.
16. An antisense oligonucleotide having a sequence complementary to
the nucleic acid according to claim 1 and that inhibits expression
of the nucleic acid.
17. A membrane or membrane preparation comprising a membrane or
portion thereof of a cell expressing a nucleic acid having a
nucleotide sequence substantially the same as SEQ ID NO 1, SEQ ID
NO 3, or SEQ ID NO 5.
18. A membrane or membrane preparation according to claim 17
wherein the cell is a mammalian cell.
19. An antibody or fragment thereof that is immunologically
reactive to a mammalian Y5 receptor.
20. An antibody or fragment according to claim 19 thereof wherein
the mammalian Y5 receptor has an amino acid sequence substantially
the same as one chosen from the group consisting of SEQ ID NO 2,
SEQ ID NO 4, and SEQ ID NO 6.
21. An antibody or fragment thereof according to claim 19 that is a
monoclonal antibody.
22. An antibody or fragment thereof according to claim 20 that is a
monoclonal antibody.
23. A cell line producing an antibody according to claim 19.
24. A cell line producing an antibody according to claim 20.
25. A cell line producing an antibody according to claim 21.
26. A cell line producing an antibody according to claim 22.
27. An epitope of a mammalian Y5 receptor wherein the epitope is
immunologically reactive to the antibody or fragment thereof
according to claim 19.
28. An epitope of a mammalian Y5 receptor wherein the epitope is
immunologically reactive to the antibody or fragment thereof
according to claim 20.
29. An epitope of a mammalian Y5 receptor wherein the epitope is
immunologically reactive to the antibody or fragment thereof
according to claim 21.
30. An epitope of a mammalian Y5 receptor wherein the epitope is
immunologically reactive to the antibody or fragment thereof
according to claim 22.
31. A method of producing a neuropeptide Y receptor comprising
culturing a cell according to claim 11 and recovering the receptor
expressed by the cell.
32. A method of producing a neuropeptide Y receptor comprising
culturing a cell according to claim 12 and recovering the receptor
expressed by the cell.
33. A method of producing a neuropeptide Y receptor comprising
culturing a cell according to claim 13 and recovering the receptor
expressed by the cell.
34. A method of producing a neuropeptide Y receptor comprising
culturing a cell according to claim 14 and recovering the receptor
expressed by the cell.
35. A method of identifying a neuropeptide Y agonist or antagonist
comprising contacting a potential agonist or antagonist molecule
with a membrane or membrane preparation according to claim 17.
36. A method of identifying a neuropeptide Y agonist or antagonist
comprising contacting a potential agonist or antagonist molecule
with a membrane or membrane preparation according to claim 18.
37. A neuropeptide Y antagonist comprising a compound identified
according to claim 36.
38. A method of suppressing the appetite of a mammal comprising
administering to the mammal an appetite suppressing amount of a
neuropeptide Y antagonist according to claim 37.
39. A method of suppressing the appetite of a mammal according to
claim 38 wherein the amount of antagonist is from about 0.01 to
about 100 mg/kg.
40. A pharmaceutical composition comprising an effective appetite
suppressing amount of an antagonist according to claim 37 together
with a pharmaceutically acceptable carrier.
41. A neuropeptide Y agonist comprising a compound identified
according to claim 36.
42. A method of stimulating the appetite of a mammal comprising
administering to the mammal an appetite stimulating amount of a
neuropeptide Y agonist according to claim 41.
43. A method of stimulating the appetite of a mammal according to
claim 42 wherein the amount of agonist is from about 0.01 to about
100 mg/kg.
44. A pharmaceutical composition comprising an effective appetite
stimulating amount of an agonist according to claim 41 together
with a pharmaceutically acceptable carrier.
45. A non-human transgenic mammal that expresses the nucleic acid
having a sequence substantially the same one chosen from the group
consisting of SEQ ID NO 1, SEQ ID NO 3, and SEQ ID NO 5.
Description
[0001] This is a continuation-in-part application of U.S.
Provisional Serial No. 60/014,969 filed on Apr. 8, 1996.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a novel neurotransmitter
Neuropeptide Y receptor, its nucleic acid sequence, and compounds,
compositions, and methods for their use.
[0004] 2. Summary of the Related Art
[0005] Neuropeptide Y (NPY) is a 36-amino acid peptide
neurotransmitter that is located throughout the central and
peripheral nervous systems. Tatemoto, Proc. Natl. Acad. Sci. USA
79, 5485 (1982); Hazlewood, Proc. Soc. Exp. Biol. Med. 202, 44
(1993). It affects a broad range of phenomena, including blood
pressure regulation, memory, anxiolysis/sedation, food and water
appetite, vascular and other smooth muscle activity, intestinal
electrolyte secretion, and urinary sodium excretion. E.g., Colmers
and Wahlestedt, The Biology of Neuropeptide Y and Related Peptides
(Humana Press, Totowa, N.J., 1993); Kalra et al., Phys. &
Behavior 50, 5 (1991).
[0006] Peptide YY (PYY) is also a 36 amino acid peptide and has
significant sequence homology (70%) to NPY. Tatemoto et al., Nature
296, 659 (1982). Its anatomical distribution is similar to that of
NPY, although it is located mainly in the endocrine cells of the
lower gastrointestinal tract. Bottcher et al., Regul. Pept. 8, 261
(1984). Like NPY, PYY stimulates feeding in rats. Morley et al.,
Brain Res. 341, 200 (1985). Along with the pancreatic polypeptide
(PP), NPY and PYY have a common tertiary structure, characterized
by the so-called PP-fold. Glover, Eur. J. Biochem. 142, 379 (1985).
Both NPY and PYY show about a 50% sequence homology with PP.
[0007] Because of their structural similarities, NPY and PYY have a
number of common receptors. At least four receptor subtypes, Y1,
Y2, Y3, and Y4/PP, have been identified. The affinity for NPY, PYY,
and various fragments thereof varies among the subtypes. See, e.g.,
Bard et al. (WO 95/17906) and references cited therein. For
example, Y1 and Y2 subtypes have high affinity for NPY and PYY.
Whereas Y1 has high affinity for (Leu.sup.31Pro.sup.34)NPY
((LP)NPY)and low affinity for (13-36)NPY, Y2 behaves oppositely. Y3
has high affinity for NPY but low affinity for PYY. Y4/PP has a
high affinity for PP but relatively low affinity for NPY.
[0008] Wahlestedt (WO 93/24515) and Larhammar et al. (J. Biol.
Chem. 267, 10935 (1992)) describe the cloning and identification of
the human Y1-type NPY/PYY receptor isolated from human fetal brain
tissue. Selbie et al. (WO 93/09227) disclosed the full length cDNA
sequence of the Y1 receptor from human hippocampus. Eva et al.
(FEBS Lett. 271, 81 (1990)) cloned the NPY Y1 receptor from rat
forebrain. Eva et al. (FEBS Lett. 314, 285 (1992)) cloned the NPY
Y1 receptor from murine genomic DNA.
[0009] The Y2-type receptor has also been cloned. Gerald et al. (WO
95/21245) disclosed the cDNA sequence of human hippocampal Y2 and
two rat Y2 clones. Rose et al. (J. Biol Chem. 270, 22661 (1995))
disclosed the cDNA sequence of the Y2 receptor from a human
neuroblastoma cell line.
[0010] Bard et al. (supra) and Lundell et al. (J. Biol Chem. 270,
29123 (1995)) described cloning the cDNA sequence of the Y4/PP
receptor from both rat spleen and human placenta.
[0011] To date, the Y3 receptor has not been cloned.
[0012] Because of the important role of NPY and PYY in a number of
physiological processes, such as feeding, there is a strong need to
further develop materials and methods for investigating the
mechanistic behavior of these compounds and for treating diseased
and other abnormal states associated with the physiological
processes in which NPY and PYY act. Specifically, the NPY
analogs/fragments that induce feeding, such as (LP)(3-36)NPY, do
not bind to the previously identified NPY/PYY receptors with
affinities consistent with the feeding response. Accordingly, there
is a need and desire to identify the NPY/PYY receptor that is
responsible for the feeding response. Antagonists to such a
receptor could be used to treat obesity and diabetes by reducing
appetite and food consumption.
SUMMARY OF THE INVENTION
[0013] The present invention provides, inter alia, novel NPY/PYY
receptor proteins. Also provided are the nucleic acid sequences
encoding these novel receptor proteins, as well as compounds and
methods for using these proteins and their nucleic acid
sequences.
[0014] The present invention provides novel proteins, nucleic
acids, and methods useful for developing and identifying compounds
for the treatment of such diseases and disorders as obesity.
Identified and disclosed herein is the protein sequence for a novel
receptor for the neurotransmitters Neuropeptide Y (NPY) and Peptide
YY (PYY) and the nucleic acid sequence encoding this receptor,
which we call the NPY Y5 (or simply "Y5") receptor. The importance
of this discovery is manifested in the effects of NPY, which
include blood pressure regulation, memory enhancement,
anxiolysis/sedation, and increased food intake. Thus, this receptor
protein is useful for screening for NPY/PYY agonist and antagonist
activity for controlling these conditions.
[0015] In one aspect of the present invention, we provide isolated
nucleic acid sequences for a novel NPY and PYY receptor, the Y5
receptor. In particular, we provide the cDNA sequences encoding for
the rat and human receptors and isoforms thereof. These nucleic
acid sequences have a variety of uses. For example, they are useful
for making vectors and for transforming cells, both of which are
ultimately useful for production of the Y5 receptor protein. They
are also useful as scientific research tools for developing nucleic
acid probes for determining receptor expression levels, e.g., to
identify diseased or otherwise abnormal states. They are useful for
developing analytical tools such as antisense oligonucleotides for
selectively inhibiting expression of the receptor gene to determine
physiological responses.
[0016] In another aspect of the present invention, we provide a
homogenous composition comprising the receptor Y5 protein. The
protein is useful for screening drugs for agonist and antagonist
activity, and, therefore, for screening for drugs useful in
regulating physiological responses associated with the Y5 receptor.
Specifically, antagonists to the Y5 receptor could be used to treat
obesity and diabetes by reducing appetite and food consumption,
whereas agonists could be used for the treatment of anorexic
conditions. The proteins are also useful for developing antibodies
for detection of the protein.
[0017] Flowing from the foregoing are a number of other aspects of
the invention, including (a) vectors, such as plasmids, comprising
the receptor Y5 nucleic acid sequence that may further comprise
additional regulatory elements, e.g., promoters, (b) transformed
cells that express the Y5 receptor, (c) nucleic acid probes, (d)
antisense oligonucleotides, (e) agonists, (f) antagonists, and (g)
transgenic mammals. Further aspects of the invention comprise
methods for making and using the foregoing compounds and
compositions.
[0018] The foregoing merely summarizes certain aspects of the
present invention and is not intended, nor should it be construed,
to limit the invention in any manner. All patents and other
publications recited herein are hereby incorporated by reference in
their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 displays the competition curves of various peptides
for [.sup.125I]PYY to Y5 receptor membranes transiently expressed
in COS-7 cells.
[0020] FIG. 2 displays saturation curves for specific binding of
[.sup.125I]PYY to Y5 receptor membranes transiently expressed in
COS-7 cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] The present invention comprises, in part, a novel NPY/PYY
receptor protein, the Y5 receptor. Particularly preferred
embodiments of the Y5 receptor are those having an amino acid
sequence substantially the same as SEQ ID NOs 2, 4, or 6. As used
herein, reference to the Y5 receptor is meant as a reference to any
protein having an amino acid sequence substantially the same as SEQ
ID NOs 2, 4, or 6. The present invention also comprises the nucleic
acid sequence encoding the Y5 protein, which nucleic acid sequences
is substantially the same as SEQ ID NOs 1, 3, or 5. Receptors SEQ
ID NOs 2 and SEQ ID NO 4 are rat Y5 receptors and appear to be
allelic variations, with SEQ ID NO 4 the most commonly occurring
and, therefore, the preferred embodiment of the rat Y5 receptor of
this invention. SEQ ID NO 6 is the human Y5 receptor and its
preferred embodiment.
[0022] As used herein, a protein "having an amino acid sequence
substantially the same as SEQ ID NO x" (where "x" is the number of
one of the protein sequences recited in the Sequence Listing) means
a protein whose amino acid sequence is the same as SEQ ID NO x or
differs only in a way such that IC.sub.50[(3-36)NPY],
IC.sub.50[(Leu.sup.31Pro.sup.34)NPY], and
IC.sub.50[(Leu.sup.31Pro.sup.34) (3-36)NPY] as determined according
to the method detailed in Example 4, infra, are less than or equal
to 30 nM. The NPY fragments (3-36)NPY, (Leu.sup.31Pro.sup.34)NPY
and (Leu.sup.31Pro.sup.34)(3-36)NPY induce a feeding response.
Those skilled in the art will appreciate that conservative
substitutions of amino acids can be made without significantly
diminishing the protein's affinity for NPY, PYY, and fragments and
analogs thereof. Other substitutions may be made that increase the
protein's affinity for these compounds. Making and identifying such
proteins is a routine matter given the teachings herein, and can be
accomplished, for example, by altering the nucleic acid sequence
encoding the protein (as disclosed herein), inserting it into a
vector, transforming a cell, expressing the nucleic acid sequence,
and measuring the binding affinity of the resulting protein, all as
taught herein.
[0023] As used herein the term "a molecule having a nucleotide
sequence substantially the same as SEQ ID NO y" (wherein "y" is the
number of one of the protein-encoding nucleotide sequences listed
in the Sequence Listing) means a nucleic acid encoding a protein
"having an amino acid sequence substantially the same as SEQ ID NO
y+1" (wherein "y+1" is the number of the amino acid sequence for
which nucleotide sequence "y" codes) as defined above. This
definition is intended to encompass natural allelic variations in
the Y5 sequence. Cloned nucleic acid provided by the present
invention may encode Y5 protein of any species of origin, including
(but not limited to), for example, mouse, rat, rabbit, cat, dog,
primate, and human. Preferably the nucleic acid provided by the
invention encodes Y5 receptors of mammalian, and most preferably,
rat or human origin.
[0024] The invention also includes nucleotide sequences encoding
chimeric proteins comprised of parts of the Y5 receptor and parts
of other related seven-transmembrane receptors.
[0025] The 6B clone (SEQ ID NO 1) (see Example 2, infra) has a 2.4
kb cDNA insert with a open reading frame from nucleotide 248 to
1582 that encodes a 445 amino acid protein (SEQ ID NO 2).
Hydrophobicity plot analysis using PEPPLOT of GCG shows that the Y5
receptor has seven transmembrane-like domains, indicating it might
be a G-protein-coupled receptor. Unlike other known subtypes of NPY
receptor family, the third intracellular loop of the Y5 receptor is
unusually long. Another novel feature of the Y5 peptide sequence is
that it has a much shorter C-terminal tail sequence than other
known members of the NPY receptor family. It is also important to
note that the Y5 sequence shows only 30-33% amino acid sequence
identity to other NPY receptors.
[0026] Nucleic acid hybridization probes provided by the invention
are DNAs consisting essentially of the nucleotide sequences
complementary to any sequence depicted in SEQ ID NOs 1, 3, and 5
that is effective in nucleic acid hybridization. Nucleic acid
probes are useful for detecting Y5 gene expression in cells and
tissues using techniques well-known in the art, including, but not
limited to, Northern blot hybridization, in situ hybridization, and
Southern hybridization to reverse transcriptase-polymerase chain
reaction product DNAs. The probes provided by the present
invention, including oligonucleotide probes derived therefrom, are
also useful for Southern hybridization of mammalian, preferably
human, genomic DNA for screening for restriction fragment length
polymorphism (RFLP) associated with certain genetic disorders. As
used herein, the term complementary means a nucleic acid having a
sequence that is sufficiently complementary in the Watson-Crick
sense to a target nucleic acid to bind to the target under
physiological conditions or experimental conditions those skilled
in the art routinely use when employing probes.
[0027] Receptor Y5 binds various fragments and analogs of NPY and
PYY with affinities different from that of the known receptors. The
rank order of binding affinity of receptor Y5 was found to be:
NPY=(LP)NPY=PYY=(3-36)NPY=(LP)(3-36)NPY>(10-36)NPY>(18-36)NPY
[0028] Table 1, infra, presents a more detailed affinity profile of
the Y5 receptor for NPY, PYY, and various fragments thereof. As
used herein, a protein having substantially the same affinity
profile as the Y5 receptor means a protein in which the IC.sub.50
of each of the peptides listed in Table 1, infra, is no more than
an order of magnitude greater than those listed in Table 1 for each
of the respective peptides as measured according to the methods
described in Example 4. Importantly, the NPY analogs/fragments that
induce feeding, such as (LP)(3-36)NPY, do not bind to the
previously identified NPY/PYY receptors with affinities consistent
with the feeding response.
[0029] The production of proteins such as receptor Y5 from cloned
genes by genetic engineering means is well known in this art. The
discussion which follows is accordingly intended as an overview of
this field, and is not intended to reflect the full state of the
art.
[0030] DNA which encodes receptor Y5 may be obtained, in view of
the instant disclosure, by chemical synthesis, by screening reverse
transcripts of mRNA from appropriate cells or cell line cultures,
by screening genomic libraries from appropriate cells, or by
combinations of these procedures, as illustrated below. Screening
of mRNA or genomic DNA may be carried out with oligonucleotide
probes generated from the Y5 gene sequence information provided
herein. Probes may be labeled with a detectable group such as a
fluorescent group, a radioactive atom or a chemiluminescent group
in accordance with known procedures and used in conventional
hybridization assays, as described in greater detail in the
Examples below. In the alternative, the Y5 gene sequence may be
obtained by use of the polymerase chain reaction (PCR) procedure,
with the PCR oligonucleotide primers being produced from the Y5
gene sequence provided herein. See U.S. Pat. No. 4,683,195 to
Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis.
[0031] Receptor Y5 may be synthesized in host cells transformed
with a recombinant expression construct comprising a nucleic acid
encoding the receptor Y5. Such a recombinant expression construct
can also be comprised of a vector that is a replicable DNA
construct. Vectors are used herein either to amplify DNA encoding
Y5 and/or to express DNA which encodes Y5. For the purposes of this
invention, a recombinant expression construct is a replicable DNA
construct in which a DNA sequence encoding Y5 is operably linked to
suitable control sequences capable of effecting the expression of
Y5 in a suitable host. The need for such control sequences will
vary depending upon the host selected and the transformation method
chosen. Generally, control sequences include a transcriptional
promoter, an optional operator sequence to control transcription, a
sequence encoding suitable mRNA ribosomal binding sites, and
sequences which control the termination of transcription and
translation. Amplification vectors do not require expression
control domains. All that is needed is the ability to replicate in
a host, usually conferred by an origin of replication, and a
selection gene to facilitate recognition of transformants. See,
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, Cold Spring Harbor Press, New York, 1989).
[0032] Vectors useful for practicing the present invention include
plasmids, viruses (including phage), retroviruses, and integratable
DNA fragments (i.e., fragments integratable into the host genome by
homologous recombination). The vector replicates and functions
independently of the host genome, or may, in some instances,
integrate into the genome itself Suitable vectors will contain
replicon and control sequences which are derived from species
compatible with the intended expression host. The vectors may be
self-replicating. Suitable vectors for the purposes of the present
invention include pBluescript, pcDNA3, and, for insect cells,
baculovirus. A preferred vector is the plasmid pcDNA3
(Invitrogen).
[0033] Construction of suitable vectors containing the desired
coding and control sequences employs standard ligation and
restriction techniques that are well understood in the art.
Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are cleaved, tailored, and relegated in the form desired.
[0034] Site-specific DNA cleavage is performed by treating with the
suitable restriction enzyme (or enzymes) under conditions that are
generally understood in the art, and the particulars of which are
specified by the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs, Product
Catalog. In general, about 1 .mu.g of plasmid or DNA sequence is
cleaved by one unit of enzyme in about 20 .mu.l of buffer solution.
Often excess of restriction enzyme is used to ensure complete
digestion of the DNA substrate. Incubation times of about one hour
to two hours at about 37.degree. C. are workable, although
variations are tolerable. After each incubation, protein is removed
by extraction with phenol/chloroform, and may be followed by ether
extraction. The nucleic acid may be recovered from aqueous
fractions by precipitation with ethanol. If desired, size
separation of the cleaved fragments may be performed by
polyacrylamide gel or agarose gel electrophoresis using standard
techniques. A general description of size separations is found in
Methods in Enzymology 65, 499-560 (1980).
[0035] Transformed host cells are cells which have been transformed
or transfected with recombinant expression constructs made using
recombinant DNA techniques and comprising mammalian Y5-encoding
sequences. Preferred host cells for transient transfection are
COS-7 cells. Transformed host cells may ordinarily express Y5, but
host cells transformed for purposes of cloning or amplifying
nucleic acid hybridization probe DNA need not express the receptor.
When expressed, the mammalian Y5 protein will typically be located
in the host cell membrane. See, Sambrook et al., ibid.
[0036] Cultures of cells derived from multicellular organisms are
desirable hosts for recombinant Y5 protein synthesis. In principal,
any higher eukaryotic cell culture is workable, whether from
vertebrate or invertebrate culture. However, mammalian cells are
preferred, as illustrated in the Examples. Propagation of such
cells in cell culture has become a routine procedure. See Tissue
Culture (Academic Press, Kruse & Patterson, Eds., 1973).
Examples of useful host cell lines are bacteria cells, insect
cells, yeast cells, human 293 cells, VERO and HeLa cells,
LMTK.sup.- cells, and WI138, BHK, COS-7, CV, and MDCK cell lines.
Human 293 cells are preferred.
[0037] The invention provides homogeneous compositions of mammalian
Y5 produced by transformed eukaryotic cells as provided herein.
Such homogeneous compositions are intended to be comprised of
mammalian Y5 protein that comprises at least 90% of the protein in
such homogenous composition. The invention also provides membrane
preparation from cells expressing Y5 as the result of
transformation with a recombinant expression construct, as
described here.
[0038] Mammalian Y5 protein made from cloned genes in accordance
with the present invention may be used for screening compounds for
Y5 agonist or antagonist activity, or for determining the amount of
a Y5 agonist or antagonist drug in a solution (e.g., blood plasma
or serum). For example, host cells may be transformed with a
recombinant expression construct of the present invention, Y5
protein expressed in those host cells, the cells lysed, and the
membranes from those cells used to screen compounds for Y5 binding
activity. Competitive binding assays in which such procedures may
be carried out are well known in the art. By selection of host
cells which do not ordinarily express Y5, pure or crude
preparations of membranes containing Y5 can be obtained. Further,
Y5 agonists and antagonists can be identified by transforming host
cells with a recombinant expression construct as provided by the
present invention. Membranes obtained from such cells (and
membranes of intact cells) can be used in binding studies wherein
the drug dissociation activity is monitored.
[0039] It is known that the neurotransmitter NPY is a regulator of
appetite. As shown herein, the various NPY analogs/fragments that
induce feeding, such as (LP)(3-36)NPY, bind with a high affinity to
the Y5 receptor. Conversely, the NPY analogs/fragments that bind to
the Y5 receptor with a lower affinity, such as (20-36)NPY, do not
elicit feeding. It is therefore evident that by contacting the Y5
receptor with agonists and antagonists, feeding can be modulated.
Accordingly, antagonists to the Y5 receptor, identified by the
methods described herein, can be used to reduce appetite and hence
treat obesity, diabetes and hyperlipidemia, and, conversely,
agonists to the Y5 receptor can be used to treat conditions such as
anorexia.
[0040] This invention provides a pharmaceutical composition
comprising an effective amount of a agonist or antagonist drug
identified by the method described herein and a pharmaceutically
acceptable carrier. Such drugs and carrier can be administered by
various routes, for example oral, subcutaneous, intramuscular,
intravenous or intracerebral. The preferred route of administration
would be oral at daily doses of about 0.01-100 mg/kg.
[0041] This invention provides a method of treating obesity,
diabetes or hyperlipidemia, wherein the abnormality is improved by
reducing the activity of Y5 receptor or blocking the binding of
ligands to a Y5 receptor, which method comprises administering an
effective amount of the antagonist-containing pharmaceutical
composition described above to suppress the subject's appetite.
Similarly, the invention also provides methods for treating
diseases and conditions resulting from underfeeding and/or a loss
of appetite, which method comprises administering an effective
amount of an agonist-containing pharmaceutical composition
described above to stimulate the subject's appetite.
[0042] The recombinant expression constructs of the present
invention are useful in molecular biology to transform cells which
do not ordinarily express Y5 to thereafter express this receptor.
Such cells are useful as intermediates for making cell membrane
preparations useful for receptor binding assays, which are in turn
useful for drug screening. Drugs identified from such receptor
assays can be used for the treatment of obesity, diabetes or
anorexia.
[0043] The recombinant expression constructs of the present
invention are also useful in gene therapy. Cloned genes of the
present invention, or fragments thereof, may also be used in gene
therapy carried out by homologous recombination or site-directed
mutagenesis. See generally Thomas & Capecchi, Cell 51, 503-512
(1987); Bertling, Bioscience Reports 7, 107-112 (1987); Smithies et
al., Nature 317, 230-234 (1985).
[0044] Oligonucleotides of the present invention are useful as
diagnostic tools for probing Y5 gene expression in tissues. For
example, tissues are probed in situ with oligonucleotide probes
carrying detectable groups by conventional autoradiographic
techniques, as explained in greater detail in the Examples below,
to investigate native expression of this receptor or pathological
conditions relating thereto. Further, chromosomes can be probed to
investigate the presence or absence of the Y5 gene, and potential
pathological conditions related thereto, as also illustrated by the
Examples below. Probes according to the invention should generally
be at least about 15 nucleotides in length to prevent binding to
random sequences, but, under the appropriate circumstances may be
smaller.
[0045] The invention also provides antibodies that are
immunologically reactive to a mammalian Y5, preferably rat or human
Y5. The antibodies provided by the invention are raised in animals
by inoculation with cells that express a mammalian Y5 or epitopes
thereof, using methods well known in the art. Animals that are used
for such inoculations include individuals from species comprising
cows, sheep, pigs, mice, rats, rabbits, hamsters, goats and
primates. Preferred animals for inoculation are rodents (including
mice, rats, hamsters) and rabbits. The most preferred animal is the
mouse.
[0046] Cells that can be used for such inoculations, or for any of
the other means used in the invention, include any cell line which
naturally expresses a mammalian Y5, or any cell or cell line that
expresses a mammalian Y5 or any epitope thereof as a result of
molecular or genetic engineering, or that has been treated to
increase the expression of a mammalian Y5 by physical, biochemical
or genetic means. Preferred cells are human cells, most preferably
HEK 293 and BHK cells that have been transformed with a recombinant
expression construct comprising a nucleic acid encoding a mammalian
Y5, preferably a rat or human Y5, and that express the mammalian Y5
gene product.
[0047] The present invention provides monoclonal antibodies that
are immunologically reactive with an epitope of mammalian Y5 or
fragment thereof and that is present on the surface of mammalian
cells, preferably human or mouse cells. These antibodies are made
using methods and techniques well known to those of skill in the
art.
[0048] Monoclonal antibodies provided by the present invention are
produced by hybridoma cell lines, that are also provided by the
invention and that are made by methods well known in the art.
Hybridoma cell lines are made by fusing individual cells of a
myeloma cell line with spleen cells derived from animals immunized
with cells expressing the Y5 receptor, preferably rat or human
cells, as described above. The myeloma cell lines used in the
invention include lines derived from myelomas of mice, rats,
hamsters, primates and humans. Preferred myeloma cell lines are
from mouse. The animals from whom spleens are obtained after
immunization are rats, mice and hamsters, preferably mice, most
preferably Balb/c mice. Spleen cells and myeloma cells are fused
using a number of methods well known in the art, including but not
limited to incubation with inactivated Sendai virus and incubation
in the presence of polyethylene glycol (PEG). The most preferred
method for cell fusion is incubation in the presence of a solution
of 45% (w/v) PEG-1450. Monoclonal antibodies produced by hybridoma
cell lines can be harvested from cell culture supernatant fluids
from in vitro cell growth; alternatively, hybridoma cells can be
injected subcutaneously and/or into the peritoneal cavity of an
animal, most preferably a mouse, and the monoclonal antibodies
obtained from blood and/or ascites fluid.
[0049] Monoclonal antibodies provided by the present invention are
also produced by recombinant genetic methods well known to those of
skill in the art, and the present invention encompasses antibodies
made by such methods that are immunologically reactive with an
epitope of a mammalian Y5.
[0050] The present invention encompasses fragments of the antibody
that are immunologically reactive with an epitope of a mammalian
Y5. Such fragments are produced by any number of methods, including
but not limited to proteolytic cleavage, chemical synthesis or
preparation of such fragments by means of genetic engineering
technology. The present invention also encompasses single-chain
antibodies that are immunologically reactive with an epitope of a
mammalian Y5 made by methods known to those of skill in the
art.
[0051] The present invention also encompasses an epitope of a
mammalian Y5 that is comprised of sequences and/or a conformation
of sequences present in the mammalian Y5 molecule. This epitope may
be naturally occurring, or may be the result of proteolytic
cleavage of the mammalian Y5 molecule and isolation of an
epitope-containing peptide or may be obtained by synthesis of an
epitope-containing peptide using methods well known to those
skilled in the art. The present invention also encompasses epitope
peptides produced as a result of genetic engineering technology and
synthesized by genetically engineered prokaryotic or eukaryotic
cells.
[0052] The invention also includes chimeric antibodies, comprised
of light chain and heavy chain peptides immunologically reactive to
an epitope that is a mammalian Y5. The chimeric antibodies embodied
in the present invention include those that are derived from
naturally occurring antibodies as well as chimeric antibodies made
by means of genetic engineering technology well known to those of
skill in the art.
[0053] Also provided by the present invention are non-human
transgenic animals grown from germ cells transformed with the Y5
nucleic acid sequence according to the invention and that express
the Y5 receptor according to the invention and offspring and
descendants thereof. Also provided are transgenic non-human mammals
comprising a homologous recombination knockout of the native Y5
receptor, as well as transgenic non-human mammals grown from germ
cells transformed with nucleic acid antisense to the Y5 nucleic
acid of the invention and offspring and descendants thereof.
Further included as part of the present invention are transgenic
animals which the native Y5 receptor has been replaced with the
human homolog. Of course, offspring and descendants of all of the
foregoing transgenic animals are also encompassed by the
invention.
[0054] Transgenic animals according to the invention can be made
using well known techniques with the nucleic acids disclosed
herein. E.g., Leder et al., U.S. Pat. Nos. 4,736,866 and 5,175,383;
Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual
(Cold Spring Harbor Laboratory (1986)); Capecchi, Science 244, 1288
(1989); Zimmer and Gruss, Nature 338, 150 (1989); Kuhn et al.,
Science 269, 1427 (1995); Katsuki et al., Science 241, 593 (1988);
Hasty et al., Nature 350, 243 (1991); Stacey et al., Mol. Cell Biol
14, 1009 (1994); Hanks et al., Science 269, 679 (1995); and Marx,
Science 269, 636 (1995). Such transgenic animals are useful for
screening for and determining the physiological effects of Y5
receptor agonists and antagonist. Consequently, such transgenic
animals are useful for developing drugs to regulate physiological
activities in which NPY and/or PYY participate.
[0055] The following Examples are provided for illustrative
purposes only and are not intended, nor should they be construed,
as limiting the invention in any manner.
EXAMPLES
Example 1
Isolation and Sequencing of Rat Y5 Receptor
[0056] Isolation of Rat Hypothalamus mRNA and Construction of cDNA
Library
[0057] Expression cloning strategy was used to clone novel NPY
receptor in rat hypothalamus cDNA library. RNA was obtained from 9
frozen rat hypothalami weighing a total of 0.87 grams. Poly(A) RNA
was isolated directly from the tissue using the Promega PolyATtract
System 1000 kit (Promega, Madison, Wis.). The hypothalami were
homogenized in 4 mL of 4 M guanidine thiocyanate-25 mM sodium
citrate, pH 7.1-2% .beta.-mercaptoethanol using a Polytron at
full-speed for approximately 1 minute. To the homogenized tissue 8
mL of 4 M guanidine thiocyanate-25 mM sodium citrate, pH 7.1-1%
.beta.-mercaptoethanol which had been preheated to 70.degree. C.
was added. After mixing thoroughly, 870 pmol biotinylated oligo(dT)
was added; the mixture was incubated at 70.degree. C. for 5
minutes. The homogenate was subjected to centrifugation at
12000.times.g for 10 minutes at room temperature; the homogenate
was transferred to a clean tube and 10.44 mL Streptavidin
MAGNESPHERE.RTM. Paramagnetic Particles (SA-PMPs) which had been
prepared as per the published protocol was added. (Promega Corp.
published protocol TM 228; Promega Corporation, Madison, Wis.). The
homogenate and SA-PMPs were incubated together for 2 minutes at
room temperature after which the homogenate was decanted while the
SA-PMP-biotinylated oligo(dT)-hypothalamic poly(A) RNA complex was
retained in the tube by a magnetic stand. The complex was washed-as
per the protocol, after which the RNA was precipitated and
resuspended in water. 25 micrograms of this poly(A) RNA was used by
Invitrogen (Invitrogen Corporation, San Diego, Calif.) to prepare a
cDNA expression library. The protocols used by Invitrogen to
prepare the cDNA library are essentially based upon the procedures
of Okayama and Berg (Molec. Cell. Biol. 2, 161 (1982)) and Gubler
and Hoffman (Gene 25, 263 (1983)) (Invitrogen Corporation
publications 130813sa and 130928sa). An oligo(dT) anchor primer was
used for reverse transcription, and the library was cloned
unidirectionally into pcDNA3 vector which contains a CMV promoter
for eukaryotic expression. The cDNA library had 5.3.times.10.sup.5
primary recombinants with an average insert size of 2.59 kb.
[0058] Isolation of a Novel Y5 Receptor cDNA Clone
[0059] The rat hypothalamus cDNA library was plated on the
LB/Ampicillin plates in pools of 1,000 independent colonies. The
plates were incubated at 37.degree. C. for about 20 hours and the
bacteria from each plate were scraped in 4-5 ml LB/Ampicillin
media. Two ml of the bacteria samples were used for plasmid
preparation and one ml of each pool was stored at -80.degree. C. in
15% glycerol.
[0060] COS-7 cells were grown in Dulbecco's Modified Eagle Medium (
DMEM, GIBCO 11965-092), 10% fetal bovine serum (GIBCO 16000-028),
and 1.times.antibiotic/antimycotic solution (GIBCO 15240-039)
(Gaithersburg, Md.). Cells were trypsinized and split at 50 to 70%
confluency.
[0061] DNA from 1300 pools was transfected into COS-7 cells for
[.sup.125I]PYY binding assays. Twenty four hours before
transfection, cells were plated into flaskette chambers (Nunc, Inc.
177453, Naperville, Ill.) at 3.times.10.sup.5 cells/flaskette
(equivalent to 3.times.10.sup.4 cells/cm.sup.2). Two .mu.g of
plasmid DNA from each pool was transfected into the cells using 10
.mu.l of Lipofectamine (GIBCO 18324-012) according to the
manufacture's protocol. Forty eight hours after transfection, the
[.sup.125I]PYY binding assay was performed in the flaskette
chamber. The cells were treated with 2 ml total binding buffer: 10
mM HEPES, 5 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2, 1.2
mM MgSO.sub.4, 150 mM NaCl, 25 mM NaHCO.sub.3, 10 mg/ml bovine
serum albumin, 0.5 mg/ml bacitracin and 0.4 mg/ml soybean trypsin
inhibitor at room temperature for 15 minutes. The cells were then
incubated with 100 pM porcine [.sup.125I]PYY (Amersham (Arlington
Heights, Ill., Specific Activity 4000 Ci/mmol) in the total binding
buffer for 90 minutes at room temperature. After binding, the cells
were washed three times with ice-cold total binding buffer without
ligand and one time with cold phosphate buffered saline (PBS).
Cells were fixed with 1% cold glutaraldehyde in PBS for 15 minutes,
washed once with cold PBS/0.5 M Tris, pH 7.5 and incubated in
PBS/0.5 M Tris, pH 7.5 for 15 minutes at 4.degree. C. After washing
one more time with cold PBS, the slides were dipped in 0.5% gelatin
at 42.degree. C. and dried under vacuum. The dried slides were
dipped in 50% photographic emulsion (Kodak (Rochester, N.Y.) NTB2)
at 42.degree. C. and exposed in the darkbox for four days at
4.degree. C. After four days of exposure, the darkbox was moved to
room temperature for one hour and slides were developed in
developer D-19 (Kodak) for three minutes at 15.degree. C. and fixed
in fixer (Kodak) for three minutes at 15.degree. C., washed in
water and air dried. Cells were stained with Diff-Quik stain set
(Baxter, McGaw Park, Ill.) and air dried. Slides were dipped into
xylenes and mounted with DPX mountant (Electron Microscopy Science,
Fort Washington, Pa.). Positive cells were identified using dark
field microscopy.
[0062] Twenty one positive pools were identified. Since the
hypothalamus expresses different subtypes of NPY receptors
including Y1 and Y2 receptors, we analyzed all the positive pools
for Y1, Y2 and Y4/PP receptors by PCR. Of the 21 positive pools
tested as described above, 12 pools contained Y1, 4 pools contained
Y2 and none contained Y4/PP. Five pools (Y217, Y555, Y589, Y861 and
Y1139) were negative by PCR analysis. The pool Y217 was subdivided
in 24 subpools of 200 colonies, then 50 colonies, and finally a
single clone, the Y217.24.13.6B clone (6B), was isolated.
[0063] DNA and Peptide Sequences Analysis
[0064] Plasmid DNA was sequenced by Lark Technologies Inc.
(Houston, Tex.) and Biotechnology Resource Laboratory of Yale
University (New Haven, Conn.) using Sequenase Kit (US Biochemical,
Cleveland, Ohio) or Applied Biosystems' automatic sequencer system
(model 373A). The peptide sequence was deduced from the long
open-reading-frame of the nucleotide sequence. DNA and peptide
sequences were analyzed using the GCG program (Genetics Computer
Group, Madison, Wis.). The results are embodied in SEQ ID NO 1 (the
nucleic acid sequence) and SEQ ID NO 2 (the amino acid
sequence).
Example 2
Localization of Rat YS Receptor in Brain and Other Tissues
[0065] Northern Blot
[0066] To study the expression level of the Y5 receptor in the rat
brain and other tissues, we did Northern blot analysis using the 6B
2.4 kb probe. A rat multiple tissue Northern blot (Clontech
Laboratories, Palo Alto, Calif.) was hybridized to the
.sup.32P-labeled rat 6B probe. The blot contains 2 .mu.g of poly
A.sup.+ RNA per lane from rat heart, brain, spleen, lung, liver,
skeletal muscle, kidney, and testis. Hybridization was carried out
in 1.times.hybridization solution containing 6.times.SSC (0.9 M
NaCl, 0.09 M Na Citrate, pH 7.0), 5.times.Denhardt's solution (0.1%
polyvinylpyrrolidone, 0.1% ficoll type 400, 0.1% bovine serum
albumin), 100 mg/ml sheared, and denatured salmon sperm DNA at
65.degree. C. The filter was washed at 65.degree. C. in
0.1.times.SSC, 0.1% SDS and exposed to Kodak XAR 5 film with two
intensifying screens. A single 2.6 kb band was detected in the
brain after overnight exposure of the blot. No bands were found
from other tissues (heart, spleen, lung, liver, skeletal muscle,
kidney and testis) in the Clontech multiple tissue Northern blot,
even after six days of exposure.
[0067] We tested 6B expression in more rat tissues and different
regions of brain. mRNA was isolated from rat whole brain, cortex,
hypothalamus, hippocampus, olfactory bulb, spleen, stomach, kidney,
small intestine, adrenal and pancreas using Fast Track Isolation
Kit (Invitrogen). Ten .mu.g of mRNA from different brain regions
and multiple tissues were run on a denaturing formaldehyde 1%
agarose gel, transferred to a Nytran membrane (Schleicher and
Schuell) and hybridized with .sup.32P-labeled 6B 2.4 kb probe and
washed at high stringency. After overnight hybridization, the
filter was washed at high stringency and exposed to X-ray film with
intensifying screens. The 6B receptor mRNA was detectable in the
brain regions examined after one day exposure, but no signal was
observed from other tissues, even after a week exposure with double
intensifying screens.
Example 3
Isolation of Two Isoforms of the Rat Y5 Receptor
[0068] Plasmid DNA from pools Y555, Y589, and Y861 described in
Example 1 were hybridized to the Y5 probe at high stringency. A
single positive clone was isolated from the Y555 pool and sequenced
as described in Example 1. Compared to the 6B DNA sequence, the
Y555 sequence (SEQ ID NO 4 has a 123 bp insert sequence located at
the 5'-untranslated region between nucleotides 239 and 240 of Y5
clone. The coding region of the clones Y555, Y589, and Y861 has the
same sequence as clone 6B, except for one nucleotide substitution
(C to T) at position 430 of the 6B clone. The nucleotide
substitution changes the amino acid proline to leucine in the first
transmembrane domain. The corresponding amino acid sequence is
given by SEQ ID NO 4.
[0069] The different isoforms of the receptor could be the allelic
variants of the same gene. To test this hypothesis, we analyzed
genomic DNA from 16 rats. The genomic DNA from each animal was used
as template for PCR analysis. A 314 bp DNA fragment that contains
the site of the nucleotide variation was amplified and sequenced.
Of the 16 DNA samples tested, 14 samples had a T at position 430
and 2 samples had a C. This result strongly suggests that the amino
acid variation is an allelic variant.
Example 4
Pharmacological Characterization of the Novel Rat NPY Receptors
[0070] Transient Transfection
[0071] Monkey kidney cells (COS-7) were maintained in T-175
cm.sup.2 flasks (NUNC) at 37.degree. C. with 5% CO.sub.2 in a
humidified atmosphere. Cells were grown in Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 2 mM glutamine, 10% fetal
bovine serum, 1 mM sodium pyruvate, and antibiotic/antimycotic.
Cells at 70% confluency were transfected with Y5 DNA using the
Lipofectamine method (GIBCO-BRL). 15 .mu.g DNA and 90 .mu.l
Lipofectamine were added to each flask. Media was completely
replaced 24 hours post transfection, and membranes were harvested
24 hours later.
[0072] Stable Expression of the Rat NPY Y5 Receptor (Clone
Y861)
[0073] A strain of the human embryonic kidney cell line 293 adapted
to grow in suspension (293S) was used for these experiments.
Approximately 1.times.10.sup.6 cells were seeded onto a 10-cm dish
24 hours prior to transfection. The rat NPY Y5 cDNA (Y861),
subcloned in the eukaryotic expression vector pcDNA3 (Invitrogen,
Carlsbad, Calif.) was first linearized with NotI and purified using
a Wizard PCR Prep kit (Promega). In preparation for transfection,
15 .mu.g of the linearized DNA were added to 500 .mu.l of DMEM cell
culture media, and 30 .mu.l of Lipofectamine (Life Sciences) were
added into a separate 500 .mu.l aliquot of DMEM. These two
solutions were mixed together and incubated for 20 minutes at room
temperature and the resulting DNA/lipid complexes were then slowly
added to the cells (which had been previously rinsed once with
serum-free DMEM) and covered with a total volume of 10 ml. Cells
were then transferred to a humidified 10% CO.sub.2 incubator and
left for 4 hours at 37.degree. C., at which time the media was
replaced with DMEM supplemented with 8% FBS. After 16 hours, cells
were trypsinized and split at a 1:15 ratio into 10-cm dishes
containing DMEM/8% FBS in the presence of 700 .mu.g/ml of G418
(selection media). When discrete colonies became apparent (after
approximately 10 days), cells were pooled and carried through 2
additional passages in selection media. Cells were then trypsinized
and diluted in preparation for cloning by limited dilution (CBLD),
such that an average of one cell was seeded in each well of a
96-well microtiter culture plate, and was inspected periodically
for the subsequent 2 to 3 weeks. After 21 days in culture under
selection conditions, those wells containing single colonies were
selected and transferred to 24-well culture plates following
trypsinization. Each of these clones was propagated until
sufficient quantities were available for testing [.sup.125I]PYY
binding activity, from which one particular clone designated E7 was
selected on the basis of its high level of binding activity.
[0074] Stable Expression of the Human NPY Y5 Receptor
[0075] 293 cells were plated onto a T75 flask one day prior to
transfection such that they were 50-70% confluent when used for the
experiment. The human NPY Y5 intronless genomic clone HG.PCR15,
containing the full length open reading frame encoding the
receptor, was first linearized with Not I and purified using a
Wizard PCR Prep kit (Promega). For each transfection, 8 .mu.g of
linearized DNA were added to 1.25 ml of Optimem culture media (Life
Sciences) and 37 .mu.l of Transfectam (Promega) were added to 1.25
ml of Optimem. These two solutions were then mixed together and
added to cells previously washed once with Optimem. After an
incubation period of 5 hours, the DNA/Transfectam mixture was
removed, cells were washed with PBS and fed with DMEM supplemented
with 10% FBS. Cells were left intact for two days, and then
switched to selection media (DMEM 10% FBS containing 350 .mu.g/ml
of G418) for 5-10 days followed by CBLD as described above. The
individual clone 293.hy5.sb.8 was selected on the basis of its high
level of [.sup.125I]PYY binding activity, using the intact cell
binding protocol from above.
[0076] Membrane Preparation
[0077] The media was removed from each flask of transfected cells,
and the cells were washed twice with 20 ml ice-cold phosphate
buffered saline. The cells were scraped from the flask in 5 ml of
Tris buffer (20 mM Tris-HCl and 5 mM EDTA, pH 7.7), and then
transferred to a centrifuge tube. Each flask was washed with an
additional 5 ml of Tris buffer and combined in the centrifuge tube.
The cells were polytroned for 2.times.10 seconds (12 mm probe,
7000-8000 rpm) and centrifuged 5 minutes (Centra 7R, International
Equipment Co., Needham Heights, Mass.) at 800 rpm and 4.degree. C.
The supernatant was then transferred to a clean centrifuge tube and
was centrifuged at 30,000.times.g for 30 minutes and 4.degree. C.
The supernatant was removed and the pellet was stored at
-80.degree. C. Protein concentration was measured using the Bio-Rad
kit pursuant to the standard manufacturer's protocol (Biorad
Laboratories, Hercules, Calif.) with bovine IgG as the
standard.
[0078] [.sup.125I]PYY Binding Assay for NPY Y5 Receptors
[0079] The binding assays were performed on GF/C Millipore
(Bedford, Mass.) 96-well plates pretreated with 0.02%
polyethylenimine (PEI) for at least 2 hours prior to use. The PEI
was aspirated from the plates on a vacuum manifold immediately
before the samples were added to the wells. All peptides, tissue
and radioligand were diluted with binding buffer (25 mM Tris, 120
mM NaCl, 5 mM KCl, 1.2 mM KH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2, 1.2
mM MgSO.sub.4, 0.1% BSA and 0.5 mg/ml bacitracin, pH 7.4). For
competition assays, increasing concentrations of peptide were
incubated with [.sup.125I]PYY and tissue. In a final volume of 200
.mu.l, samples consisted of: membrane protein (i.e., 2.5-15 or
10-30 .mu.g membrane protein for rat Y5 or human Y5, respectively);
75-100 pM [.sup.125I]PYY NEN-DuPont (Boston, Mass.); peptide
dilution or binding buffer. Nonspecific binding was defined by 1
.mu.M PYY, NPY, PYY, (2-36)NPY, (10-36)NPY, (LP)(3-36)NPY and
(32D-Trp)NPY were synthesized at Bayer Corp. (West Haven, Conn.).
All other peptides were purchased from either Peninsula (Belmont,
Calif.) or Bachem (Torrance, Calif.).
[0080] For saturation experiments, increasing concentrations of
[.sup.125I]PYY were incubated with membrane and 1 .mu.M PYY. After
a 2 hour incubation at room temperature with constant mixing, the
samples were aspirated on a vacuum manifold. The wells were washed
with three 200 .mu.l aliquots of ice-cold binding buffer. The
individual wells were punched into 12.times.75 mm plastic tubes,
and counted on a Wallac (Gaithersburg, Md.) gamma counter. Binding
data were analyzed using the nonlinear regression curve-fitting
program RS/1 (BBN Software Products Corp., Cambridge, Mass.).
[0081] Binding Assays for Rat Y2. Y1. and Y4/PP1 Receptors
[0082] The binding buffer for rat Y2 binding was Krebs/Ringer
bicarbonate (Sigma K-4002, S-8875), pH 7.4, containing 0.01% bovine
serum albumin (BSA-Sigma A-2153) and 0.005% bacitracin. 0.85-1
.mu.g of protein and 25 pM [.sup.125I]PYY are added to each well.
Nonspecific binding is defined by 1 .mu.M NPY.
[0083] The binding buffer for rat Y1 and rat Y4/PP1 binding
consisted of 137 mM NaCl, 5.4 mM KCl, 0.44 mM KH.sub.2PO.sub.4,
1.26 mM CaCl.sub.2, 0.81 mM MgSO.sub.4, 20 mM HEPES, 1 mM
dithiothreitol (DTT), 0.1% bacitracin, 100 mg/l streptomycin
sulfate, 1 mg/l aprotinin, 10 mg/ml soybean trypsin inhibitor and
0.3% BSA, pH 7.4. For rat Y1 binding, .about.5-15 .mu.g of protein
and 50 pM [.sup.125I]PYY were added to each well, and nonspecific
binding was defined by 1 .mu.M NPY. For the rat Y4/PP1 binding
assay, .about.1-2 .mu.g of protein and 50 pM rat [.sup.125I]PP (NEN
DuPont, Boston, Mass.) were added to each well, and 1 .mu.M rat PP
was used to define nonspecific binding.
[0084] In Vitro Functional Assay--Measurement of
Forskolin-Stimulated Adenylate Cyclase
[0085] Rat Y5
[0086] (Reference: Gordon et al., J. Neurochem. 55, 506, 1990)
Suspension cells stably expressing the Y5 receptor (approximately
400,000 per sample) were resuspended in serum-free DMEM containing
10 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX). 1 uM
forskolin was added to the cells. The assay was stopped by
transferring the samples into a boiling water bath for 3 minutes.
After a 3 minute centrifugation at 14,000.times.g, an aliquot of
each sample was quantitated for cAMP levels by radioimmunoassay
(NEN DuPont, Mass.).
[0087] Human Y5
[0088] Monolayer cells stably expressing the Y5 receptor were
pre-rinsed with Wash buffer (pH 7.2: 137 mM NaCl, 2.7 mM KCl, 0.9
mM CaCl.sub.2, 0.5 mM MgCl.sub.2, 6.5 mM Na.sub.2HPO.sub.4, 1.5 mM
KH.sub.2PO.sub.4). Cells were then incubated for 10 minutes at
37.degree. C. in Assay buffer (pH 7.4: Wash buffer+10 mM HEPES, 10
mg/ml BSA, 0.5 mg/ml bacitracin, 0.4 mg/ml soybean trypsin
inhibitor). After addition of fresh buffer and 100 .mu.M IBMX, the
cells were incubated for 10 minutes at 37.degree. C. The reaction
was started with the addition of peptide and 1-10 .mu.M forskolin.
After a 20 minute incubation at 37.degree. C., the reaction was
terminated by discarding the buffer and adding 65% ethanol to each
well. The supernatant was then transferred to microfuge tubes and
the extraction step was repeated once more. After evaporation of
the ethanol from the samples, the amount of cAMP was assayed using
by radioimmunoassay (NEN DuPont: Boston, Mass.).
[0089] In Vivo Pharmacology Procedures
[0090] Adult male Wistar rats were surgically implanted with a
chronic intracerebral ventricular (ICV) cannula (Plastic Products,
Roanoke, Va.) using a stereotaxic instrument. Several days after
the surgery, 1-6 mmoles of each peptide (or saline) was injected
into the lateral ventricle of 4-12 rats in a volume of 5-10 .mu.l.
The quantity of rodent chow consumed in a 2 hour period was
measured.
[0091] In Vitro and In Vivo Pharmacology Results
[0092] FIG. 1 presents the competition curves of various peptides
for [.sup.125I]PYY binding to Y5 receptor membranes transiently
expressed in COS-7 cells. Each point is the average value of
triplicate determinations from a representative experiment.
IC.sub.50 values corresponding to 50% inhibition of specific
binding were determined using nonlinear regression analysis.
K.sub.i values were calculated from the IC.sub.50 values using the
Cheng-Prusoff correction, such that
K.sub.i=IC.sub.50/(1.+-.(L/K.sub.- d)), where L is the radioligand
concentration and K.sub.d is the dissociation constant. The results
for transiently expressed Y5 clones are presented in Table 1, and
Table 2 contains data for stably expressed Y5 clones.
1 TABLE 1 NPY Y5 BINDING AFFINITIES (K.sub.i .+-. SEM, nM) RAT Y555
RAT 6B RAT Y861 HUMAN PEPTIDE Clone* Clone Clone Clone r/hNPY 0.53
.+-. 0.06 0.49 .+-. 0.03 0.50 .+-. 0.06 0.73 .+-. 0.09 rPYY 1.1
(1.2, 0.95) 0.48 .+-. 0.09 1.0 .+-. 0.13 1.3 .+-. 0.14 h(LP)PYY 2.5
.+-. 0.5 0.57 .+-. 0.01 1.8 .+-. 0.09 1.7 .+-. 0.3 r/h(LP) 0.96
(1.0, 0.92) 0.31 0.55 .+-. 0.11 0.97 .+-. 0.36 NPY p(LP)NPY ND 0.64
.+-. 0.07 0.47 0.88 .+-. 0.11 r/h(2-36) 0.81 (0.61, 1) 0.65 1.2
.+-. 0.07 1.2 .+-. 0.15 NPY p(3-36) 3.6 .+-. 0.4 1.9 .+-. 0.27 2.0
(1.8, 2.3) 10.4 .+-. 2.0 NPY r/h(3-36) ND 0.49 2.1 (2.7, 1.6) 3.8
.+-. 0.48 NPY r(3-36) 6.2 .+-. 1.1 1.4 .+-. 0.10 4.2 .+-. 0.47 10
.+-. 3.4 PYY r/h(10-36) 35 4.9 (6.0, 3.8) 34 .+-. 2.8 110 (110,
109) NPY p(13-36) 40 (38, 41) 7.7 (7.9, 7.5) 22 (25, 19) 56 .+-. 7
NPY r(13-36) 73 11 .+-. 1.0 86 .+-. 19 77 (89, 65) NPY p(18-36) 303
194 .+-. 88 206 .+-. 61 618 .+-. 85 NPY r/h(20-36) 636 330 .+-. 31
587 >1000 NPY r/h(22-36) >1000 >1000 >1000 >1000 NPY
r/h(26-36) >1000 >1000 >1000 >1000 NPY (1-24) ND
>1000 >1000 >1000 NPY BIBP3226 ND >1000 >1000
>1000 hPP ND ND 4.0 .+-. 0.29 11 (15, 6.2) rPP ND 62 296 .+-. 47
436 (582, 290) *IC.sub.50 values (nM)
[0093]
2 TABLE 2 K.sub.i Values (nM; Average .+-. SEM) Peptide
293.hY5.sb.8 293S.Y861.2 rPYY 1.3 .+-. 0.2 0.71 .+-. 0.1 hPYY 1.1
.+-. 0.2 1.06 .+-. 0.2 (3-36)PYY 4.5 .+-. 0.7 3.6 .+-. 0.4
(13-36)PYY 24 .+-. 2.0 29 .+-. 4 h(LP)PYY 1.3 .+-. 0.1 0.76 .+-.
0.1 r/hNPY 0.79 .+-. 0.1 0.86 .+-. 0.07 p(LP)NPY 1.2 .+-. 0.4 0.67
.+-. 0.04 h/r(LP)NPY 0.89 .+-. 0.1 0.67 .+-. 0.04 (LP)(3-36)NPY 3.1
.+-. 0.6 2.9 .+-. 0.9 (2-36)NPY 1.4 .+-. 0.03 0.83 .+-. 0.1
h(3-36)NPY 3.5 .+-. 0.4 1.4 .+-. 0.4 (10-36)NPY 14 .+-. 2.7 15 .+-.
4.7 p(13-36)NPY 8.7 .+-. 1.6 8.8 .+-. 2.0 p(18-36)NPY 144 .+-. 18
61 .+-. 13 (20-36)NPY 429 .+-. 133 108 .+-. 16 (22-36)NPY >900
>930 (26-36)NPY >900 >930 (1-24)NPY >900 >930
(32D-Trp)NPY 7.3 .+-. 0.8 4.2 .+-. 1.0 hPP 3.7 .+-. 1.6 2.5 .+-.
0.5 rPP 286 .+-. 77 203 .+-. 44
[0094] K.sub.i values for various peptides for [.sup.125I]PYY
binding to the transiently expressed rat 6B, Y861 and Y555 receptor
clones as well as the human Y5 receptor. The averages.+-.standard
error of the mean (SEM) represent values from at least three
independent experiments. Two independent experiments are
represented by the average, followed by the individual values in
parentheses. Remaining values without SEM are from a single
experiment. Peptide species in Table 1 (and Table 2, infra) are
indicated with the following prefixes: r=rat, h=human, p=porcine,
r/h=rat=human. ND=not determined.
[0095] The rank order of the affinities of the peptides tested is
as follows:
NPY.about.PYY.about.(LP)PYY.about.(LP)NPY.about.(2-36)NPY.about.(3-36)PYY.-
about.(LP)(3-36)NPY.about.(3-36)NPY>(32D-Trp)NPY>(10-36)NPY.about.(1-
3-36)NPY>(18-36)NPY>(20-36)NPY>>(22-36)NPY,
(26-36)NPY
[0096] In Table 3, the pharmacological profile of the standard
peptides is expanded for the other cloned NPY receptors to further
illustrate the novel nature of the Y5 receptor pharmacology. In
addition, the in vivo feeding response of some of these peptides is
listed for comparison. The data shown are representative of the
average of at least two independent experiments, as described in
the methods. Feeding of rats injected (ICV) with saline was<3
g/2 hours.
[0097] Table 4 shows the EC.sub.50 values for same standard
peptides at the rat and human Y5 receptor.
[0098] C-terminal fragment (3-36)NPY binds preferentially to Y2
receptors, while (LP)NPY has lower affinity. Conversely, (LP)NPY
has high affinity for the Y1 receptor, while (3-36)NPY and the
C-terminal fragments are much weaker. When considering the rat
Y4/PP1 receptor, rat PP has very high affinity as compared to NPY,
PYY, (LP)NPY, and (13-36)NPY. In the in vivo feeding model,
(LP)NPY, which has high affinity for Y1 and low affinity for Y2,
and (3-36)NPY, which has a high affinity for Y2, but not Y1, all
stimulate feeding in rats. Rat PP does not induce much feeding when
administered to rats. This in vivo profile matches the in vitro
pharmacological profile outlined in Table 2 for the Y5
receptor.
[0099] In addition, while (LP)(3-36)NPY (a custom peptide
synthesized at Bayer) has weak affinity for Y1, Y2 and Y4/PP1, it
stimulates feeding in rats. Importantly, (LP)(3-36)NPY has high
affinity for the Y5 receptor (Table 2). These data are further
evidence that the Y5 receptor is linked to feeding.
3TABLE 3 IC.sub.50 VALUE (nM) Rat Y1 Rat Y2 Rat Y4/PP1 Rat Y5
Feeding PEPTIDE (clone) (clone) (clone) (Y861) (g/2 h) r/hNPY 0.13
0.24 >1000 0.45 >5 rPYY 0.43 0.079 630 0.9 >5 h(Leu.sup.31
0.57 116 ND 2.0 >5 Pro.sup.34) PYY p(Leu.sup.31 0.15 150 4.3
0.63 >5 Pro.sup.34)NPY r/h(2-36) 47 0.50 >1000 1.3 >5 NPY
p(3-36)NPY 45 0.67 >1000 2.2 >5 r/h(Leu.sup.31 44 154 20 3.4
>5 Pro.sup.34) (3-36)NPY hPP 40 >1000 0.065 4.9 >5
(32DTrp) >1000 26 ND 7.0 ND NPY r/h(10-36) 148 0.42 >1000 34
<3 NPY rPP 843 >1000 0.071 325 <3 p(18-36) 287 0.34 159
326 <3 NPY (20-36)NPY 435 0.64 ND 638 <3 (22-36)NPY >1000
0.89 ND >1000 <3 (26-36)NPY >1000 84 ND >1000 <3
(1-24)NPY >1000 >1000 ND >1000 <3
[0100] The pharmacological profile for the 6B (and Y861 and Y555)
receptor clones is distinct from Y1 receptors (where
PYY.about.NPY.about.(LP)NPY&g-
t;(3-36)NPY>(13-36)NPY.about.(18-36)NPY>(LP)(3-36)NPY), as
well as Y2 receptors (where
PYY.about.NPY.about.(13-36)NPY.about.(18-36)NPY.about-
.(3-36)NPY>>(LP)NPY.about.(LP)(3-36)NPY). The Y5 receptor is
also different from the pancreatic polypeptide (PP) receptor
(Y4/PP) since [.sup.125I]PP (rat) does not bind to it.
[0101] Although the rank order of affinities is essentially the
same when comparing 6B to Y861 and Y555, subtle differences do
exist in the IC.sub.50 values. It appears that Y861 and Y555 have
slightly lower affinities (approximately 2- to 3-fold) for PYY and
other PYY analogs, as compared to 6B. In addition, (10-36)NPY and
(13-36) have 2- to 4-fold lower affinity for Y861 and Y555.
[0102] Nonlinear regression analysis of saturation data for the Y5
receptor yielded a K.sub.d value of 0.27 nM and a receptor density
(B.sub.max) of about 140 fmol/mg protein in these transiently
transfected cells.
[0103] FIG. 2 presents the saturation curve for specific binding of
[.sup.125I]PYY to Y5 receptor membranes transiently expressed in
COS-7 cells. Membranes were incubated with concentrations of
[.sup.125I]PYY ranging from 0.05 to 5 nM, in the presence or
absence of 1 .mu.M PYY. Each point represents the average value of
triplicate determinations at each concentration tested. The inset
in FIG. 2 shows the corresponding Rosenthal plot of the data.
4 TABLE 4 EC.sub.50 Values, nM (n Value) Peptide 293S.Y861.2
293.hY5.sb.8 r/hNPY 6.3 .+-. 1.9 (3) 0.3 (1) rPYY 6.5 (2) ND
r/h(2-36)NPY 21 (2) ND r/h(3-36)NPY ND 6 (1) r/h(LeuPro)(3-36)NPY
31 .+-. 39 (3) 23 .+-. 11 (3) (32D-Trp)NPY 24 (1) 33 (1) hPP 1 (1)
5 (1) rPP 112 (1) >1000 (1)
Example 5
Isolation of Human Y5 Receptor
[0104] Isolation of Human Genomic Clone
[0105] Polymerase chain reaction (PCR) was used to amplify a 375
base pair (bp) coding region of the rat Y5 cDNA clone. The primers
for the PCR were:
[0106] (+) 5'-TAGGGAACCTGGCCTCCTCC-3' (SEQ ID NO 5) (nucleotides
487-506),
[0107] (-) 5'-TCAGAGGGCCATGACTCAAC-3' (SEQ ID NO 6) (nucleotides
843-862).
[0108] The PCR product was cloned into pCRII vector (Invitrogen)
and sequenced. After confirmation by sequencing, the insert was
purified from the low melting gel and labeled with
digoxigenin-11-dUTP using the random primed method (Boehringer
Mannheim, Indianapolis, Ind.). The labeled probe was used to screen
human genomic library.
[0109] 1.times.10.sup.6 independent recombinants were screened from
the library. Filter hybridization was carried out in the
hybridization buffer containing 6.times.SSC, 0.1%
N-lauroylsarcosine, 0.02% sodium dodecyl sulfate (SDS), 3% blocking
reagent (Boehringer Mannheim) and 30% formamide at 37.degree. C.
overnight. The filters were washed at 37.degree. C. in
0.1.times.SSC, 0.1% SDS and the positive clones were identified by
CSPD detection kit according to the manufacturer's protocol
(Boehringer Mannheim).
[0110] Two positive clones (HG11A and HG19) were isolated from the
library. The positive clones were subcloned into pBluescript vector
(Stratagene). One clone, h11a, was analyzed by restriction mapping
and plasmid Southern blot. Two EcoRV fragments, 2.4 kb and 0.4 kb,
were hybridized by the rat Y5 probe. These two DNA fragments were
subcloned and sequenced from both ends. DNA sequence analysis was
performed using GCG program. The coding region of the human Y5
genomic clone was identified by DNA sequence analysis. This region
was amplified by PCR using genomic clone h11A as template and
subcloned into pcDNA3 expression vector (Invitrogen) for further
studies. The h11A clone has the nucleic acid coding sequence given
by SEQ ID NO 5 and the protein that it encodes has the amino acid
sequence given by SEQ ID NO 6.
[0111] The human Y5 DNA coding region was used to search the
sequence similarities in the gene bank. The Y5 coding sequence from
nucleotide 821 to the stop codon at position 1338 is nearly
identical, but in an opposite orientation, to part of the human
NPY-Y1 gene (Ball et al, J. Biol. Chem. 270, 30102 (1995)). The
identical sequence covered the 1C exon promoter, exon 1C, and part
of the intron sequences of the NPY-Y1 receptor in an opposite
orientation. Compared to the published nucleotide sequence, the Y5
coding region has a T insertion at position 1226 and a TG insertion
at positions 1235 and 1236.
Sequence CWU 1
1
* * * * *