WO2001085791A1

WO2001085791A1 - Nucleic acid sequences for novel gpcrs

Info

Publication number: WO2001085791A1
Application number: PCT/US2001/015332
Authority: WO
Inventors: Joseph P. Brown; Margaret Miller; Glenna Burmer; Christine Fabre-Suver; David Pritchard
Original assignee: LifeSpan BioSciences Inc
Current assignee: LifeSpan BioSciences Inc
Priority date: 2000-05-11
Filing date: 2001-05-11
Publication date: 2001-11-15
Anticipated expiration: 2002-11-11
Also published as: WO2001085791A8; AU2001261476A1

Abstract

The present invention is directed to isolated nucleic acid encoding G protein-coupled receptors and the proteins encoded thereby. Included are methods of using the disclosed nucleic acid and protein.

Description

NUCLEIC ACID SEQUENCES FOR NOVEL GPCRs

BACKGROUND OF THE INVENTION Many physiologically important events are mediated by the binding of guanine nucleotide-binding regulatory proteins (G proteins) to G protein-coupled receptors (GPCRs). These events include vasodilation, stimulation or decrease in heart rate, bronchodilation, stimulation of endocrine secretions and enhancement of gut peristalsis, development, mitogenesis, cell proliferation and oncogenesis.

Guanine nucleotide-binding proteins are a family of proteins that transduce signals from numerous cell surface receptors to downstream intracellular effector molecules. G proteins are typically heterotrimeric proteins consisting of a guanyl- nucleotide binding alpha subunit, a beta and a gamma subunits, the latter two being tightly associated under physiological conditions (for a review, see, e.g., Con lin et al, Cell 73:631-641 (1993)). Each subunit is encoded by a separate gene. G proteins commonly cycle between two forms, depending on whether GDP or GTP is bound to the alpha subunit. Upon binding of a ligand to a G protein-coupled receptor, the GDP molecule bound to the alpha subunit is exchanged for a GTP molecule resulting in the dissociation of the α subunit from the β and γ subunits. The free alpha subunit and the beta-gamma complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways, for example by binding to and activating adenyl cyclase. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena.

The different members of the G protein coupled receptors super-family share a number of functional and structural characteristics. In particular, as described above, GPCRs have the ability to stimulate the exchange of bound GDP for GTP on associated G proteins alpha subunits in response to agonist binding. Structurally, GPCRs typically contain seven hydrophobic transmembrane segments that are suggested to be transmembrane helices of 20-30 amino acids connected by extracellular or cytoplasmic loops (see, e.g., Kobilka et al, Science 240:1310 (1988); Maggio et al, FEBS Lett. 319:195 (1993); Maggio et al, Proc. Natl Acad. Sci USA 90:3103 (1993); Ridge et al, Proc. Natl Sci USA 91:3204 (1995); Schonenberg et al, J. Biol. Chem. 270:18000 (1995); Huang et al, J. Biol. Chem. 256:3802 (1981); Popot et al, J. Mol. Biol 198:655 (1987); Kahn and Engelman, Biochemistry 31:6144 (1992); Schoneberg et al, EMBOJ. 15:1283 (1996); Wong et al, J. Biol Chem. 265:6219 (1990); Monnot et al, J. Biol Chem. 271:1507 (1996); Gudermann et al, Annu. Rev. Neurosci. 20:399 (1997); Osuga et al, J. Biol. Chem. 272:25006 (1997); Lefkowitz et al, J. Biol Chem. 263:4993-4996 (1988); Panayotou and Waterfield, Curr. Opinion Cell Biol 1:167-176 (1989); and G Protein-Coupled Receptor Database, http://www.gcrdb.uthscsa.edu). In addition to G proteins, many enzymes, such as, for example, adenylate cyclase, cGMP phosphodiesterase and phospholipase C, can act as effectors for GPCRs' signal transduction (>yee, e.g., Kinnamon & Margolskee, Curr. Opin. Neurobiol. 6:506-513 (1996)).

A large variety of molecules have been shown to be ligands for GPCRs. Identified ligands include, for example, purines, nucleotides and melatonin (e.g., adenosine, cAMP, NTPs, etc), bio genie amines (e.g., adrenaline, dopamine, l istamine, acetylcholine, noradrenaline, serotonin, etc), peptides (e.g., angiotensin, calcitonin, chemokine, Corticotropin Releasing Factor, galanin, Growth Hormone Releasing

Hormone, Gastric Inhibitory Peptride, Glucagon, Neuropeptide Y, Neurotensin, Opoiod, Thrombin, Secretin, Somatostatin, Thyrotropin Releasing Hormone, Vasopressin, Vasoactive Intestinal Peptide, etc), lipids and lipid-based compounds (e.g., cannabinoids, Platelet Activating Factor, etc), excitatory amino acids and ions (e.g., glutamate, calcium, GABA, etc), toxins, etc. In addition, there are many "orphan" G protein-coupled receptors (e.g., some olfactory G protein-coupled receptors) for which ligands have not been identified.

G protein-coupled receptors thus play a central role in transducing numerous signals and regulating cellular metabolism. Accordingly, GPCRs have been implicated in a large number of diseases, such as, Alzheimer's disease, rheumatoid arthritis, osteoarthritis, osteoporosis, amyotrophic lateral sclerosis, multiple sclerosis and atherosclerosis, asthma, depression, epilepsy, schizophrenia, Parkinson's disease, a number of sarcomas (e.g., chondrosarcoma, Ewing's sarcoma, osteosarcoma, etc) and carcinomas (e.g., basal cell carcinoma, breast carcinoma, embryonal carcinoma, ovarian carcinoma, renal cell carcinoma, lung adenocarcinoma, lung small cell carcinoma, pancreatic carcinoma, prostate carcinoma, transitional carcinoma of the bladder, squamous cell carcinoma, thyroid carcinoma, etc), psoriasis, cardiomyopathy, Crohn's disease, Duchenne muscular dystrophy, glioblastoma multiform, Hodgkin's disease, lymphorna, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, seminoma, tuberculosis, tonsil, ulcerative colitis, etc. While many GPCRs have been identified, many more remain to be discovered. In addition, the specific GPCRs involved in the different biological processes, and in particular diseases, are not known.

Galanin is a widely distributed 28 amino acid peptide hormone which has been shown to regulate a variety of biological processes, including, for example, hormone release, neurotransmitter release, nociception, feeding behavior, cognitive function and reproductive behavior. Galanin signaling has been shown to modulate the release of a variety of neurotransmitters, including, but not limited to, acetylcholine, norepinephrine, serotonin and dopamine (see, e.g., Bartfai Crit. Rev. Neurobiol 7:229 (1993)). Cumulative evidence suggests that galanin acts as an inhibitory cosecreted peptide. Galanin has been postulated to impair secretion of neurotransmitters by acting at the pre-synaptic autoreceptors as well as at the post-synaptic action site of these neurotransmitters. In particular, galanin inhibits acetylcholine release into the ventral hippocampus. Galanin may thus impair memory and learning by inhibiting the cholinergic function.

Galanin is to date the only neurotransmitter that has been shown to be upregulated in Alzheimer's disease. In addition, a variety of experiments, including the central injection of galanin and the generation of transgenic mice, have shown that the overexpression and/or oversecretion of galanin impairs performance of memory and learning tasks. These results indicate that the hypertrophy of galanin pathways contributes to the cognitive deficits in Alzheimer's disease.

Galanin has further been shown to inhibit the release of vasopressin and insulin, while it stimulates the release of growth hormone, prolactin and luteinizing hormone. Galanin has been shown to play a role in the control of fat metabolism, and body adiposity, which may be mediated by its effect on insulin. Galanin inhibits insulin secretion and, conversely, insulin injection inhibits central galanin expression. Galanin acts within the medial preoptic area and paraventricular nucleus to modulate fat intake and fat metabolism, but the specific subtype of galanin receptors involved in this function are not known. Galanin also acts within the supraoptic nucleus and paraventricular nucleus to modulate fluid balance. In addition, galanin regulates feeding behavior.

Galanin may exert neurotrophic and/or neuroprotective actions within the central nervous system. Treatment of rats with galanin has been shown to reduce behavioral impairments following brain injury. Galanin gene expression is upregulated in injured neurons and this may contribute to cell survival. Despite the substantial loss of cells within the locus ceruleus, the percentage of noradrenergic neurons that coexpress galanin mRNA is increased in Alzheimer's disease supporting the idea that galanin may exert a neuroprotective effect.

Galanin is co-localized with gonadotropin-releasing hormone (GnRH) in the medial preoptic region of several species. The pattern of coexpression exhibits sexual dimorphism in rats. In both rats and monkeys, gonadal hormones regulate galanin expression in GnRH cells. Galanin, acting within the anterior pituitary, plays a role in the regulation of luteinizing hormone release. Galanin facilitates sex behavior via actions within the medial preoptic regions.

Under normal conditions, galanin has potent antinociceptive effects. After peripheral nerve injury the inhibitory control exerted by endogenous galanin is increased. During inflammation, galanin expression within the dorsal horn is increased. Endogenous galanin appears to play an enhanced antinociceptive role in chronic pain or neuropathic or inflammatory origin.

Galanin has been indicated in the etiology of depression. Galanin is colocalized within the serotoninergic and noradrenergic systems. An increase in the amount of galanin released from ascending noradrenergic neurons into the ventral tegmental area has been proposed to decrease dopamine release and thereby decrease motor activation and anhedonia, two major symptoms of depression. The receptors involved in these functions are not known.

Galanin has also been shown to control gastrointestinal and cardiovascular actions. For example, in the guinea pig ileum, galanin administration inhibits neurally induced smooth muscle contractility probably via its ability to reduce acetylcholine release. In addition, galanin inhibits somatostatin and gastrin release. Galanin also decreases blood flow following injection into the mesenteric arteriole, as well as sodium and chloride net absorption.

Galanin thus plays an important role in a large variety of physiological processes.

The effects of galanin are mediated via G-protein coupled receptors for which three types have been cloned, GALR1, GALR2 and GALR3 (see, e.g., Howard et al, FEBS letter, 405:285-290 (1997); Bloomquist et al, Biochem. Biophys. Res. Commun. 243:474-479 (1998); WO 98/15570; WO 99/31130; WO 97/46681; WO 97/26853). For most of the biological processes regulated by galanin, the specific receptors involved in these functions are not known.

Identifying additional G protein-coupled receptors would allow insight into the role of the each receptor in the different biological processes in which GPCR- mediated signaling is involved. There is a strong need in the art for diagnostic and therapeutic tools for detection and treatment of the numerous diseases and disorders involving GPCR-mediated signaling. In addition, identifying additional receptors for galanin would allow insight into the role of the each receptor in the different biological processes in which galanin is involved. Moreover, there is a strong need in the art for diagnostic and therapeutic tools for detection and treatment of the numerous diseases and disorders involving galanin signaling. This invention addresses these and other needs.

SUMMARY OF THE INVENTION The present invention provides polypeptides having at least 70%>, 75%>, 80%), 85%), 90%), 95%) or more identity with the polypeptides encoded by the nucleic acid molecules having a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1. In one embodiment, the polypeptides of the invention are encoded by a nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1. In other embodiments, the polypeptides of the present invention comprise a region of 15 amino acids or more, optionally 30 amino acids or more, having at least 80%, preferably at least 85%>, and most preferably 90% or more, identity with a region of 15 amino acids or more, optionally 30 amino acids or more, from a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1. In some embodiments, the nucleic acids molecules encoding the polypeptides of the invention are operably linked to a heterologous promoter. The present invention also provides expression vectors comprising the nucleic acid molecules encoding the polypeptides of the invention, as well as host cells comprising the expression vectors. In one embodiment, the host cell is a mammalian cell. The present invention is also directed to nucleic acid probes that specifically hybridize with the nucleic acid molecules encoding the described polypeptides. The probes can be DNA or RNA. Antisense nucleic acid molecules that specifically hybridize to the nucleic acid sequences encoding the polypeptides of the invention are also provided. In another aspect, antibodies that specifically bind to the polypeptides of the invention are also provided. The antibodies can be monoclonal or polyclonal.

The antibodies and nucleic acid probes described above can be used to detect the presence of the polypeptides of the invention or of the nucleic acid molecules encoding the described polypeptides. They can be used to diagnose a variety of diseases and disorders in which G protein-coupled receptors are involved, such as, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcmoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, etc.

The present invention is also directed to methods for identifying compounds that modulate the expression of one or more polypeptides of the invention, the methods comprising culturing a cell in the presence of a modulator to form a first cell culture, contacting RNA or cDNA from the first cell culture with at least one probe, each probe comprising a polynucleotide sequence encoding a polypeptide of the invention, and determining whether the amount of the probe(s) which hybridizes to the RNA or cDNA from the first cell culture is increased or decreased relative to the amount of the probe(s) which hybridizes to RNA or cDNA from a second cell culture grown in the absence of the modulator.

In addition, the present invention provides methods for identifying compounds that modulate the activity of one or more polypeptides of the invention, the methods comprising culturing cells expressing at least one polypeptide of interest in the presence of a compound, measuring the activity of the polypeptide(s) or second messenger activity and determining whether the activity is increased or decreased relative to the activity of the polypeptide(s) or second messenger activity from a second cell culture grown in the absence of the modulator.

The compounds identified using the methods of the present invention can be modulators, activators, repressors, agonists or antagonists and have therapeutic uses for treating a variety of disorders and/or diseases in which G protein-coupled receptors have been implicated, such as, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcmoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, etc.

The present invention provides is directed to polypeptides having at least 80%o identity, optionally at least 85%> identity, with the polypeptide encoded by the nucleic acid molecule having the nucleotide sequence set forth in SEQ LO NO: 1. In one embodiment, the polypeptide of the present invention is the polypeptide encoded by the sequence set forth in SEQ ID NO:l. In other embodiments, the polypeptides of the present invention comprise a region of 15 amino acids or more, optionally 30 amino acids or more, having at least 80%, preferably at least 85% and most preferably 90% or more identity with a region of 15 amino acids or more, optionally 30 amino acids or more, from the polypeptide encoded by the nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:l. Vectors comprising the nucleic acids encoding the polypeptides of the invention, and host cells comprising the expression vectors are also provided. In some embodiments, the nucleic acid molecules encoding the polypeptides of the invention are operably linked to a heterologous promoter, hi some embodiments, the host cell is a mammalian cell.

The present invention is also directed to nucleic acid probes that specifically hybridize with the nucleic acid molecules encoding the polypeptides of the invention. The probes can be DNA or RNA. Antisense nucleic acid molecules that specifically hybridize to the nucleic acid molecules encoding the polypeptides of the invention are also provided.

In another aspect, antibodies that specifically bind to the polypeptides of the invention are also provided. The antibodies can be monoclonal or polyclonal. The nucleic acid probes and antibodies described above can be used to detect the presence of the nucleic acid molecules encoding the polypeptides of the invention. They can be used to diagnose a variety of diseases and disorders in which galanin is involved, such as, cognition and memory disorders, anorexia, hormonal release disorders, cardiovascular activity disorders, pain perception disorders, obesity, diabetes, Alzheimer's disease, etc.

The present invention is also directed to methods for identifying compounds that modulate the expression of the polypeptides of the invention, comprising culturing a cell in the presence of a modulator to form a first cell culture, contacting RNA or cDNA from the first cell culture with a probe which comprises a polynucleotide sequence encoding the polypeptide of the invention, and determining whether the amount of the probe which hybridizes to the RNA or cDNA from the first cell culture is increased or decreased relative to the amount of the probe which hybridizes to RNA or cDNA from a second cell culture grown in the absence of the modulator. In addition, the present invention provides a method for identifying compounds that modulate the activity of the polypeptides of the invention, comprising culturing cells expressing the polypeptide of interest in the presence of a compound, measuring the activity of the polypeptide or second messenger activity and determining whether the activity is increased or decreased relative to the activity of the polypeptide or second messenger activity from a second cell culture grown in the absence of the modulator.

The compounds identified using the methods of the present invention can be modulators, activators, repressors, agonists or antagonists and have therapeutic uses for treating a variety of disorders and/or diseases in which galanin has been implicated. For example, compounds that decrease the expression (repressors) or activity

(antagonists) of the polypeptides of the invention can be used, e.g., to treat obesity, diabetes, hyperlipidemia, stroke, cognitive disorders, Alzheimer's disease, and/or endocrine disorders. Compounds that increase expression (activators) or activity (agonists) of the polypeptides of the invention can be used, for example, to treat anorexia and to decrease noniception.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

I. INTRODUCTION The present invention is directed to novel G protein-coupled receptors (GPCRs) that are useful for treating and diagnosing a number of diseases and disorders, including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcmoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, etc. The present invention also provides methods for identifying modulators of G protein-coupled receptor-mediated signaling. Such modulators are useful for treating the above-listed and other diseases and disorders.

In some aspects, the present invention is directed to new galanin receptors that are useful for treating and diagnosing a number of diseases and disorders, including, but not limited to, Alzheimer's disease, learning and memory disorders, hormonal problems, fat metabolism disorders, feeding disorders, pain perception disorders, diabetes, depression, etc. The present invention also provides methods for identifying modulators of galanin signaling. Such modulators are useful for treating the above-listed and other diseases and disorders.

The invention provides novel G protein-coupled receptors, as well as vectors and cells to express these novel GPCRs, including, e.g., galanin receptors. Probes and antibodies that can be used to detect the GPCRs of the invention are also provided, as well as antisense polynucleotides. The probes and antibodies are useful for diagnostic purposes. In addition, the nucleic acids encoding the polypeptides of the invention, antisense polynucleotides and polypeptides of the invention are useful for gene therapy applications. The present invention also provides nucleic acid molecules encoding the polypeptides of the invention operably linked to a heterologous promoter that drives expression of the protein encoded by the nucleic acid sequence.

The invention further provides methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of these novel G protein-coupled receptors. Such modulators of the activity of the GPCRs are useful for pharmacological and genetic modulation of the signaling pathways in which GPCRs are involved. These methods of screening can be used to identify high affinity agonists and antagonists of GPCRs' activity. These modulatory compounds can then be used in pharmaceutical industry to regulate G protein-coupled receptor-mediated signaling to treat a variety of diseases or disorders. Thus, the invention provides assays for GPCR- mediated signaling modulation, where the G protein-coupled receptors of the invention or other molecules located downstream of the G protein coupled receptor act as direct or indirect reporter molecules for the effect of modulators on GPCR-mediated signaling. G protein-coupled receptors can be used in assays, e.g., to measure changes in ligand binding, transcription, signal transduction, receptor-ligand interactions, second messenger concentrations, in vitro, in vivo, and ex vivo.

In some embodiments, the present invention provides novel galanin receptors (GAL4), as well as vectors and cells to express the galanin receptors. Probes and antibodies that can be used to detect the galanin receptors of the invention are also provided, as well as antisense polynucleotides. The probes and antibodies are useful for diagnostic purposes. In addition, the nucleic acids encoding the polypeptides of the invention, antisense polynucleotides and polypeptides of the invention are useful for gene therapy applications.

In some aspects, the invention further provides methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of these novel galanin receptors. Such modulators of the activity of the galanin receptors are useful for pharmacological and genetic modulation of the galanin signaling pathways. These methods of screening can be used to identify high affinity agonists and antagonists of galanin receptors' activity. These modulatory compounds can then be used in pharmaceutical industry to regulate galanin signaling to treat a variety of diseases or disorders. Thus, the invention provides assays for galanin signaling modulation, where the galanin receptors of the invention or other molecules located downstream in the galanin signaling pathway act as direct or indirect reporter molecules for the effect of modulators on galanin signaling. Galanin receptors can be used in assays, e.g. , to measure changes in ligand binding, transcription, signal transduction, receptor-ligand interactions, second messenger concentrations, in vitro, in vivo, and ex vivo.

II. DEFINITIONS "Amplification primers" are oligonucleotides comprising either natural or analog nucleotides that can serve as the basis for the amplification of a selected nucleic acid sequence. They include, for example, both polymerase chain reaction primers and ligase chain reaction oligonucleotides. "Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (VH) refer to these light and heavy chains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2; a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv). "Biological samples" refers to any tissue or liquid sample having genomic DNA or other nucleic acids (e.g., mRNA) or proteins. It refers to samples of cells or tissue from a normal healthy individual as well as samples of cells or tissue from a subject suspected of having, e.g., Alzheimer's disease, rheumatoid arthritis, osteoarthritis, osteoporosis, amyotrophic lateral sclerosis, multiple sclerosis and atherosclerosis, asthma, depression, epilepsy, schizophrenia, Parkinson's disease, a sarcoma (e.g., chondrosarcoma, Ewing's sarcoma, osteosarcoma, etc), a carcinoma (e.g., basal cell carcinoma, breast carcinoma, embryonal carcinoma, ovarian carcinoma, renal cell carcinoma, lung adenocarcmoma, lung small cell carcinoma, pancreatic carcinoma, prostate carcinoma, transitional carcinoma of the bladder, squamous cell carcinoma, thyroid carcinoma, etc.), psoriasis, cardiomyopathy, Crohn's disease, Duchenne muscular dystrophy, glioblastoma multiform, Hodgkin's disease, lymphoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, seminoma, tuberculosis, tonsil, ulcerative colitis, or any other disease or disorder in which G protein- coupled receptors are involved, as well as learning and/or memory disorders, diabetes, pain perception disorders, anorexia, obesity, hormonal release problems, or any other disease or disorder in which galanin is involved.. The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85%. pure, more preferably at least 95%> pure, and most preferably at least 99% pure.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al (1992); Rossolini et al, Mol Cell. Probes 8:91- 98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3^rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistiγ Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 50 to 350 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

The term "similarity," or percent "similarity," in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined in the 8 conservative amino acid substitutions defined above (i.e., 60%, optionally 65%>, 70%o, 75%, 80%, 85%o, 90%>, or 95%> similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially similar." Optionally, this identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is at least about 75-100 amino acids in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol Biol 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WT), or by manual alignment and visual inspection (see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al. (1984) Nuc. -Acids Res. 12:387-395).

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence. The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50%) of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50%> of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 65°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min.

As used herein a "nucleic acid probe" is defined as a nucleic acid capable of binding to a target nucleic acid (e.g., a nucleic acid encoding a galanin receptor) of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.

Nucleic acid probes can be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers

A "labeled nucleic acid probe" is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be determined by detecting the presence of the label bound to the probe. The phrase "a nucleic acid sequence encoding" refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a transacting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell. The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enliancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.

"Inhibitors," "activators," and "modulators" of G protein-coupled receptors expression or of G protein-coupled receptors' activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for G protein-coupled receptors expression or G protein-mediated signaling, e.g., ligands, agonists, antagonists, and their homologs and mimetics. Inhibitors are compounds that, e.g., inhibit expression of a G protein-coupled receptor or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate the activity of a G protein-coupled receptor, e.g., antagonists. Activators are compounds that, e.g., induce or activate the expression of a G protein-coupled receptor or bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up-regulate the activity of G protein-coupled receptors, e.g., agonists. Modulators include compounds that, e.g., alter the interaction of a receptor with extracellular proteins that bind activators or inhibitors, G proteins, and kinases. Modulators include genetically modified versions of G protein-coupled receptors, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays for inhibitors, activators and modulators include, e.g., expressing a G protein-coupled receptor in cells or cell membranes, applying putative modulator compounds, in the presence or absence of a GPCR ligand (such as galanin, where appropriate) and then determining the functional effects on G protein-mediated signaling, as described above. Samples or assays comprising G protein-coupled receptors that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative G protein-coupled receptor activity value of 100%. Inhibition of a G protein-coupled receptor is achieved when the G protein-coupled receptor activity value relative to the control is about 80%>, optionally 50% or 25-0%>. Activation of a G protein-coupled receptor is achieved when the G protein-coupled receptor activity value relative to the control is 110%, optionally 150%>, optionally 200- 500%, or 1000-3000% higher.

III. GENERAL RECOMBINANT NUCLEIC ACIDS METHODS FOR USE WITH THE INVENTION In numerous embodiments of the present invention, nucleic acids encoding the GPCRs of interest will be isolated and cloned using recombinant methods. Such embodiments are used, e.g., to isolate GPCR-encoding polynucleotides for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from GPCRs, to monitor GPCR gene expression, for the isolation or detection of GPCR sequences in different species, for diagnostic purposes in a patient, e.g., to detect mutations in GPCRs, etc. In one embodiment, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a rat, a mouse, etc. In addition, recombinant expression of a GPCR of interest in eukaryotic cells, is useful for making cell membrane preparations that can be used for receptor binding assays. Receptor binding assays are used, in particular, for screening for modulators of the activity of GPCRs. A. General Recombinant Nucleic Acids Methods

The numerous applications of the present invention involving the cloning, synthesis, maintenance, mutagenesis, and other manipulations of nucleic acid sequences can be performed using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al, Current Protocols in Molecular Biology (1994).

Nucleotide sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis or, alternatively, from published DNA sequences.

Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22(20):1859-1862 (1981), using an automated synthesizer, as described in Needham Van Devanter et al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is, for example, by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Reanier, J Chrom. 255:137-149 (1983).

The nucleic acids described here, or fragments thereof, can be used as hybridization probes for genomic or cDNA libraries to isolate the corresponding complete gene (including regulatory and promoter regions, exons and introns) or cDNAs, in particular cDNA clones corresponding to full-length transcripts. The probes may also be used to isolate other genes and cDNAs which have a high sequence similarity to the gene of interest or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. • The sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert, Methods in Enzymology 65:499-560 (1980). The sequence can be confirmed after the assembly of the oligonucleotide fragments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double- stranded templates of Wallace et al, Gene 16:21-26 (1981). Southern blot hybridization techniques can be carried out according to Southern et al, J. Mol. Biol. 98:503 (1975).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding the Desired Proteins

In general, the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode copy DNA (cDNA) or genomic DNA. The particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences provided herein (e.g., the sequences set forth in Table 1), which provides a reference for PCR primers and defines suitable regions for isolating G protein-coupled receptors specific probes. Alternatively, where the sequence is cloned into an expression library, the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against the G protein-coupled receptor of interest. Methods for making and screening genomic and cDNA libraries are well- known to those of skill in the art (see, e.g., Gubler and Hoffman, Gene 25:263-269 (1983); Benton and Davis, Science 196:180-182 (1977); and Sambrook, supra).

Briefly, to make the cDNA library, one should choose a source that is rich in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. For a genomic library, the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al, Proc. Natl. Acad. Sci. USA 72:3961-3965 (1975).

An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template. Suitable primers can be designed from specific GPCRs, e.g., the sequences described in Table 1. This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a G protein-coupled receptor of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and 4,683,202). Genes amplified by a

PCR reaction can be purified, e.g., from agarose gels, and cloned into an appropriate vector.

Appropriate primers and probes for identifying the genes encoding the G protein-coupled receptors of the invention from mammalian tissues can be derived from the sequences provided herein, in particular the sequences set forth in Table 1. For a general overview of PCR, see, Innis et al, PCR Protocols: A Guide to Methods and

Applications, Academic Press, San Diego (1990).

Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.

A gene encoding a G protein-coupled receptor of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes, using standard methods well-known to those of skill in the art, or eukaryotes as described infra.

C. Expression in Eukaryotes

Standard eukaryotic transfection methods are used to produce eukaryotic cell lines, e.g., yeast, insect, or mammalian cell lines, which express large quantities of the G protein-coupled receptors of the invention which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619-17622, (1989); and Guide to Protein Purification, in Vol. 182 of Methods in Enzymology (Deutscher ed., 1990)).

Transformations of eukaryotic cells are performed according to standard techniques as described by Morrison, J Bad, 132:349-351 (1977), or by Clark-Curtiss and Curtiss, Methods in Enzymology, 101 :347-362 R. Wu et al (Eds) Academic Press, NY (1983).

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure utilized be capable of successfully introducing at least one gene into the host cell which is capable of expressing the protein.

The particular eukaryotic expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic cells may be used. Expression vectors containing regulatory elements from eukaryotic viruses are typically used. Suitable vectors for use in the present invention include, but are not limited to, SV40 vectors, vectors derived from bovine papilloma virus or from the Epstein Barr virus and baculovirus vectors, and any other vector allowing expression of proteins under the direction of the SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhednn promoter, or other promoters shown effective for expression in eukaryotic cells.

The vectors usually include selectable markers which result in gene amplification, such as, e.g., thymidine kinase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase), adenosine deaminase, dihydrofolate reductase, asparagine synthetase and ouabain selection. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as, e.g., using a baculovirus vector in insect cells, with a target protein encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The expression vector of the present invention will typically contain both prokaryotic sequences that facilitate the cloning of the vector in bacteria as well as one or more eukaryotic transcription units that are expressed only in eukaryotic cells, such as mammalian cells. The vector may or may not comprise a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the transfected DNA integrates into the genome of the transfected cell, where the promoter directs expression of the desired gene. The expression vector is typically constructed from elements derived from different, well characterized viral or mammalian genes. For a general discussion of the expression of cloned genes in cultured mammalian cells, see, Sambrook et al, supra, Ch. 16.

The prokaryotic elements that are typically included in the mammalian expression vector include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells.

The expression vector contains a eukaryotic transcription unit or expression cassette that contains all the elements required for the expression of the DNA encoding the G protein-coupled receptors of interest in eukaryotic cells. A typical expression cassette contains a promoter operably linked to the DNA sequence encoding the G protein-coupled receptor and signals required for efficient polyadenylation of the transcript. The DNA sequence encoding the protein may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell. Such signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25- 30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

Enliancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues (see, Enhancers and Eukaryotic Expression, Cold Spring Harbor Pres, Cold Spring Harbor, NY (1983)).

In the construction of the expression cassette, the promoter is preferably positioned at about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, some variation in this distance can, however, be accommodated without loss of promoter function.

In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.

If the mRNA encoded by the structural gene is to be efficiently translated, polyadenylation sequences are also commonly added to the vector construct. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40, or a partial genomic copy of a gene already resident on the expression vector.

In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned genes or to facilitate the identification of cells that carry the transfected DNA. For instance, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The cDNA encoding the protein of interest can be ligated to various expression vectors for use in transforming host cell cultures. The vectors typically contain gene sequences to initiate transcription and translation of the G protein-coupled receptor gene. These sequences need to be compatible with the selected host cell. In addition, the vectors preferably contain a marker to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or metallothionein. Additionally, a vector might contain a replicative origin.

Cells of mammalian origin are illustrative of cell cultures useful for the production of, for example, a G protein-coupled receptor of interest. Mammalian cell systems often will be in the form of monolayers of cells, although mammalian cell suspensions may also be used. Illustrative examples of mammalian cell lines include VERO and HeLa cells, NTH 3T3, COS, Chinese hamster ovary (CHO), WI38, BHK, COS-7 or MDCK cell lines.

As indicated above, the vector, e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the gene sequence encoding the G protein-coupled receptor of interest. These sequences are refened to as expression control sequences. Illustrative expression control sequences are described, e.g., in Berman et αl, Science, 222:524-527 (1983); Thomsen et αl, Proc. Nαtl. Acαd. Sci. 81 :659-663 (1984); and Brinster et αl., Nature 296:39-42 (1982). The cloning vector containing the expression control sequences is cleaved using restriction enzymes, adjusted in size as necessary or desirable and ligated with sequences encoding the G protein-coupled receptor by means well- known in the art.

When higher animal host cells are employed, polyadenylation or transcription terminator sequences from known mammalian genes need to be incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al, J. Virol 45:773-781 (1983)).

Additionally, gene sequences to control replication in the host cell may be incoφorated into the vector such as those found in bovine papilloma virus type- vectors (see, Saveria-Campo, "Bovine Papilloma virus DNA a Eukaryotic Cloning Vector" In: DNA Cloning Vol.1I: a Practical Approach (Glover Ed.), IRL Press, Arlington, Virginia pp. 213-238 (1985)).

The transformed cells are cultured by means well-known in the art. For example, such means are published in Biochemical Methods in Cell Culture and Virology, Kuchler, Dowden, Hutchinson and Ross, Inc. (1977). The expressed protein is isolated from cells grown as suspensions or as monolayers. The latter are recovered by well- known mechanical, chemical or enzymatic means.

IV. PURIFICATION OF THE PROTEINS FOR USE WITH THE INVENTION After expression, the proteins of the present invention can be purified to substantial purity by standard techniques, including selective precipitation with substances as ammonium sulfate, column chromatography, immunopurification methods, and other methods known to those of skill in the art (see, e.g., Scopes Protein Purification: Principles and Practice, Springer- Verlag, NY (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).

A number of conventional procedures can be employed when a recombinant protein is being purified. For example, proteins having established molecular adhesion properties can be reversibly fused to the subject protein. With the appropriate ligand, a G protein-coupled receptor of interest, for example, can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, the G protein-coupled receptors of the invention can be purified using immunoaffinity columns.

A. Purification of Proteins from Recombinant Bacteria

When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter refened to as inclusion bodies) typically involves the extraction, separation and or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1%> Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al, and Sambrook et al, both supra, and will be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.

Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%>, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate- forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of ineversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not ineversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al, supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20%> sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well-known to those of skill in the art.

B. Standard Protein Separation Techniques For Purifying Proteins 1. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The prefened salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%>. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well-known to those of skill in the art and can be used to fractionate complex protein mixtures.

2. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cutoff than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be cl romatographed as described below.

3. Column Cfiromatographv

The proteins of interest can also.be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well-known in the art.

It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).

V. DETECTION OF GENE EXPRESSION OF THE GPCRs

The polypeptides of the present invention and the polynucleotides encoding them can be employed as research reagents and materials for discovery of treatments and diagnostics to human disease. It will be readily apparent to those of skill in the art that although the following discussion is directed to methods for detecting nucleic acids encoding a G protein-coupled receptor, similar methods can be used to detect nucleic acids associated with, e.g., Alzheimer's disease, depression, specific carcinomas and sarcomas, or any disease or disorder in which GPCR-mediated signaling is involved. In aspects involving, e.g., a galanin receptor, similar methods can be used to detect nucleic acids associated with, e.g., Alzheimer's disease, learning and memory disorders, reproduction and sex behavior disorders, feeding disorders, fat metabolism and body adiposity, regulation of neurotransmitter release, pain perception, depression, regulation of hormone release, cardiovascular actions regulation, or any disease or disorder in which galanin signaling is involved.

As should be apparent to those of skill in the art, the invention is based, at least in part, in the identification of novel G protein-coupled receptors, including a novel galanin receptor (GAL4). Accordingly, the present invention also includes methods for detecting the presence, alteration or absence of nucleic acids (e.g., DNA or RNA) encoding such G protein-coupled receptors in a physiological specimen in order to determine the presence of, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcmoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, etc., associated with mutations created in the sequences encoding the GPCRs that modify the expression and/or activity of the receptors, including those disorders aassociated with mutations created in the sequences encoding the galanin receptor that modify the activity of the receptor, including cognitive deficit, Alzheimer' s disease, reproductive disorder, fat metabolism disorder, inhibition of neurotransmitter release, pain perception disorder, depression, hormone release disorder, decrease in blood flow, etc. Any tissue having cells bearing the genome of an individual, or RNA encoding the GPCRs can be used as well as biopsies of suspect tissue. It is also possible and prefened in some circumstances to conduct assays on cells that are isolated under microscopic visualization. A particularly useful method is the microdissection technique described in WO 95/23960. The cells isolated by microscopic visualization can be used in any of the assays described herein including both genomic and immunological based assays. This invention provides methods of genotyping family members in which relatives are diagnosed with, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcmoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, Alzheimer's disease, depression, fat metabolism disorders, anorexia, stroke, diabetes, etc. Conventional methods of genotyping are known to those of skill in the art. The probes are capable of binding to a target nucleic acid (e.g., a nucleic acid encoding a G protein-coupled receptor of interest). By assaying for the presence or absence of the probe, one can detect the presence or absence of the target nucleic acid in a sample. Preferably, non-hybridizing probe and target nucleic acids are removed (e.g., by washing) prior to detecting the presence of the probe. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g. , from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a G protein-coupled receptor of the invention.

The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization fonnats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins, Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al, Nature, 223:582-587 (1969). Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.

The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theoiγ of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, pp. 9-20, Burdon and van Knippenberg Eds., Elsevier (1985)).

The probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like. Other labels include, e.g., ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to ϊmmunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in Haugland, Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).

In general, a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis. Most typically, the amount of, for example, a G protein-coupled receptor

RNA is measured by quantitating the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantitating labels are well-known to those of skill in the art.

In prefened embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed anangement.

A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer anays (VLSIPS™), available from Affymetrix, Inc. in Santa Clara, CA, can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al, Science, 251:767-777 (1991); Sheldon et al, Clinical

Chemistry 39(4):718-719 (1993); and Kozal et al, Nature Medicine 2(7):753-759 (1996). Thus, in one embodiment, the invention provides methods of detecting expression levels of the G protein-coupled receptors of the invention in combination with other G protein- coupled receptors and other nucleic acids known to be involved in regulating, e.g., Alzheimer's disease, depression, feeding behavior, diabetes, obesity, stroke, cognition and memory, hormone release, amyotrophic lateral sclerosis, asthma, atherosclerosis, basal cell carcinoma, breast carcinoma, cardiomyopathy, chondrosarcoma, COPD, Crohn's disease, depression, Duchenne muscular dystrophy, embryonal carcinoma, epilepsy, Ewing's sarcoma, glioblastoma multiform, Hodgkin's disease, lymphoma, lung adenocarcinoma, lung small cell carcinoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, multiple sclerosis, osteoarthritis, osteoporosis, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, Parkinson's disease, prostate carcinoma, psoriasis, rhabdomyosarcoma, renal cell carcinoma, rheumatoid arthritis, schizophrenia, seminoma, squamous cell carcinoma, tuberculosis, thyroid carcinoma, tonsil, transitional carcinoma of the bladder, ulcerative colitis, etc., in which nucleic acids (e.g., RNA from a cell culture) are hybridized to an anay of nucleic acids that are known to be associated with the above-listed diseases and disorders. Thus, in one embodiment, the invention provides methods for detecting the expression levels of nucleic acids encoding the G protein-coupled receptors of the invention, in which nucleic acids (e.g., RNA from a cell culture) are hybridized to an anay of nucleic acids that are known to be associated with the above-listed diseases and disorders in which GPCRs have been implicated. In a second embodiment, the invention provides methods for detecting the expression levels of nucleic acids encoding the galanin receptors of the invention, in which nucleic acids (e.g., RNA from a cell culture) are hybridized to an array of nucleic acids that are known to be associated with Alzheimer's disease, depression, fat metabolism disorders, feeding disorders, hormonal disorders, etc. For example, in the assay described supra, oligonucleotides which hybridize to a plurality of nucleic acids encoding either G protein-coupled receptors or other molecules known to be involved in the above-mentioned diseases and disorders are optionally synthesized on a DNA chip (such chips are available from Affymetrix) and the RNA from a biological sample, such as a cell culture, is hybridized to the chip for simultaneous analysis of multiple nucleic acids. The nucleic acids encoding the G protein-coupled receptors that are present in the sample which is assayed are detected at specific positions on the chip. Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes). One prefened example uses an antibody that recognizes DNA- RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al, Analytical

Biochemistry 181:153-162 (1989); Bogulavski et al, J. Immunol. Methods 89:123-130 (1986); Prooijen-Knegt, Exp. Cell Res. 141:397-407 (1982); Rudkin, Nature 265:472-473 (1976); Stollar. PNAS 65:993-1000 (1970); Ballard, Mol Immunol 19:793-799 (1982); Pisetsky and Caster, Mol. Immunol 19:645-650 (1982); Viscidi et al, J. Clin. Microbial. 41:199-209 (1988); and Kiney et al, J. Clin. Microbiol. 27:6-12 (1989) describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, MD). In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies which are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (ed), Fundamental Immunology, Third Edition Raven Press, Ltd., NY (1993); Coligan, Current Protocols in Immunology Wiley/Greene, NY (1991); Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY (1989); Stites et al. (eds.), Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY, (1986); and Kohler and Milstein, Nature 256:495-497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al, Science 246:1275-1281 (1989); and Ward et al, Nature 341 :544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_D of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.

The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.

The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBAθ, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation. A prefened embodiment is the use of allelic specific amplifications. In the case of PCR, the amplification primers are designed to bind to a portion of, for example, a gene encoding a G protein-coupled receptor protein, but the terminal base at the 3' end is used to discriminate between the mutant and wild-type forms of the G protein-coupled receptor gene. If the terminal base matches the point mutation or the wild-type, polymerase dependent three prime extension can proceed and an amplification product is detected. This method for detecting point mutations or polymorphisms is described in detail by Sommer et al, inMayo Clin. Proc. 64:1361-1372 (1989). By using appropriate controls, one can develop a kit having both positive and negative amplification products. The products can be detected using specific probes or by simply detecting their presence or absence. A variation of the PCR method uses LCR where the point of discrimination, i.e., either the point mutation or the wild-type bases fall between the LCR oligonucleotides. The ligation of the oligonucleotides becomes the means for discriminating between the mutant and wild-type forms of the gene encoding the G protein-coupled receptor.

An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well-known and are generally described in Angerer et al, Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.

VI. IMMUNOLOGICAL DETECTION OF THE GPCRs

In numerous embodiments of the present invention, antibodies that specifically bind to the G protein-coupled receptors of the invention will be used. Such antibodies have numerous applications, including for the modulation of the activity of the G protein-coupled receptors and for immunoassays to detect the G protein-coupled receptors of the invention, as well as variants, derivatives, fragments, etc. thereof. Immunoassays can be used to qualitatively or quantitatively analyze the proteins of interest. A general overview of the applicable technology can be found in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., NY (1988). Immunoassays for detecting target G protein-coupled receptor proteins are useful for diagnosing any disease or disorder in which GPCR-mediated signaling has been involved such as, e.g., Alzheimer's disease, depression, specific sarcomas and carcinomas, Parkinson's disease, psoriasis, rheumatoid arthritis, schizophrenia, tuberculosis, learning and memory disorders, diabetes, reproduction and sex behavior disorders, anorexia, fat metabolism and body adiposity disorders, regulation of neurotransmitter release, pain perception, depression, regulation of hormone release, cardiovascular actions regulation, etc. In some embodiments, the antibodies of the present invention specifically bind to the G protein-coupled receptors of the invention and do not bind to other G protein-coupled receptors or to G protein-coupled receptors from a different species, such as mouse, rat, etc. (identified GPCRs are listed in public databases, such as SwissProt, see http://www.expasy.ch/sprot/sprot-top.html, or GenBank, see http://www.ncbi.nlm.nih.gov/; see also G protein coupled receptor Database, http://www.gcrdb.uthscsa.edu). In some embodiments, the antibodies of the present invention specifically bind to the galanin receptors of the invention and do not bind to other galanin receptors, such as GALR1, GALR2 and GALR3 (see, e.g., SwissProt accession numbers P47211, 043603, and O60755 for the sequences of the human GALRl, GALR2 and GALR3, respectively) or to galanin receptors from a different species (see, e.g., SwissProt accession numbers P56479, 088854, 088853, for the sequences of the mouse GALRl, GALR2, and GALR3, respectively, and accession numbers Q62805, O08726, and 088626, for the sequences of the rat GALRl, GALR2, and GALR3, respectively).

A. Antibodies to Target Proteins

Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest are known to those of skill in the art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et al, supra and references cited therein; Goding, supra; and Kohler and Milstein, Nature 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors (see, Huse et al, supra; and Ward et al, supra). For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.

Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10⁴ or greater are selected and tested for their cross-reactivity against non-G protein-coupled receptor proteins or even other homologous proteins from other organisms, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_D of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.

A number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the prefened immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determimng the titer of reactivity to the G protein-coupled receptor of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra). Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (See, Kohler and Milstein, Eur. J. Immunol 6:511-519 (1976)). Alternative methods of immortalization include, e.g., transformation with Epstein Ban Virus, oncogenes, or retroviruses, or other methods well-known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, supra.

Once target protein specific antibodies are available, the protein can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general, see, Stites, supra. Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio, Enzyme Immunoassay, CRC Press, Boca Raton, Florida (1980); Tijssen, supra; and Harlow and Lane, supra. Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum which was raised to the protein partially encoded by a sequence described herein (e.g., a sequence selected from the sequences set forth in Table 1) or a fragment thereof. This antiserum is selected to have low cross-reactivity against non-G protein-coupled receptor proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.

Polyclonal antibodies that specifically bind to a G protein-coupled receptor of interest from a particular species can be made by subtracting out cross-reactive antibodies using G protein-coupled receptor homologs. In an analogous fashion, antibodies specific to a particular G protein-coupled receptor (e.g., a G protein-coupled receptor encoded by a sequence set forth in Table 1) can be obtained in an organism with multiple G protein-coupled receptors genes by subtracting out cross-reactive antibodies using other G protein-coupled receptors.

Polyclonal antibodies that specifically bind to a galanin receptor of interest from a particular species can be made by subtracting out cross-reactive antibodies using galanin receptor homologs. In an analogous fashion, antibodies specific to a particular galanin receptor (e.g., the galanin receptors of the invention) can be obtained in an organism with multiple galanin receptor genes by subtracting out cross-reactive antibodies using other galanin receptors, such as GALRl, GALR2 and GALR3. B. Immunological Binding Assays

In a prefened embodiment, a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai, Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a G protein-coupled receptor of the invention or antigenic subsequences thereof). The capture agent is a moiety that specifically binds to the analyte. In a prefened embodiment, the capture agent is an antibody that specifically binds, for example, a GPCR of the invention. The antibody (e.g., anti-GPCR antibody) may be produced by any of a number of means well-known to those of skill in the art and as described above.

Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled GPCR polypeptide or a labeled anti-GPCR antibody. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex. In a prefened embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval et al. J. Immunol. I ll: 1401-1406 (1973); and Akersrrom et al, J. Immunol. 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.

1. Non-competitive Assay Formats Immunoassays for detecting proteins of interest from tissue samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured. In one prefened "sandwich" assay, for example, the capture agent (e.g., anti-GPCR antibodies) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the G protein-coupled receptor present in the test sample. The G protein-coupled receptor thus immobilized is then bound by a labeling agent, such as a second anti-GPCR antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

2. Competitive Assay Formats

In competitive assays, the amount of target protein (analyte) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (i.e., a GPCR of interest) displaced (or competed away) from a capture agent (i.e., anti- GPCR antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, the protein of interest is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds to the GPCR of interest. The amount of GPCR bound to the antibody is inversely proportional to the concentration of GPCR present in the sample. In a particularly prefened embodiment, the antibody is immobilized on a solid substrate. The amount of the GPCR bound to the antibody may be determined either by measuring the amount of subject protein present in a GPCR protein/antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein. The amount of GPCR protein may be detected by providing a labeled GPCR protein molecule.

A hapten inhibition assay is another prefened competitive assay. In this assay, a known analyte, in this case the target protein, is immobilized on a solid substrate. A known amount of anti-GPCR antibody is added to the sample, and the sample is then contacted with the immobilized target. In this case, the amount of anti-GPCR antibody bound to the immobilized GPCR is inversely proportional to the amount of GPCR protein present in the sample. Again, the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.

Immunoassays in the competitive binding format can be used for cross- reactivity determinations. For example, the protein encoded by the sequences described herein can be immobilized on a solid support. Proteins are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein encoded by any of the sequences described herein. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50%> of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein. 3. Other Assay Formats

In a particularly preferred embodiment, Western blot (immunoblot) analysis is used to detect and quantify the presence of a G protein-coupled receptor of the invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest. For example, the anti-GPCR antibodies specifically bind to the G protein-coupled receptor on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest. Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al, Amer. Clin. Prod. Rev. 5:34-41 (1986)).

4. Reduction of Non-Specific Binding One of skill in the art will appreciate that it is often desirable to use nonspecific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of nonspecific binding to the substrate. Means of reducing such non-specific binding are well- known to those of skill in the art. Typically, this involves coating the substrate with a proteinaceous composition. In particular, protein compositions, such as bovine serum albumin (BSA), nonfat powdered milk and gelatin, are widely used.

5. Labels

The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well- developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired component of the assay according to methods well-known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904).

Means of detecting labels are well-known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.

VII. SCREENING FOR MODULATORS OF THE GPCRs OF THE INVENTION

The invention also provides methods for identifying compounds that modulate signaling mediated by the G protein-coupled receptors of the invention. These compounds include both those that modulate the expression and those that modulate the activity of the G protein-coupled receptors of the invention. Furthermore, these compounds may modulate the expression and/or activity of one or of various G protein- coupled receptors of the invention, and optionally of all the G protein-coupled receptors of the invention. In addition, the identified compounds can also modulate, e.g., the development of Alzheimer's disease, rheumatoid arthritis, osteoarthritis, osteoporosis, amyotrophic lateral sclerosis, multiple sclerosis and atherosclerosis, asthma, depression, epilepsy, schizophrenia, Parkinson's disease, sarcomas such as, chondrosarcoma, Ewing's sarcoma, and osteosarcoma, carcinomas such as, basal cell carcinoma, breast carcinoma, embryonal carcinoma, ovarian carcinoma, renal cell carcinoma, lung adenocarcinoma, lung small cell carcinoma, pancreatic carcinoma, prostate carcinoma, transitional carcinoma of the bladder, squamous cell carcinoma, and thyroid carcinoma, psoriasis, cardiomyopathy, Crohn's disease, Duchenne muscular dystrophy, glioblastoma multiform, Hodgkin's disease, lymphoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, seminoma, tuberculosis, tonsil, ulcerative colitis, learning and memory processes, reproduction and sex behavior, feeding behavior, fat metabolism and body adiposity, neurotransmitter release, pain perception, depression, hormone release, cardiovascular actions, or any other disease or disorder involving GPCR-mediated signaling.

A. Screening for Modulators of the G Protein-Coupled Receptors

The present invention provides methods for identifying compounds that increase or decrease the expression level or the activity of one or more G protein-coupled receptors of interest. Compounds that are identified as modulators of the expression or activity of one or more G protein-coupled receptors of the invention using the methods described herein find use both in vitro and in vivo. For example, one can treat cell cultures with the modulators in experiments designed to determine the mechanisms by which GPCR-mediated signaling is regulated. Compounds that modulate the activity of the G protein-coupled receptors are useful for studying, for example, the mechanisms that lead to depression, Alzheimer's disease, specific sarcomas and carcinomas, other cancers such as lymphomas and melanomas, psoriasis, cardiomyopathies, etc. Compounds that modulate the activity of the galanin receptor are useful for studying, for example, the mechanisms that lead to growth hormone release, depression or fat accumulation, neurotransmitter or insulin release. The methods for isolating compounds that modulate the expression of the

G protein-coupled receptors of the invention typically involve culturing a cell in the presence of a potential modulator to form a first cell culture. RNA (or cDNA) from the first cell culture is contacted with one or more probes, each probe comprising a polynucleotide sequence encoding a G protein-coupled receptor of the invention (e.g., a nucleotide sequence selected from the group of sequences set forth in Table 1). The amount of the probe(s) which hybridizes to the RNA (or cDNA) from the first cell culture is determined. Typically, one determines whether the amount of the probe(s) which hybridizes to the RNA (or cDNA) is increased or decreased relative to the amount of the probe(s) which hybridizes to RNA (or cDNA) from a second cell culture grown in the absence of the modulator.

The G protein-coupled receptors of the invention and their alleles and polymorphic variants mediate signaling in different pathways involving a variety of ligands. The activity of G protein-coupled receptor polypeptides can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding (e.g., radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like. Furthermore, such assays can be used to test for inhibitors and activators of the G protein-coupled receptors of the invention. Modulators can also be genetically altered versions of the present G protein- coupled receptors. Such modulators of GPCR-mediated signaling activity are useful for treating a variety of diseases and disorders described herein. For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology vols. 237 and 238 (1994) and volume 96 (1983); Bourne et αl, Nature

10:349:117-27 (1991); Bourne et al, Nature 348:125-32 (1990); Pitcher et al, Annu. Rev. Biochem. 67:653-92 (1998).

The G protein-coupled receptors of the assay will typically be polypeptides having identity with polypeptides encoded by a nucleic acid molecule having a nucleotide sequence selected from the sequences set forth in Table 1, or conservatively modified variants thereof.

Generally, the amino acid sequence identity will be at least 70%, 75%, 80%, 85%, 90%, 95%o or more identity and further will not be identical to the sequences for known GPCRs (for sequences of identified GPCRs, see, e.g., http://www.gcrdb.uthscsa.edu; http://www.ncbi.nlm.nih.gov; and http://www.expasy.ch/sprot/sprot.top.html). With regard to galanin receptors, the amino acid sequences of the invention will not be identical to the sequences for GALRl, GALR2 or GALR3 (see, e.g., SwissProt accession numbers P47211, O43603, and O60755 for the sequences of the human GALRl, GALR2 and GALR3, respectively). Optionally, the polypeptide(s) of the assays will comprise a domain of a G protein-coupled receptor, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like. The polypeptides of the present invention may also be polypeptides comprising a region of 15 amino acids or more, optionally 30 amino acids or more, having at least 80%, preferably at least 85%_>, and most preferably 90% or more, identity with a region of 15 amino acids or more, optionally 30 amino acids or more, from a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1, and having substantially the same biological activity. Either the G protein-coupled receptor protein or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.

Modulators of the activity of G protein-coupled receptors are tested using G protein-coupled receptors polypeptides as described above, either recombinant or naturally occurring. The proteins can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring. For example, neurons, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein. G protein-mediated signaling can also be examined in vitro with soluble or solid state reactions, using a full-length G protein-coupled receptor or a chimeric molecule such as an extracellular domain or transmembrane region, or combination thereof, of a G protein- coupled receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain and/or transmembrane region covalently linked to the transmembrane and/or cytoplasmic domain of a G protein-coupled receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding. In numerous embodiments, a chimeric receptor will be made that comprises all or part of a G protein-coupled receptor polypeptide as well as an additional sequence that facilitates the localization of the G protein-coupled receptor to the membrane. Ligand binding to a G protein-coupled receptor, a domain thereof, or a chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape), chromatographic, or solubility properties. G protein-coupled receptor-G protein interactions can also be examined. For example, binding of the G protein to the receptor or its release from the receptor can be examined. For example, in the absence of GTP, an activator will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways. Such an assay can be modified to search for inhibitors, e.g., by adding an activator to the G protein-coupled receptor and G protein in the absence of GTP, which form a tight complex, and then screen for inhibitors by looking at dissociation of the G protein-coupled receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.

In some embodiments, G protein-coupled receptors-ligand interactions are monitored as a function of G protein-coupled receptors activation.

An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins. Target enzymes and effector proteins for G protein-coupled receptors that can be used in the context of the present invention are known to those of skill in the art.

In some embodiments, a G protein-coupled receptor polypeptide is expressed in a eukaryotic cell as a chimeric receptor with a heterologous, chaperone sequence that facilitates its maturation and targeting through the secretory pathway. Chimeric G protein-coupled receptors can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, the cells comprise a functional G protein that is capable of coupling the chimeric receptor to an intracellular signaling pathway or to a signaling protein. Activation of such chimeric receptors in such cells can be detected using any standard method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell.

In addition, activated G protein-coupled receptors become substrates for kinases. Phosphorylation of the G protein-coupled receptors of the invention can thus also be measured as a means to detect activation of the receptors. Phosphorylation may be detected by assaying the transfer of ³²P from gamma-labeled GTP to the receptor with a scintillation counter.

Samples or assays that are treated with a potential G protein-coupled receptor inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation. Such assays may be carried out in the presence of ligand, and modulation of the ligand-dependent activation is monitored. Control samples (untreated with activators or inhibitors) are assigned a relative G protein- coupled receptor activity value of 100. Inhibition of a G protein-coupled receptor protein is achieved when the G protein-coupled receptor activity value relative to the control is about 90%o, optionally 50%, optionally 25-0%. Activation of a G protein-coupled receptor protein is achieved when the G protein-coupled receptor activity value relative to the control is 110%, optionally 150%, 200-500%), or 1000-2000% or more.

Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing a G protein- coupled receptor of interest. One means to determine changes in cellular polarization is by measuring changes in cunent (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the "cell-attached" mode, the "inside- out" mode, and the "whole cell" mode (see, e.g., Ackerman et al, New Engl J. Med. 336: 1575-1595 (1997)). Whole cell cunents are conveniently determined using the standard methodology (see, e.g., Hamil et al, PFlugers. Archiv. 391:85 (1981). Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergand-Bogind et al, J. Membrane Biol 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al, J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al, J. Membrane Biology 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM. The effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above, and other parameters known to those of skill in the art. Any suitable physiological change that affects G protein-coupled receptor activity can be used to assess the influence of a test compound on the G protein-coupled receptors of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers, changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca²⁺, IP3, cGMP, or cAMP. Prefened assays for G protein-coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G protein-coupled receptors as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog. For G protein-coupled receptors, promiscuous G proteins can be used in the assay of choice (Wilkie et al. , Proc. Natl.

Acad. Sci. USA 88:10049-10053 (1991)). Such promiscuous G proteins allow coupling of a wide range of receptors.

Other assays to determine the activity of G protein-coupled receptors, can involve measuring changes in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, that occur due to the activation or inhibition of enzymes such as adenylate cyclase upon activation of the receptor.

In one embodiment, the changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) may be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al, Am. J. Resp. Cell and Mol. Biol. 11 : 159- 164 (1994) may be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Patent No. 4,115,538.

In another embodiment, transcription levels can be measured to assess the effects of a test compound on signal transduction. A host cell containing a G protein- coupled receptor of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be detected using northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using reporter gene may be used as described in U.S. Patent No. 5,436,128. The reporter genes can be, e.g., chloramphenicol acetyltransferase, luciferase, β-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili and Spector, Nature Biotechnology 15:961-964 (1997)). The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with the amount of transcription in a substantially identical cell that lacks the protein of interest. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the protein of interest.

Any other method that allows to determine the effect of a compounds on the activity of a G protein-coupled receptor of interest can also be used in the context of the present invention (for articles disclosing methods for determining the activity of G protein-coupled receptors, see, e.g., Fisone et al, Brain Res. 568:279-84 (1991); Ogren et al, Ann. NY Acad. Sci. 863:342-63 (1998); Wang et al, Neuropeptides 33:197-205 (1999)).

B. Modulators of the Activity of the G Protein-Coupled Receptors of the Invention The compounds tested as modulators of the G protein-coupled receptors of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions of a G protein-coupled receptor gene. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one prefened embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well- known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991); and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to, peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel et al, Berger et al, and Sambrook et al, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pynolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506,337; benzodiazepines, 5,288,514, and the like), etc. Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment, the invention provides soluble assays using molecules such as a domain, such as a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc., a domain that is covalently linked to a heterologous protein to create a chimeric molecule, a G protein-coupled receptor, or a cell or tissue expressing a G protein-coupled receptor, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, G protein-coupled receptor, or cell or tissue expressing the G protein-coupled receptor is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day. Assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.

The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the G protein-coupled receptor of interest) is attached to the solid support by interaction of the tag and the tag binder. A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).

Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott and Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages. Tag binders are fixed to solid substrates using any of a variety of methods cunently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer anays is well described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al, J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al, Science 251:767-777 (1991); Sheldon et al, Clinical Chemistry 39(4):718-719 (1993); and Kozal et al, Nature Medicine 2(7):753759 (1996) (all describing anays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of the G protein-coupled receptors of the invention. Control reactions that measure the G protein-coupled receptor activity of the cell in a reaction that does not include a potential modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in a prefened embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions which do not include a modulator provide a background level of binding activity.

In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a lαiown activator of the G protein-coupled receptors of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of a G protein-coupled receptor determined according to the methods herein. Second, a known inhibitor of the G protein-coupled receptors of the invention can be added, and the resulting decrease in signal for the expression or activity of a G protein-coupled receptor similarly detected. It will be appreciated that modulators can also be combined with activators or inhibitors to find modulators which inhibit the increase or decrease that is otherwise caused by the presence of the known modulator of the G protein-coupled receptor.

D. Computer-Based Assays Yet another assay for compounds that modulate the activity of G protein- coupled receptors involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of a G protein-coupled receptor based on the structural information encoded by its amino acid sequence. The input amino acid sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein. The models of the protein structure are then examined to identify regions of the structure that have the ability to bind, e.g., ligands. These regions are then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues (or conesponding nucleic acid sequences encoding a G protein-coupled receptor) into the computer system. The nucleotide sequence encoding the GPCR can be any sequence encoding a polypeptide having at least 30%, optionally at least 40%>, 50%, 60%, 70%>, 80%), 90%) or more identity with a polypeptide encoded by a nucleic acid molecule having a sequence selected from the group consisting of the sequences set forth in Table 1, and conservatively modified versions thereof. The amino acid sequences encoded by the nucleic acid sequences provided herein represent the primary sequences or subsequences of the proteins, which encode the structural information of the proteins. At least 10 residues of an amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structures of the protein of interest. The software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are refened to as "energy terms" and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program uses these tenns encoded by the primary structure or amino acid sequence to create the secondary structural model.

The tertiary structure of the protein encoded by the secondary structure is then fonned on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like. Once the structure has been generated, potential ligand-binding regions are identified by the computer system. Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of the G protein-coupled receptor to identify ligands that bind to the protein. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.

Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of genes encoding the G protein-coupled receptors of the invention. Such mutations can be associated with disease states or genetic traits. As described above, GeneChip™ and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated G protein-coupled receptor genes involves receiving input of a first amino acid sequence of a G protein-coupled receptor (or of a first nucleic acid sequence encoding a GPCR of the invention), e.g., any amino acid sequence having at least 30%, optionally at least 40%, 50%, 60%, 70%, 80%, 90% or more identity with a polypeptide encoded by a nucleic acid molecule having a sequence selected from the group consisting of the sequences set forth in Table 1, or conservatively modified versions thereof, or alternatively any amino acid sequence comprising a region of 15 amino acids or more, optionally 30 amino acids or more, having at least 80%, preferably at least 85%>, and most preferably 90%> or more, identity with a region of 15 amino acids or more, optionally 30 amino acids or more, from a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence. The second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified. Such sequences can represent allelic differences in various G protein-coupled receptor genes, and mutations associated with disease states and genetic traits.

VIII. COMPOSITIONS, KITS AND INTEGRATED SYSTEMS The invention provides compositions, kits and integrated systems for practicing the assays described herein using nucleic acids encoding the G protein-coupled receptors of the invention, or the G protein-coupled receptors proteins themselves, anti-G protein-coupled receptors antibodies, etc.

The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more nucleic acids encoding a G protein-coupled receptor immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of a G protein-coupled receptor of the invention can also be included in the assay compositions. The invention also provides kits for carrying out the assays of the invention. The kits typically include a probe that comprises a polynucleotide sequence encoding a G protein-coupled receptor, and a label for detecting the presence of the probe. The kits may include several polynucleotide sequences encoding G protein- coupled receptors of the invention. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the G protein-coupled receptors of the invention, or on activity of the G protein-coupled receptors of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of G protein- coupled receptors, a robotic armature for mixing kit components or the like.

The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of the G protein-coupled receptors of the invention. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety. A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, MA) automated robot using a Microlab 2200 (Hamilton; Reno, NV) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous STAT binding assays. Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or Pentium chip-compatible DOS , OS2^® WINDOWS^®, WINDOWS NT^®, WINDOWS95^® or WLNDOWS98^® based computers), MACINTOSH^®, or UNIX^® based (e.g., SUN^® work station) computers.

One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an anay of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels conesponding to regions of the specimen (e.g., individual hybridization sites on an anay of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.

IX. GENE THERAPY APPLICATIONS

A variety of human diseases can be treated by therapeutic approaches that involve stably introducing a gene into a human cell such that the gene is transcribed and the gene product is produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, including those in which the defect is in a single gene. Gene therapy is also useful for treatment of acquired diseases and other conditions. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases, see, Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932 (1993).

In the context of the present invention, gene therapy can be used for treating a variety of disorders and/or diseases in which G protein-coupled receptor- mediated signaling has been implicated. For example, introduction by gene therapy of polynucleotides encoding a G protein-coupled receptor of the invention can be used to treat, e.g., Alzheimer's disease, rheumatoid arthritis, osteoarthritis, osteoporosis, amyotrophic lateral sclerosis, multiple sclerosis and atherosclerosis, asthma, depression, epilepsy, schizophrenia, Parkinson's disease, a number of sarcomas (e.g., chondrosarcoma, Ewing's sarcoma, osteosarcoma, etc.) and carcinomas (e.g., basal cell carcinoma, breast carcinoma, embryonal carcinoma, ovarian carcinoma, renal cell carcinoma, lung adenocarcinoma, lung small cell carcinoma, pancreatic carcinoma, prostate carcinoma, transitional carcinoma of the bladder, squamous cell carcinoma, thyroid carcinoma, etc.), psoriasis, cardiomyopathy, Crohn's disease, Duchenne muscular dystrophy, glioblastoma multiform, Hodgkin's disease, lymphoma, macular degeneration, malignant fibrous histiocytoma, melanoma, meningioma, mesothelioma, seminoma, tuberculosis, tonsil, ulcerative colitis, etc. Introduction by gene therapy of polynucleotides encoding a galanin receptor of the invention can be used to treat, e.g., anorexia, to induce nerve regeneration and to decrease noniception. In addition, antisense polynucleotides can also be administered using gene therapy to treat, e.g., obesity, diabetes

A. Vectors for Gene Delivery

For delivery to a cell or organism, the nucleic acids of the invention can be incorporated into a vector. Examples of vectors used for such purposes include expression plasmids capable of directing the expression of the nucleic acids in the target cell. In other instances, the vector is a viral vector system wherein the nucleic acids are incorporated into a viral genome that is capable of transfecting the target cell, hi a prefened embodiment, the nucleic acids can be operably linked to expression and control sequences that can direct expression of the gene in the desired target host cells. Thus, one can achieve expression of the nucleic acid under appropriate conditions in the target cell.

B. Gene Delivery Systems

Viral vector systems useful in the expression of the nucleic acids include, for example, naturally occurring or recombinant viral vector systems. Depending upon the particular application, suitable viral vectors include replication competent, replication deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genome of human or bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated virus, minute virus of mice (MVM), HIV, sindbis virus, and retro viruses (including, but not limited to, Rous sarcoma virus), and MoMLV. Typically, the genes of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral DNA, followed by infection of a sensitive host cell and expression of the gene of interest.

As used herein, "gene delivery system" refers to any means for the delivery of a nucleic acid of the invention to a target cell. In some embodiments of the invention, nucleic acids are conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and intemalization of the endosome) through an appropriate linking moiety, such as a DNA linking moiety (see, e.g., Wu et al, J. Biol Chem. 263:14621-14624 (1988); and WO 92/06180). For example, nucleic acids can be linked through a polylysine moiety to asialo-oromucocid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.

Similarly, viral envelopes used for packaging gene constructs that include the nucleic acids of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (see, e.g., WO 93/20221; WO 93/14188; and WO 94/06923). In some embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis (Curiel et al, Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments, molecular conjugates of the instant invention can include microtubule inliibitors (WO 94/06922), synthetic peptides mimicking influenza virus hemagglutinin (Plank et al, J. Biol. Chem. 269:12918-12924 (1994)), and nuclear localization signals such as SV40 T antigen (WO 93/19768).

Retro viral vectors are also useful for introducing the nucleic acids of the invention into target cells or organisms. Retroviral vectors are produced by genetically manipulating retroviruses. The viral genome of retroviruses is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transduced cells with a high degree of stability and efficiency. The integrated DNA copy is refened to as a provirus and is inherited by daughter cells as is any other gene. The wild type retroviral genome and the proviral DNA have three genes, the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site) (see, Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al, Cell 33:153-159 (1983); Cone and Mulligan, Proc. Natl Acad. Sci. U.S.A. 81:6349- 6353 (1984)).

The design of retroviral vectors is well-known to those of ordinary skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well-known in the art and are used to construct retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including, e.g., European Patent Application EPA 0 178 220; U.S. Patent No. 4,405,712; Gilboa, Biotechniques 4:504-512 (1986); Mann et al, Cell 33:153- 159 (1983); Cone and Mulligan, Proc. Natl Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al, Biotechniques 6:608-614 (1988); Miller et al, Biotechniques 7:981-990 (1989); Miller (1992) supra; Mulligan (1993), supra; and WO 92/07943.

The retroviral vector particles are prepared by recombinantly inserting the desired nucleotide sequence into a retro virus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line. The resultant retroviral vector particle is incapable of replication in the host cell but is capable of integrating into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient is capable of producing, for example, a G protein-coupled receptor of interest and thus restore the cells to a normal phenotype.

Packaging cell lines that are used to prepare the retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging, but which are incapable of producing infectious virions. The defective retroviral vectors that are used, on the other hand, lack these structural genes but encode the remaining proteins necessary for packaging. To prepare a packaging cell line, one can construct an infectious clone of a desired retrovirus in which the packaging site has been deleted. Cells comprising this construct will express all structural viral proteins, but the introduced DNA will be incapable of being packaged. Alternatively, packaging cell lines can be produced by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins, h these cells, the gag, pol, and env genes can be derived from the same or different retroviruses. A number of packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al, J. Virol 65:2220-2224 (1991)). Examples of other packaging cell lines are described in Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.

Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used. Alternatively, amphotropic or xenotropic envelope proteins, such as those produced by PA317 and GPX packaging cell lines may be used to package the retroviral vectors. In some embodiments of the invention, an antisense nucleic acid is administered which hybridizes to a gene encoding a G protein-coupled receptor of the invention or to a transcript thereof. The antisense nucleic acid can be provided as an antisense oligonucleotide (see, e.g. , Murayama et al, Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid can also be provided; such genes can be introduced into cells by methods known to those of skill in the art. For example, one can introduce a gene that encodes an antisense nucleic acid in a viral vector, such as, for example, in hepatitis B virus (see, e.g., Ji et al, J. Viral Hepat. 4:167-173 (1997)), in adeno-associated virus (see, e.g., Xiao et al, Brain Res. 756:76-83 (1997)), or in other systems including, but not limited, to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al, Ann. NY Acad. Sci. 811:299-308 (1997)), a "peptide vector" (see, e.g., Vidal et al, CR Acad. Sci III 32:279-287 (1997)), as a gene in an episomal or plasmid vector (see, e.g., Cooper et al, Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al, Hum Gene Ther. 8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see, e.g., Niidome et al, J. Biol. Chem. 272:15307-15312

(1997)), as "naked DNA" (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466), in lipidic vector systems (see, e.g., Lee et al, Crit Rev Ther Drug Carrier Syst. 14: 173-206 (1997)), polymer coated liposomes (U.S. Patent Nos. 5,213,804 and 5,013,556), cationic liposomes (Epand et al, U.S. Patent Nos. 5,283,185; 5,578,475; 5,279,833; and 5,334,761), gas filled microspheres (U.S. Patent No. 5,542,935), ligand-targeted encapsulated macromolecules (U.S. Patent Nos. 5,108,921; 5,521,291; 5,554,386; and 5,166,320).

C. Pharmaceutical Formulations

When used for pharmaceutical purposes, the vectors used for gene therapy are fonnulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al, Biochemistry 5:467 (1966).

The compositions can additionally include a stabilizer, enhancer or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the nucleic acids of the invention and any associated vector. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well-known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

D. Administration of Formulations

The formulations of the invention can be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan. In some embodiments of the invention, the nucleic acids of the invention are formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations. Exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in, e.g., U.S. Patent No. 5,346,701. E. Methods of Treatment

The gene therapy formulations of the invention are typically administered to a cell. The cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture. The cell can be provided in vivo, ex vivo, or in vitro. The formulations can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods. In some embodiments of the invention, the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics. In further embodiments, the nucleic acids are taken up directly by the tissue of interest. In some embodiments of the invention, the nucleic acids of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient. Examples of e vivo administration of therapeutic gene constructs include Nolta et al, Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al, Seminars in Oncology 23 (l):46-65 (1996); Raper et al, Annals of Surgery 223(2):116-26 (1996); Dalesandro et al, J. Thorac. Cardi. Surg. 11(2):416-22 (1996); and Makarov et al, Proc. Natl. Acad. Sci. USA 93(l):402-6 (1996).

X. ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS

Modulators of the G protein-coupled receptors of the present invention can be administered directly to the mammalian subject for modulation of G protein-coupled receptor signaling in vivo. Administration is by any of the routes normally used for introducing a modulator compound into contact with the tissue to be treated and well- known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington, Pharmaceutical Sciences, 17^th ed. 1985)).

The modulators of the expression or activity of the G protein-coupled receptors of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via ' inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi- dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part a of prepared food or drug.

The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time. The dose will be determined by the efficacy of the particular modulators employed and the condition of the subject, as well as the body weight or surface area of the area to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject. For administration, the GPCR modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the inhibitor at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Table 1 below indicates, by identification in the "LifeSpan Cluster ID" column, sequences encoding putative human G protein-coupled receptors that were identified by low-stringency protein- and DNA-based blast searches of publicly available databases. "Ace. No" indicates the accession number of the sequence in the database from which the sequence of each putative receptor was identified. The type of database from which the sequence was identified and the length of the sequence in base-pairs (bp) are indicated in the "Database type" and the "Sequence Length" columns, respectively. The sequence is shown in the "Sequence" column. The column designated "LS Cluster Name and/or Representative Sequence (SEQ ID NO) provides the name of LifeSpan's gene cluster for the sequence as well as the sequence ID of another representative sequence for the cluster, if available. These representative sequences are provided in the sequence listing following Table 1. Table 1 further shows information about the closest homolog of the sequence. The name, accession number and length of the closest homolog are shown in the "Homolog Name," "Homolog Accession No." and "Len" columns, respectively. Length is given in number of amino acids unless otherwise indicated. The table also indicates the position ("From" and "To" columns) and length ("Aligned") of the region of significant identity between the sequence of interest and its closest homolog, as well as the percent identity ("Percent") over the described region. Table 1

LS

LS Cluster Cluster H>: N me and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative (4cc. No.

(Original) Sequence

LG NO. (SEQ ID NO)

2₂315 AC006087 Genomic 1237 CAATGGCATG TGTGTTCAGA GGGCGGAATC CTGGGGACAC TGTGTGAAGG j GPR92 P43657 P2Y 344 17 303 i 288 37

Clone ' I AGACAGAGA GTGGGAGTCG GAGGGTCGGA GCAGCCCCTG ACTGGCGGCA Puπnoreceptor LG5261 I J TCCGGCCTGG TGGCGTCGGT GGTGACGGCG GACCTTTCGG ATTGCGCGAG ι SEQ ID 5

CGCCGCCCGC GTCCCGTTGG TGGCCGAGGT CCTGGCCCGG TGCGGAGTGC [ NO 3)

CCAGGCCGCG CAGGGTGTTG CGGAAGCCCT CGGCGCTAAA GTAGTACACC I

AGCGGGTCCA GCACGCAGTT GGCGCCGGCC AGCAGCACCA TCACCATCAG , ¹ CACCCCGCGC ACGCGATCGC GGGCAGGCAC GCTGGCCGCC ACCAGCTTGC

TCCGCAGCAG CCCGTAGACC GCCAGCGTGC TGTTGTAGGG CACGAAGCAC iAGCAGGAAGA TGACGAGGTT AGCCAGCAGG AGGCGCACGG TCTTCCGCCG > CCGCTGGCTC TGCGTGGCGT CGGGGCGCGC CAGCGTCCAG AAGACTCGGC I CCGACGAGTA GACCACCGCC GCCAGGGGCA GCAGGAAGCC CAGCGCCTCG ^! GCCAGCAGCA CGAGGGGCAG CAGCCTGCCT TTCCACAGCT CGTCGCTGAA j GCTCTCGAAG CATAGGCGCA CCTCGAGGTC CCGGTAGCGG CAACGCGAGG

GCCTGTGCAC GCGGGCGGCG GGCACGGCAA ACACCAGGAT GAGCGCCCAC .ACGCCCAGGC AGAGCAGCCG CGCCACGCGG GGCCGCCGCA GGTGGCGCAG j I TCGCAGCGGG TGCACGATGG CGGCGTAGCG GTCCACGTTG ATGAGCATCA j ' GGAAGATGCA GCTGCCGTAC ATGTTCATCT GGAAGATGGC GCCCGTCGTC

TGGCACAGGA GGTCGGGGAA GGGCCAGTGG TGCAGTGCGT AGTAGGAGAG j 'ACGAACGGGC AGCGAGAGGG TGAAGAGCAG GTCGCTGGCC GCCAGGTTAC

ACATGTACAC GCTCACCACC GAGTGCACGC GCAGCGCGCG CAGGAAGACC ¹ CAGAGGGCTA GCGCGTTGAG GGGGAGCCCG GCAGCCAGCA CCAAGCTGTA ' 1 GACCACCAAG TGCAGGCGGT GGGTAGGTCG GTAGTCAGGA CACGGGAGAA ' CAGAACTGTT GGTTGAGGAG CTGTTGGCTA ACATCGTGCC AAAGTGGGAT I

TGGGAGCTAG GCTGGGGATG CCATGGAGCA CACCAGAATC ATGGCATGGC I (ATTCACCTCC GGGGCTGGGG CCTAGAGGCT GTACAGA

LS

LS Cluster Cluster U): Name and

Database Sequence RepreHomolog

Current Ace No Sequence Type Homolog Name sentative Len. From To Aligned Percent Length IAcc.No.

(Original) Sequence

LG NO. (SEQ ID NO)

• 54602 AA012849 Dbest 447 TCCACATCTT CACACTCCTT CAGGATCAAA AACCTAAGCC ACATGACTGG Phermone Q62855 Pheromone j 310 ( 35 182 142 i 34

ATGAGCCGTC ACTTGGCCTT CATTCGGGTA GTGATGGTCC TCACTGTAGT i receptor receptor VN6, rat I GGATGTTTTG CCTCCAGATA TGCTTGAATC ACTGCATTTT GGGAATAACT I (PHRET) j TCAAATGCAA GTCCTTGATC TAAATAAACA GAGTGACGAG GGGCCTATGT j >ATCTATACCA CCTGTCTCCT GAGTATACAC CAGGCCAGCA TAATCAGCCT I SEQ ID I CAGCAACTTC TGGTTGGAAA GCTTTAAACA TAAATTTACA AATAACATTG ' NO 5 I TCAGTGTCCT CTTTTTTCTT TTTTGTTCCC TCAATTTGTC TTTCAGTAGT ' GACATAATAT TCTTCACTGT GGCTTCTTCC ATTGTGACCC AGACCAATCT i iACTTAAGGTC CGCAAATACT GCTCACGTTC TCCCATGAAG TCCATCA j 55728 AI024852 Dbest 505 j TTTTTTCATT TAGATAACAT TTTATTTGTT AGAGCAAGAT TTGGCAGACT ETL I 094867 j KIAA0768 872 405 548 145 53 I CATTTCAAC AGCTTAAACA TCCAAAACAA CAGGGGACAT TTTTGAACAA I protein Protein

TCTGTAATAT TCTTCTTGAA TCTTTCTAGA TAAAACACAC AGGAATAAAA I [Fragment] I AATGAACAT CCCCTGGAAA GCATTGCTGA CTGTGAAGAβ GTAAGCTGTA SEQ ID IACCACTGATG CGTGCACAAC ATGGAGAACC CCAAAGATCC AGGTGGTGCC NO 6 ' GAGAAGGAAC AGAAGAGCGA GGGCTCCTCT TGCACAAGAC CTTATGTTCT ' CAAAGCAACT AACTTCTGGT TTCAACCCTG CAGTGTGACG AAAAACTTTG ^! I TATATGATGA CTCCAAAAGC CAAGAGATTA ACAAGAATGA TTAGGCATGC

TGGTCCTATA AAACTCCAAA TAAAGTGTGT TTCGGTGCTA AGCCAACATA f CTTNTGTTGT GCCATAATAT CTGTATCCΓA GTGCΓGCCGA AAATCCAACT j IACCAC

1 160221 T19393 j Dbest _f 379 I GCACATTCGT CCTCCTAACT ^"CGACTTTCTT CCTGACAATA GGCCCTGCAG ' GPR27 AF027955 G protein- ' 2679 ' 2548 I 2626 79 93

(121660) ! < f TCTTTTTGTA GCGGTACTGA CGTCTTTTAT TCCATGTGTG GTTCCTTTTT , coupled receptor j bp TTCTTTTTCT ATAAAGGCTG TACTAATTTT CTTCATGCAA CGTTTCCTAA SEQ ID j (GPR27), Mus AGACCATGGC CAGTTTTCTA CAGAAGCTAT TTTTGACAAC CTCAAGTGGC ' NO 7 musculus '

_I ATTACATTTT GCAGTGAAGT AGAGGAACCT AGGGGGACTT CTTCACAAGT ' TAGATTTCTT GAGGATCTTC TGTTGGAAGC AGGAGAAAGT GGGGGGTGGG j GGGAAGTTGT CCGAAATGCC CTCTGAATTG CCGGCTGCAG GGTCCTTGTG

I CTGCGCTGGT TCTTTGAAAG TCTCAGTGT

LS

Cluster Name and RepreHomolog

Sequence Homolog Name Len. From To Aligned Percent sentative l&cc. No. Sequence (SEQ ID NO)

[AATGCTAAAA GTACTGGTTG GCTCTGTAGG ACCCTCAGAA TCAAAAGGAA , C-C P32248 C-C Chemokine 378 21 156 139 48 ₍ACTCCTCCAC ACTTTGTCTC TGTCTTCTCC AGGACCCATA TTTCTTGGCC chemokine I I receptor type 7 JACTTTCATAA CGTAGTTTTT GAAAGATGCT CCCATAAAAA CATAAAGGAT > receptor ' precursor j TGGGTTGAGG CAGCTGTGAA AGAGTGCGAT GCTTTCTGTG ACTTGGATGG ' 11 1 CGATGTCCAT GCGTTTGCTC ATGTTGCAGC TGGTGATCAG GGAGTAGATG j :ATGTCTATGG CTCGGCAGAA CTTGACAATG TTATAAGGCA GTTGAGTGAC SEQ ID ' j AATGAAAACT ATAACGACTG TGAGCAGAAC TTTTAGGGGT CGAGATATTT NO 34 | ! TAATGTTTGG CATCTTCATG AGTGTCCTTG CTGTGATAAA GTAGCACACC I CCCATAATAA GAAAGGGTAC TACAAATCCA ATGCAGATCT CTAGCATTTG ₍ jAATCAATGCT TTCATTGATG TTCCTAGGTA GCGGGGGAAA ATGGGAATGC [ACCTAGCATT GTCATTTACT GTATAAAAAA CCAGCTGGGG TATGCTCAGC IAAGATGGCAG CCATCCAGAC ACAGAAACAG ATGATCCAGC ATGGTTTTCC J CACTCCTGAT TGGCTGGGGA CTTTAGTTAC TGCCACATAT CTGTCTATGC g TGATACAAGC CAGAAACTGC ATTCCAGAGA CAAAGTTTAG TGTGTACAAG 1 GCTGAAGTTA TTTTGCACAT TATTTTCCCT AAAACCCACC CATGAACTGC I ATTAACAGCC CAAAAAGGCA GAGTGAATAG AAGGAGTAAA TCTGCTACAG CCAAATTCAG GATGTACACA TCTGTTTTGG TTCTCTGTTT CTTGTAATAG GCATAAATTG CCACTACCAT GGAATTGCCT GCAAGTCCAA TGACGAAAAC TATTGTGAGG AATACAGGGA GGAAAACTTT TGCAAATTCT CTGACATCTT CTTTGATACA GATCAGTTCA TATTGACTGT AGTCATAAGT GCCATTCATT ! CATTTTCCT CATAATAATA ATCTGTTGAC TGGTTCTGTT CCAAAGCCAT ! 1 1 I GGCTCCAAT

TTTTTGAAGT TTTCATTCAT AAATGCATAG ACAATGGGAT TACAGATGGA 1 ^< .

Q9Y5X5 G protein 1 522 260 { i 334 68 33

GTTGGAAAAT CCAATAATTT GCACGATAGC AAAAATCATC TTGATTGTGA coupled receptoorr

CATCATCATA TTCCTTTTCA AAATTACTGT ATTCAATCAT CATATGGACA SEQ ID

ACATGGAATG GTGCCCAGCA CACAGCAAAG AGAGCCACCA CTGTCACCAT NO 8 i

CATAATGACA GCTCGTTTCT TCTTCCATAA GAGGCAGGAG GAAGAGGATG

ACAAGGATGA AGGTGGTGTA GATCTTCTGG TGCACAGGGC TGGTCCACTC

^: TTCTAAGCAG CAGATGTGTT CCTTTTCATA TAGGAAGTCA TATTTGATCT !

^' CAAGTTGTTG CACGTGCCAC ATGGGTGATC CTACGATGAC TGCCACCAGC

I CAGACCACAC CTAGCATTGT GAAAGCCCTT CG

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative |Acc. No.

(Original) Sequence

LG NO. (SEQ ID NO)

160324 AI090920 Dbest 455 ACTTTGCCTT CCAGCTTTTT GTTGTTTTTT CTGTCCTTAC TTTTGGACTT I GPR86 Q15391 I ORF, complete 338 84 230 147 43

TCTATAAGAA TCATATACTT TTTTTGCAAT AACCACATAA AACACAAGCA j , cds

1 TTAGGATAAA AACAGTCCAG AAAATAAACT GGCATATGTT ATTTACCATT J SEQ ID

TGATGCCATT TCAGCCCCAG AGGCCCCTTT AAGGAAGCAC ACTTTTTCAC j NO 9 jAGACGATGGT GTTGCTTCCT TGTTGCTCAA GATCATATTT GGCAGGGAGA '

I TGAAGAACAA AAAGAACCAG ATGAAGATTG AGACCGTTTT TGCAAAAACA I

GGTTTTTTTA GAAAAATATT TCTCAAAGGT CTGATGATCT TGAGGAATCT

GTCAAAGGCT ATGAGCGCTA ACAGCACGAT GCCCACATAC ATGGTCTCAT

AAAATATCAC CGAAGAAAAA CGACACACAA AAGCTCTGAG CTGCCAGGGT I

GCCAG j 160435 AA804531 Dbest 599 AACTGGAAGG GCAGCCGTCT GCCGCCCACG AACACCTTCT CAAGCACTTT I P2Y P55085 j Proteinase- ' 397 6₂ 172 111 ₃8 GAGTGACCAC GGCTTGCAAG CTGGTGGCTG GCCCCCCGAG TCCCGGGCTC Purmo- activated receptor TGAGGCACGG CCGTCGACTT AAGCGTTGCA TCCTGTTACC TGGAGACCCT ceptor 8 I 2 precursor i CTGAGCTCTC ACCTGCTACT TCTGCCGCTG CTTCTGCACA GAGCCCGGGC I GAGGACCCCT CCAGGATGCA GGTCCCGAAC AGCACCGGCC CGGACAACGC . GACGCTGCAG ATGCTGCGGA ACCCGGCGAT CGCGGTGGCC CTGCCCGTGG | TGTACTCGCT GGTGGCGGCG GTCAGCATCC CGGGCAACCT CTTCTCTCTG TGGGTGCTGT GCCGGCGCAT GGGGCCCAGA TCCCCGTCGG TCATCTTCAT j GATCAACCTG AGCGTCACGG ACCTGATGCT GGCCAGCGTG TTGCCTTTCC ' AAATCTACTA CCATTGCAAC CGCCACCACT GGGTATTCGG GGTGCTGCTT j TGCAACGTGG TGACCGTGGC CTTTTACGCA AACATGTAGT TCAGCATUCT CAGCATGANC TGTATCAGCG TGGAGGCTTC CTTGGGGGTC TGTAACGCT J i 190711 j AA88₃367 Dbest 400 TTTAAAGGTC AGCCTCTTTG TATAGAAGCG GTGATGGGCG ATATCAAGTA . GPR85 Q9Y5N1 Histarmne H3 445 41 134 95 34

¹ (160444) ITCTGGTGACA CTGATGCAGA AGAGCATGAA AGCAGTGTGG AAACAGGACA | receptor

AAACCCCCAG AAAGGCAATC ACTTTGCAAG TCAGAGTCCC ATAAGTCCAG ι SEQ ID j GTAGAGCCAT TTTTGACAGA GTTGAACACA AATGGGAAAC AAATTGCAGA ' NO 36 j TCTGAGGATA TCTGAACAGC AAAGATCCAA CAGGAAGTAG TAAGGTGCTC i j TATGCAAGGT CTTATCTTTC ACTAGCAAAA TGGAGATCAG GAGGTTGCCC '

JACCACGCTGA CTCCTATTAT GAAACCCAAG GAAGTCAGTT TCAGAAAGGC , ,TGTTAGAGGC GAGAGATTTT GCAAAATGTT GTCAGCTGCA TGGCTATAGT '

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Homolog Name Len. From To Aligned Perce Type Length Sequence sentative lA.cc. No.

(Original) Sequence

LGNO. (SEQH) NO)

189876 1AP000808 ι I G¹ enomic 1113 GGGCATGGGC TCGCACAGGT GGGAAGCACC TGTGGGCGGC TCTCAAGCCC j P04201 MAS proto- i 325 306 I 297 35 Clone 'CCATCTCATT GGTGCCCACG GTGGGCGTCT CCCCACCTTC CAGCTCGGGC j I oncogene

LG1543 I TCCTCGCGAA GCGCCTGTTG GAGCACAGTC CCCAGGGACC TGGTGGGCAG , SEQ ID S I CCTGTGGCTC CTCCGGCTGC CCACCAGGAA GTAGATGACG GGGTTGGCGC NO 15 I TGCTGCTTAC GGACGAGGAG AGGCGTGACA AGCTGAAGCA CAGGACCTGC j SATCTCGGGCG GCAGGCTCAA CCAGTAGAGC ACAAACCAGT AGATGCTCAG |AGGCAGGGAA CAGATGAGGA ACACCAGGAC AGAGGCCAGG ACCACCACGA jACAGCCGTGT GGGCTGCCGC CGCCACTGCT GGGAGCTCCT CCGCACCCAG | jACAAAGAGGG TCAGGCTGGA CAGAGTCATC ACTGGGGTTA AGACCCCCAT I JGATGAGGGCG GCCTGGACCA TGTCCACCCT GAAGCACCGA TCTTCATTGA | ATTTCAAGAA CTTGCTGCAG AAGGAAGAGG TCAACCCGTT CATCAGGAGA , , CAGAGTGTCC ACAGCAGGCC ACACACCCAG GCTGACAGGT GCCTGGGCCG , GTGACACTTG AACCAGATAG GGAAGAGGAC AGAGAGACAG CGCTGGGTGC , , TGATGGCCGT CAGCAGGCTC AGGCCCACTG TGTAGGCAAA GTACATCAGT j CTCTTCATCA GCTCGTGGAC CTTGTCAGTG GTATTGACCA GGGGCTGGGT TTCCAGGCTG AGCGTGGAAG CCATGCTGAA GAGGAAGAGG AGGTCGGCTG ^!CCGCCAGGTT GAGGATATAG ATGCAGAAGG GGTTCCTGTG CATTCGAAAG fCCCAGCAGCC AGATCACCAT GCTGTTGCCT GCCATCCCGC ACAGGCAGGT ^GAACATGGCC AGGGAGCTCA GCACCAGGTA GGCCGTGTGC ACTGTGCTCC ,CTCTGGAATA GTTTAGGGCT GACTCCACGG TCCCACTGCT ATTCAAAGTC TGGTTCATCC CTACGAGAGG AAGATGTACC AATGTGAAAT TCTGTGTTGC ■ j TGGGACCACG GGGGACCCCT GGGTGCCCCT CGAATTTCCA GCTTCAGAGC

(TCTCCCCTCC AGG I I

189878 . AC016362 Genomic 504 CCTGGCAGTG CCGATGTTCC GATACTGGάA~CAGCAGCAGG~TGCCGGAAGG .P08173 Muscanmc 479 369 479 111 92

Clone TCTTTTTAAA GGTGGCGTTG CACAGAGCAT AGCAGGCAGG GTTGATGGTG acetylcholine LG1143 _f CTGTTGACGT AGCAGAGCCA GTAGCCAATG GACCACACCG GGTCAGGGAT j I receptor M4 'GCAGCTCTGG CAGAAGGTGT TCACCAGGAC CATGACGTTG GAGGCGTCC '

CGGTGAGGAT GAAAGCTAAC ANAATGGCAA AGATCGGTCG TGGCACTTTG CGCTCCCGGG CCCGCATCTG CCGCTTCTTG CGCACCTGGG TGCGAGCGAT i GCTAGCGAAC TTGCGGGCCA CGTTGGCCGC AGGCGCATGC CAGNCGGCGT j

GGGAGGGACA ATCTCAGGGC TGGCACACAC TCATGGGCTG GCTGGCTTCG J TCAAATTTTG GATCTTGGAC CATCTGGGAG GCTTGGTTGA AGGCCCCCGG >

CTCGGACTTG CGGGCATGAA TCCAGGCCTT ACTCTAHAGG ATCCCCCCCT CTCC I i

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative kcc No.

(Original) Sequence

LG NO. (SEQH) NO)

189884 AC011375 Genomic , 1137 AATCTCTGCC TTCTCAGTTT TCCCTTTGCC AGAGGAGGGA GAGCTGGGTT Q9NS07 GPR SALPR 469 81 444 326 28

I Clone TCTCTTTTTC TGGTATGGAT GCTGGGGATT CTGGAGATGG AACCTTGTCA

I LG608 GGAAGACCCT CTGAGTTGCC AGCTGGTGTT TCCTGAGACT CTGAGACAGT SEQ ID TGGAGGTTTT TTGGTTATCA TCCATTTCCA TACACCTTTC AAGCCTTCCC NO 40 TGAACTCTTC CGACATCACA AGAAAAATGA GAGGATTTGC TGAAGAGATG GAAAACATCA AGACTTGAGA CAGGGCTATG AAACCTTGTG GTGGGGCCGG Ammo GCCTGCAGCC TTCAGATGCC ATACCCACAG CCAAGCTACC CATTCGGGGA acid GCCACAAGAG AGCAGAGATG ATGGCAATGC TCAGCAGCAT CACTGTGACT sequence TGCTTTGAGC GTATCTGGTT TCTAAGATTT TGAGTCTTAG TTCCTCGTTT SEQ ID TTTACATTGG TCATAAGCTC TCCAGAAATA AAAGCTGGCA AAAAATAATG NO 18 GAAGGCCAAA TGCCAGGAGT GGGTAGAGCT TACCAAACAT CGACATAAAC TCTTCAGCCA CAGCTGGTAC ATCCACGAGG CACATTTCCA CACCTTCATG ATGCCTGATG GTGCTAAAGA ACCATTCCGG CAGGGGTAAC AGGCTAGCCA CAGTCCAGAT GGCCACCAGC ACTGACCAGA TGGTGTAGTT GTGGATACTC ACTTGCTTGG CTGGGTCACT TGCATACATG AAGCATACTT TGGCCACCAC AACGATTGTC AGGCTCTTGG CTGCCATGCA TGTGTGGATA AACCAGTCAG AGGACTTGCA GACAAACCAG CCTAGATCCC AAACACTTTT GGAGTACGCC GTAGCTCGGA TAGGTGCAGA AAACAGCAGG AGGGAGAGAT CAGCCAGGCT GAGATTCAGA ATCAGGGAGT GGATCATGGA TGGCTTTCCT TTCCAAGCAT TGTGAAGGAG GATGCCAATC ACACACAGGT TTCCCACGAA GCCCACCAGG CAGACAGCCA CCAAGAGAGC CGGGATGATG GTTCTCCAGT CCTGGGAATC AGAGGGCAGG TACCCTCCGG CAAAGTGGAG GTGAGCAAAG GACACATTCA TGCTGCTGGA GTTAGAGTCT GCAAAGGCAG CTGCCAG

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative lAcc. No.

(Original) Sequence

LG NO. (SEQH) NO)

189885 C011402 i Genomic , 963 ^! TCTGCTCTTG ACATCTTTTC CATGATCATC TACACAGTGA CTTTCTTCCT j SEQ ID P21462 ( FMET-LEU-PHE 350 26 62 37 56 Clone AGGCTTGGCT GGCAATGGCC TTGTCATTTG GGTAGTTGGA TTCCACATGT I NO 19 receptor 1 LG5574 , CCTGCACAGT CAACACGGTG TGGGTGGTAC CTCAACCTGC CCGTGGCTGA

CTTCATCATC ATCTTCCCAC TGCTTCTCCA GCTGGTTATG GTAGCTCTGT ,

AACCCTTTGG CCAGCTGCTC TGTAAACTCA ATAGCACCAT GTCTATTTTT I jAACTTTCTGG CCAGTGTCTT CCTTCTGACC CTCATCTCCA TGGACCACTG

ACTTGTGATC CTGTGGCCAA TCTAGTCCTG GAACAATTGC ACACCAGCAA

AGGCAACTCT GGGGCCCTTG AGGACCTGGC TTTTGGCAAT TTGTTTCTCT

GTTCCCTACT TGATCTTCAA GGAAACTCGT GGTGGAAAGT GTCACCCTCT

• TTGTACAACC AGTATGATCT GCAGAATGAA ACTCAAGGAA GTCACCAACT

' TTGGAAAGAG ATTATCATTC CATGGCACCA AACGCTGGTC ACAACAGCCC

IACTTTTTCTT TGGCTTCTTT CTCCCTCTGG CTATCATCAC TGGCTACTAC |

ATCCTTGTAG CCTTGAAGTT AAGAGAAAGG CAGCTGGTTA AGTTTAGCTG 'ACCCTTCCAG GTCCTTGCGA CTGTGGTAAC CACCTTCTTC CTCTGCTGGT I 'TGCCCTTGCA AGTGTCCCTG TGGCTGGACT TCACATCATT TCGGGAAGAC j lAGAGAGGGCC TGAACCAGGT GGCCTTACTC CTAAGACCCC TGGCCTTGTC j

I TATGGCCTTT ATCAACAGCT GTCTCAATCC AGTTCTCTAT GTCTTCATTG

GGCATGATTT CTGGGAGCAC TTGCTCCACT CCCTGCTAGC TGCCTTAGAA

■ CGGGCACTTA GTGAGGAGCC AGATAGTGCC TGAATCCCAG CTCCCAGGCA I

JGATGAGTCCT TTA _^

189886 AC016189 ! Genomic 330 ^""' GGGGTCTACC TCATGGCCTG^" TGTGAGCGTG GACCATTACC CAGCTGTGGT SEQ ED I P32249 I EBV-mduced G 361 118 227 110 40 LG1121 Clone CTGTGCCCAC TGGGGCCCGC GCCTCCGCAC GGCTGGCCGC GCCAGGCTGG NO 20 ' protem-coupled

TCTGCGTGGC CATCTGGACC TTGGTGCTGC TGCAGACGAT GCCCTTGCTC receptor 2

TTGATGCCCA TGACCAAGCC GCTGGTGGGC AAGCTGGCCT GCATGGAGTA j CAGCAGCATG GAGTCAGTCC TCGGGCTGCC CCTCATGGTC CTGGTGGCCT

' TTGCCATTGG CTTCTGTGGG CCAGTGGGGA TCATCCTGTC CTGCTATATG

AAGATCACCT GGAAGCTGGG CAGCACAGCT

LS

LS Cluster Cluster

ID: '(: _Ά Name and

Database Sequence RepreHomolog

Current Aec. No Sequence Homolog Name Len. From Type Length sentative Ace. No. To Aligned Percent

(Original) Sequence

LG O. (SEQH) NO)

189887 'AC011457 I Genomic 537 GATGCATCCA TGTAACCCAG TACTGGAGGT CAAGATGGAG ACAATCTCCA AAD14370 Calcium 266 39 , 210 , 179 1 31

1 LG626 I | Clone CAGCCACCAT GGCCTTTCCC TGAGTGCTAC TATAACTCAG GAGGAAAGCC _f | receptor|CaR 1 1 ' '

ACCCAGATAC TGCAGAACAC CAACATGCTG AAGGTGAGAC ATTTGGTTTC j protein ι 1 '

1 i ATTGAAGATG TCAGGCGGGT TACTTTCCAG GAAGACTACA GCAAAGGTCC 1 (fragment) | ,

1 CCAGAACCAA GAAGCCCAAA TAGCTCAAGA CACAGTAGAA AACAGTGATA 1 ! ! |

1 GAGCCCCCAT TACACTGGAT GATGATTATA CCAGGACTAT GGGTCTTTGT 1

1 1 CTAGGGAGGA AGGAGATGTT TCCAACTAGA TTCCAGAGAT GGTCACTTGG ' | ' 1 j I ATGAGGGGGC AAAGGTGACA ACAGAATGAG ATTTTAGGGT ACCCAACAGG , 1 i 1 ^!

I ACCTTGGGTC CTGGCTGGCC TTGTAGCTTT CCCATCCTGC TAGCTGGCCT

1 ! i | S i

TGTAGCTTGG AAGGCCATGA CCACAGTGAA AGTTTTGGCT GAAGCAGAAG j

_| ACACAGCCAC TGTGAACTGA ACTGAAAAGG TGGCCTG j ' 1 1 I ;

189888 AC010896 Genomic 1317 CCAGCCCCAG GTGAGGCCAA AGATGGGTGT AAGAATGAGC AGGGCTTTGA (094858 KIAA0758 j 986 ι 431 874 439 . 26 ; LG5533 i Clone TCACCCCCAG CAGAGCTTGG CGCTTCTCTG CTGGGGGTCC CTCTGACAGC J I protein 1 1 1 GAAGGTCTCA GCAACTTCAG CATGGCCATG GCTAGTACCA GCCCATTCAC [Fragment] ' '

1 > GCCTATGATG GCCAGCACTG GCCCACGAAG GTGTATAACG CCCCTCCCTT j 1 1

1s 1 CCCATCCAAC CAGCATTCCC CCTCCCTCAG GTATTGCCCT TGAGGTAGGT . j

AGAGCCCCAG GGTGACACCT GCCAACCCCA GTGGGCACAG GTAGCCCAGG < ¹

! i s ^! !

AGCACCATGA GGGGGAGAAC TCGGTGCTTT GCCAGCTGGT GAAAGACAAA 1

GAGCAGCTGG TGGGCCAACA CCAGGGCCTG CGCCACATCC AGAAAAAGGT ■ j 1 ' t GGCCAGGTAG AGGAAATGAC AGAGGAAGGC GGCACAAGGC AGAGCGGGCT j i

TCGGGGCCCT GGAGAGAGGA ATGGGGCGCC CAGGAAGCAA GTGTCTGCGG ι ' _J ! !

CCAGCAAGCA GAACACCATG TTGAGCAGGG CGGCGTGGCG GAAATAGGAG | < I ' ' ^s

ATCTTGTTCC GCACCACGAC TCTCCACACC AGCCAGTACA CACCCAGGCA ι

I j CACAAGCAGC GCCAGTATGG AAGCTCCCAA GCCCACTTGA GTCAGCAGCG j ' ¹ 1 1

I CCAGAGCGGG TTCTTCCGGA ACAGTCTGTG GGGACATGAG GACGGAGAAG , 1 ¹

' 1 i GCAGTGAGGT GCTGGCAGAG GCACTGAGCA GTGGGGCTGG CACTGGCCAC | '

CTGTGCCTGG CACCCTTCTT TGGACCAACC CCCCCTGCCC TGGAAGAGAC J I

TGTGATCCCA GAAGACACAG TGAGGGGAAC CATCTGTGTT CCCAAAGTCC i 1

| ATGATGACCT CTCCCTGGCT GAAGGCCCGG TCACCTGCCA TGATGGAAAT 1 | 1 ' ' f GACAAGGACC AGGCCAGGAG TGGCATAGAG GGAATCCCCC AGCCCTTGTC . | ,

CATAGTTTGA GGGCAGAAGG TGGTCCAGTT TTCGCAGCAC CAGGCTAGTA | I ¹ 1 ,

ATACTTATTT CAGTTCCATT ACGGACCAAT GGGGCCAGTG AGTGCCTGGG _t

| AATCTGAGCC TGCAGTGGGG GCCGAGTAGG GAAGGAGATG CTGTAGTCAG | 1 ! !

CAGGAAACGT GGGTCCAAAC AGCTGGCTCT GCAGCAGCAC ATTGGGTAAG j ! , 1

¹ 1 CTGAAGGCGA AGGGGTGGTC CTGTGGGCAC AGGCTGCATG CCAGGGTCTC ¹ 1

' 1 ! CACAGCCAGC AGGAGAGTCG AGCCTGCCCA GGGCTTCCGG GCTTGGGCCA J |

1 j GGGTCCACAG AGACCTGGTG TCCATATCTA GGACCTTGTC TGTGGCAATC ' I i

I I AGGAGATTCT GCAGCAC , J ¹ 1 .

*

LS

LS Cluster Cluster

ID: Name and

Database Sequence

Current Ace. No Sequence RepreHomolog h Homolog Name Len. From To Aligned Perce Type Lengt sentative Ace. No. <

(Original) Sequence

LG NO. (SEQH) NO)

189885 AC011352 Genomic 954 GACATCTTTT CCATGATCAT CTACACAGTG ACTTTCTTCC TAGGCTTGGC P21462 FMET-LEU-PHE 350 26 62 37 56

(189892) Clone TGGCAATGGC CTTGTCATTT GGGTAGTTGG ATTCCACATG TCCTGCACAG 1 receptor

LG606 TCAACACGGT GTGGGTGGTA CCTCAACCTG CCCGTGGCTG ACTTCATCAT SEQ ID CATCTTCCCA CTGCTTCTCC AGCTGGTTAT GGTAGCTCTG TAACCCTTTG j NO 19 GCCAGCTGCT CTGTAAACTC AATAGCACCA TGTCTATTTT TAACTTTCTG GCCAGTGTCT TCCTTCTGAC CCTCATCTCC ATGGACCACT GACTTGTGAT | CCTGTGGCCA ATCTAGTCCT GGAACAATTG CACACCAGCA AAGGCAACTC f TGGGGCCCTT GAGGACCTGG CTTTTGGCAA TTTGTTTCTC TGTTCCCTAC TTGATCTTCA AGGAAACTCG TGGTGGAAAG TGTCACCCTC TTTGTACAAC 1

CAGTATGATC TGCAGAATGA AACTCAAGGA AGTCACCAAC TTTGGAAAGA i GATTATCATT CCATGGCACC AAACGCTGGT CACAACAGCC CACTTTTTCT j ' TTGGCTTCTT TCTCCCTCTG GCTATCATCA CTGGCTACTA CATCCTTGTA ^!

GCCTTGAAGT TAAGAGAAAG GCAGCTGGTT AAGTTTAGCT GACCCTTCCA j

GGTCCTTGC ACTGTGGTAA CCACCTTCTT CCTCTGCTGG TTGCCCTTGC 'AAGTGTCCCT GTGGCTGGAC TTCACATCAT TTCGGGAAGA CAGAGAGGGC j ^! CTGAACCAGG TGGCCTTACT CCTAAGACCC CTGGCCTTGT CTATGGCCTT I [ TATCAACAGC TGTCTCAATC CAGTTCTCTA TGTCTTCATT GGGCATGATT j

TCTGGGAGCA CTTGCTCCAC TCCCTGCTAG CTGCCTTAGA ACGGGCACTT JAGTGAGGAGC CAGATAGTGC CTGAATCCCA GCTCCCAGGC AGATGAGTCC j

TTTA '

189893 AGO 11647 Genomic 720 lAATGGCCACT TGGGATGTG GTCCTCCTGG AGGTAAGGAA GAGGGCACAC O94910 KIAA0821 1474 i 212 ¹ 447 240 33 Clone jTTCTACCAAT GCTCTGCCTA GCCCTGGGGT CTCAGGGTCT GGAAGCCTCG protein

LG699 'TTAGGTTTGA CAGGTTTTGG TGGAGCATGA GCCCCAGAGG GCCTCCTGGT SEQ ID GGGTGGTCTT GGTAGCCTGG TCTTCATTGT CAGGAAGGTG AGGTCATAAT NO 43 TTATCAGGTA ACCATCACTG AAGACATACG GCTTGTGGTC TGAGGGGCAG TAGTTGATGC CCTGCAGCTT TTCCAGCATC TTGTCCAACA GGATGCTGAG GCGGCGCTCC TGCCCGGTGG TGGTGTCAAA AGCATAGAAG ATCTCCTCTT GGTGGGTGTT CAGTGAGTGT AAGGCATAGA GCACCCCACA GGCCATGAAG GCCCCTGACA GGGCTGGCTT GTACTGGCTG GTACGCCAGG TTTTCTCCAC TTCTAGGGTG CTAGCGTTGA GACGACTCAC AACCAGGTTG CCCTTGCTCT CCTCAGTGGC ATACAGAACC CACAGCCCCT TCTCATCACC AGCAAAATCT

JAAGTCCTTCC AGGGCACACC AGCACAGGAA AAGCGGTTGT TATAGGTGGC _jACCAGGCAGC AGACGCCACA GCACCAGTGT GTTGGAGGAA AGGTCCATTT TGGCCATGTC ACTTGTGCCA CAGTAGTTAA AGTACATAAA GTTCTTGTAC

JACAACGTTTC CACTGCCATC

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative lAcc. No.

(Original) Sequence

LG NO. (SEQH) NO)

190701 SAL121834 I Genomic . 660 GCTTGCATAA GCATAGACAG ATATGTGGCA GTAACTAATG TCCCCAGCCA C-C P49238 Probable G 355 123 304 182 38 (189894) l 1 Clone IATCAGGAGTG GGAAAACCAT GCTGGATCAT CTGTTTCTGT GTCTGGATGG chemoprotem-coupled

LG1446 ICTGCCATCTT GCTGAGCATA CCCCAGCTGG TTTTTTATAC AGTAAATGAC kine receptor GPR13 AATGCTAGGT GCATTCCCAT TTTCCCCCGC TACCTAGGAA CATCAATGAA receptor AGCATTGATT CAAATGCTAG AGATCTGCAT TGGATTTGTA GTACCCTTTC 11 TTATTATGGG GGTGTGCTAC TTTATCACGG CAAGGACACT CATGAAGATG jCCAAACATTA AAATATCTCG ACCCCTAAAA GTTCTGCTCA CAGTCGTTAT SEQ ID SAGTTTTCATT GTCACTCAAC TGCCTTATAA CATTGTCAAG TTCTGCCGAG NO 34 CCATAGACAT CATCTACTCC CTGATCACCA GCTGCAACAT GAGCAAACGC jATGGACATCG CGATCCAAGT CACAGAAAGC ATTGCACTCT TTCACAGCTG < ^S CCTCAACCCA ATCCTTTATG TTTTTATGGG AGCATCTTTC AAAAACTACG jTTATGAAAGT GGCCAAGAAA TATGGGTCCT GGAGAAGACA GAGACAAAGT 'GTGGAGGAGT TTCCTTTTGA TTCTGAGGGT CCTACAGAGC CAACCAGTAC ( iT_TTAGC_ATT

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. Type Length sentative lAcc. No. From To Aligned Perce

(Original) Sequence

LGNO. (SEQ H) NO)

189901 'AC013396 I Genomic 1218 GCCCTTACCC CCACAGCGCT GCAGCCCTGC AGCTGGCCCT CAGCCCTGGG j P49019 Probable G 387 18 301 280 43

Clone jAGGAGCCTTC CTTTTCCAGA GAGACCTCGC CCTGCACTTT CAGCTTCCCT j protem-coupled LG895 ', ATGGCCTCCG CCTTCCTAGA GGCCTCCCGG TAGCGCCACT GCCTGGAGGG j SEQ ID I receptor HM74 TTGGTAGGAG CTCTCGTCGC TCACTGGGCC CTGCCGGCCC CGCGTGAGGC ' NO 25 CCAGCAAGGC CCGGCTCTGG TGGAGGAAGT TGGGGCTAGA GAAGCAGTAG 'AGCACGGGGT CCAGGACACT GTTGAGGTAG GTGAAGGCCA GGGAGCCATG I GAAGAGCTGT GTGCAGAGGT CCAGGGATCG GCAGGCGGAC AGCCAGAAAG j JCCACCATGGA AGCCATGCCA AAGATGATGC TGGGCAAGAA GCAGATGGTG ' TAGACGGCCA CCACCATGGC CAGCACACGC ATGGCCCTCT GCGGGCCTGC j i CTGCCCGCCC AGACCACGGT TCCGGATGGT GAGCCCAATG CTCACAATAG > 'CAAAGAGGAT GAGCGCCAGT GGCAGGAAGA ACTCCAGCAG GTACAGTGCC TGGTGCCAGC GGAGCGAGGC CGAGGGCTTC GTGCCCACCC TGTAGCTGAG | iGCAGGAGGGG CCGGAGAAGG TGCTCAGGAG CAGGTGCCCG TTGAGGAGCA I GGATGCCCAC CCAGAGTCCC CCGGCCACCC GGGCAGCTGC CCCCACGGAA ' (GCACGGCTCA GCACGTGGTG GGGCTGCACC ACCTTCAGGT GCGGTTGAGI i TGCGATGGCT GTGAGGAAGA CAACGCTGGC CGTGCGGTTG GTGGACAGCA j TGAAGAGGTT GACTTTGCAG GCAGCAGCCC CAAAGCGCCA GGTCTCATGG ' AGGAGGTAGT AGTCCACGCG GAGGGGCAGG TTGCTGATCA GGAGGAAGTC AGCGGCCACC AGGCTGACCA GGAACACCGT GTTGGAGGTC CAGGGCCGCG j ' TGTGGATGCA GAAGATGAAG AGGGCCAAAC TGTTCCCCAC CAGGCCCAGG ' ACAAACTCCA GGGCCAGGAT TGGTGCCAGG AAGGCAGACA CCAGCGAGGA AGAGGTGGGG TGGCAGGGCC CTCCAGAGGA CCCCCCCACA GTGGTAAAGG | CAGAGGGAGC AGAGGAGGGT GAGGGAGAGA AGGAGGGAGG GAGAACAGAG GAGGAGAGAG AGGGAGATGG AGAGCTCAGG TTATGAAGTT CCATGGGCTG I CTTGGGCCAT GGGCCTGA

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

(Original) Sequence

LG O. (SEQlD NO)

190188 AC018896 Genomic ' 1005 ACAGGCCCCT TCAAGCCCTG TGAGTACCTC TTTGAAAGCT GGGGCATCCG 075473 Orphan G 907 546 872 325 50

! Clone CCTGGCCGTG TGGGCCATCG TGTTGCTCTC CGTGCTCTGC AATGGACTGG protem-coupled LG5982 TGCTGCTGAC CGTGTTCGCT GGCGGGCCTG TCCCCCTGCC CCCGGTCAAG SEQ ID receptor HG38

TTTGTGGTAG GTGCGATTGC AGGCGCCAAC ACCTTGACTG GCATTTCCTG NO 26

TGGCCTTCTA GCCTCAGTCG ATGCCCTGAC CTTTGGTCAG TTCTCTGAGT

ACGGAGCCCG CTGGGAGACG GGGCTAGGCT GCCGGGCCAC TGGCTTCCTG

¹ GCAGTACTTG GGTCGGAGGC ATCGGTGCTG CTGCTCACTC TGGCCGCAGT

, GCAGTGCAGC GTCTCCGTCT CCTGTGTCCG GGCCTATGGG AAGTCCCCCT

CCCTGGGCAG CGTTCGAGCA GGGGTCCTAG GCTGCCTGGC ACTGGCAGGG

CTGGCCGCCG CGCTGCCCCT GGCCTCAGTG GGAGAATACG GGGCCTCCCC

'ACTCTGCCTG CCCTACGCGC CACCTGAGGG TCAGCCAGCA GCCCTGGGCT jTCACCGTGGC CCTGGTGATG ATGAACTCCT TCTGTTTCCT GGTCGTGGCC

GGTGCCTACA TCAAACTGTA CTGTGACCTG CCGCGGGGCG ACTTTGAGGC I CGTGTGGGAC TGCGCCATGG TGAGGCACGT GGCCTGGCTC ATCTTCGCAG JACGGGCTCCT CTACTGTCCC GTGGCCTTCC TCAGCTTTGC CTCCATGCTG i GGCCTCTTCC CTGTCACGCC CGAGGCCGTC AAGTCTGTCC TGCTGGTGGT S GCTGCCCCTG CCTGCCTGCC TCAACCCACT GCTGTACCTG CTCTTCAACC j i CCCACTTCCG GGATGACCTT CGGCGGCTTC GGCCCCGCGC AGGGGACTCA

GGGCCCCTAG CCTATGCTGC GGCCGGGGAG CTGGAGAAGA GCTCCTGTGA j TTCTACCCAG GCCCTGGTAG CCTTCTCTGA TGTGGATCTC ATTCTGGAAG j

CTTCT I i

190408 I AC008969 Genomic 1 813 I ACATTTGGG GCAAAGATGC CACCCAGCAG CCCTGCCATG GAAGCCAGGAΓ P41594 Metabotropic i 1212 744 i 822 I 81 35 clone j TGGAAAAGAT CTCCACGGCC ACAGTGGACT TGCCCTGTGC GCTGTGGTAC I glutamate LG5392 iAGGGGCAGGA AGGTTGTCCA GAAGCTGCAG AACAGCAGCA CGCTGAAGGT receptor 5 j GAGGAACTTG GACTTGTTGA AGGCGTCTGG CAGACCCCTG GCCAGGAAGG I precursor CTACAAAGAA GGTGCCCCCA GCCAGGAGGC CCAGGTAGCC CAGCACACAG GAGAAAGCGA CAGCAGAGCC CTCTCAGCAC TGGATGACAA TGTGGCTGGG CTCTGAGGCC ATGTCCCCAT CTGGGAATGG TGGGGAAGTG CCCAGCCAGA I I TGCCACAGAG AACAACCTGC ACCAAGGAAG CAGCAAGGAC CACCGAGGTG l (GAAGCGCCAG GTCCCAGGCA CACCCAGACC CTGTCACCTG GTGACCCTGA j iAGGCCAGGAC GGAAGAGACA GCCATGGTGA ACACAACAGC AACTGTGGTC j TGGTAGAGGC GGCAGGTGGC AGCTGTGGGA CAACCAAGGC AAGGCAAGGG j ACAGAGGGCA CAGAGGGTCA GGGAGGTGAG CGGCGTGCAG CTGAGAGCTC I j TGTTGTTGGC CCCGACCACA GGTGTGTCTC GGTGCTTCAG AACAGTCTGA j GGACCAGCAC TGCCAGGCTG GCCAGCATGA GCGCCACCAA GGCGAGCATG i _jAGTCCCAGGG GGTCGTCAAA GGCCAGGAAG GTCTCTGTCC TGGGCAGGCA GCCATCTCTG GTGCGGCTTG AGTACTGCTC CTCTGGGCAC AGAAGACATC ' , TCTTCGTACC TGT

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Perce Type Length sentative Ace. No.

(Original) Sequence

LG NO. (SEQH) NO)

' 189889 [AC016856 Genomic 947 TGCTTGACCA CGGAAAAATT TCCAAAAGCT TCCCCACTCT GTTGTCTGAC , P41180 Extracellular 1078 577 i 828 246 36 i (190417) 1 clone CCAGTTTCTC TTTCTCCCTT CAGATAGTGT TGCATGTCAG AAGTGCTCTG ' calcium-sensing

LG5881 , ACAACCAGTG GCCCAATGTG CAGAAGGGCG AGTGCATCCC CAAAACCCTT , SEQ ID receptor ' ' ι

GACTTCTTGT TCTATCACAA GCCCCTTGAC ACAGCGTTGG CTGTCTGCAC < NO 21 precursor j 1 j

AGCCCTGCTC TTTCTCCTTG CCCTGGCCAT CTTAGGCATC TTCGTCTGAC |

| ! 1 i 1 i

ACCACCACAC TCCCATCATC CGAGCCAACA ACTGCCAGCT CAGCTATCTC ι

CTGCTGTCCT CCTTGGCCCT CAGCTTCCTC TGCCCCTTCA TGTTCATTGG | 1 i

CCACCCAGAC CCCATCACTT GTGCTGTGCA CCAGGCAGAT TTTGGGGTCA * ' i 1

CCTTCATGGT CTGCACATCC ACTGTGCTGG CCAAGACCAT CGTGGTGGTG ¹ 1 I 1

1 GCAGCCTTCC ATGCCACCCA GGCAGACACC CAGCTTAGGG GGTGGGCGGG

¹ GACAGTCCTC CTCAGCACCA TCCTCACTGT TCCCTGACCC AGGCAGCCTT I I ' ¹ 1

GTGTGCACTC TGGGTGACCA GATGGCCCCC TCAGCCTGTG AAACTCTACA i ¹ 1 ^• '

¹ GAACCCTGGC CCACAGTGAC TGTAAAGTGT GATAAAGGCT CCTTGGAACT | I 1 I 1 I !

1 TCTCTTGGAA CTGGGCTACT TGAGTTTGCT AGATCTGGTC AGCTTGCTGG j

TGACCTTCCC CACCTGCCGG CTGCCTGACA CCTTCAATGA AGCAAAGCAT j

ATCACTCTCA GCATGTTGTC TGCTCCTGTG TCTGGGTGTC CTTCATACCT 1 1 ' j ^l 1 •

1 GCCCACATGC ATGCCCACAG CAAAGACACC ATGGCCATGG AGGTCTTTGT | S i 1 1

CATCTTGGCA TCAGCAGGAG GCCTCATGTC CTCCCTCTTC TTTTCCAAAT | 1 '

¹ 1 GCTACATCAT CCTTCTCCAT CCTGAAAAGA ACACAAAAGA CCAAATG J 1 1 '

1 190418 IAC020641 Genomic 840 TTTTAAAATG GAGCCATATG CTTGGCGGAA TTGGCGGTTC ATGGCTGCAT ' GPR 84 P28566 5-hydroxy- j 365 j 109 ₍ 360 < 280 25

| clone AGAGCACAGG GTTGATGCAA CCATTGAGCC AGGTGAGGTT GGCAGCAAGC 1 EX33 fryptamme IE . j \

LG6080 i ATGTGGACCA CCCGGGGAGC CTGGACTCTG GCATCCAGAA TGTTGAGCAG i receptor CAAGAAGGGG ATGTAGCTCA GGGCAAAGCA GAGGAACACA GCAAAACACA « SEQ ID I !

TTCGAGTCAC CTTCCCAAAT TCCGATGAAG AATCCGGAGC TCTTCTGGCT 1 NO 29 i

1

| CCTTTAATTG GCTGGGCTTT GGCAGATGCT TCTGGAGGGC TTTTCTCTGC j

' CATCTGCTTA GCTCTCTTGC TGTTGATCTG GTCTCCCACT TCTGATGAGT 1 ' i l

CCCCTTCCAG GGTCTGGGTG GTGGCAGCAC TGACTGGCTC AGATGAAATC •

CCCTCACTGG GTCCTCCTGA TGCTAACCTG CTGTCCAGCT CCTGGAAACG j

ACCAGGCATG GCCTCATCAG TCCTGGCCAC ATGGTTGGAβ TGGATGCTTG ¹ i 1

CCTGTCGCAA CTTGTATTGG TCCAGTGCCT GTGCTGCTCG TTTGACCTGG ' 1 .

CGGTGGATGA GGCAATAGAA GATGCCAAC CTGCTGAGCC CAAGCACAAA . 1

! A

GTAGATGCCC ATGAGGATGG TGGTGTAAGG CCGGCCTCGG ATGCGGTCAA ^! |

| AGCTGCAGGT GCAGACTACA GGTACCAGGA TATAAATAGG CCAGAGGGGA 1 1 i ! ' I '

GCAAAGCTGG CCACGCCCAC AACCCAGGTG CTCACCAGTG CCAGCACTAT ' 1 »

CCCCTTGGCA CTGAAAACTT GGGGAAAAAG CTTAGGGTGG GCAATGAGGA I

I GGTAGCGTCC CAGTGCGATG AGGCAGAGGG TCAGGATGGA ! „ 1 ' .

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No olog Name Type Length Sequence Hom Len. From To Aligned Perce sentative lAcc. No.

(Original) Sequence

LG NO. (SEQH) NO)

190419 AC021089 Genomic 867 CTTTGCTTCA GAGCTAAACC _AGTT_TTTCΓ_Γ CTCTCCACAG CAAATATCTT 014694 CCR5 receptor I 333 22 304 270 27 clone GACAGTGATC ATCCTCTCCC AGCTGGTGGC AAGAAGACAG AAGTCCTCCT (fragment) LG6171 ACAACTATCT CTTGGCACTC GCTGCTGCCG ACATCTTGGT CCTCTTTTTC SEQ ID ATAGTGTTTG TGGACTTCCT GTTGGAAGAT TTCATCTTGA ACATGCAGAT NO 30

I GCCTCAGGTC CCCGACAAGA TCATAGAAGT GCTGGAATTC TCATCCATCC ACACCTCCAT ATGGATTACT GTACCGTTAA CCATTGACAG GTATATCGCT GTCTGCCACC CGCTCAAGTA CCACACGGTC TCATACCCAG CCCGCACCCG

JGAAAGTCATT GTAAGTGTTT ACATCACCTG CTTCCTGACC AGCATCCCCT ATTACTGGTG GCCCAACATC TGGACTGAAG ACTACATCAG CACCTCTGTG

I CATCACGTCC TCATCTGGAT CCACTGCTTC ACCGTCTACC TGGTGCCCTG CTCCATCTTC TTCATCTTGA ACTCAATCAT TGTGTACAAG CTCAGGAGGA

JAGAGCAATTT TCGTCTCCGT GGCTACTCCA CGGGGAAGAC CACCGCCATC

I TTGTTCACCA TTACCTCCAT CTTTGCCACA CTTTGGGCCC CCCGCATCAT

' CATGATTCTT TACCACCTCT ATGGGGCGCC CATCCAGAAC CGCTGGCTGG JTACACATCAT GTCCGACATT GCCAACATGC ΓAGCCCTTCΓ GAACACAGCC IATCAACTTCT TCCTCTACTG CTTCATCAGC AAGCGGTTCC GCACCATGGC I AGCCGCCACG CTCAAGGCTT TCTTCAAGTG CCAGAAGCAA CCTGTACAGT TCTACACCAA TCATAAC

LS

LS Cluster Cluster ID: Name and

Database Sequence RepreHomolog

Current Ace. No Sequence Homolog Name Len. From To Aligned Percent Type Length sentative lAcc. No.

(Original) Sequence

LG O. (SEQH) NO)

190427 'AL137118 ' Genomic ' 1026 TTCCTTTCTC AACCACACAC TAACAGGGAA AACACACTTT GTCTTTGCCT Cystem-yl Q9Y271 Cystemyl 337 17 311 291 39 I clone TCTGTGGATG GCCTTTTCTG AGTGCAGACT TTAGTCTGTC CTTAAAATTC | leuko- j j leukotπene

LG6807 TCCCCAGCAA AGTAATAGAG CAGAGGATTG AAGCAGGCAT TGGCTGCTGC tπene receptor 1

) CAAGGCCAGT GTGATAACCA AAGCTTTATG CAGTCTGTCT TTGCATAAAC J CysLT2 |

I CCACTTTCCA TGTCGTCAAG TGGACGGTCC TCAGTGTGTG ATAGGGCAGG ! receptor j

¹ AAACACAAGA AGAAGATGAT CAAGGTGATG ATGATGGTGG TCAGTGCCTT

I CCTGTGAGAA ACCCGCAGCC CCGATTCTGG GACCTCCACT TTTAACAGAA [ SEQ ID J

I CCCGAATGAT CAGCAGATAA CAGATGCTGA GTGTGAAAAA TGGCAGCAGG NO 31 I j CAGCCCACCA CCAAGGCAAT ATAGTTCATG GTCTGCAGCT TAGCAATTTT ' , j ATAGAGATTC AGCTCTAAGC ATGATGTGAC ACTGCCGTTC TGCTCAGAGC

I CACTGTCCAG GAGCATTATT GAGGAAGCCA TGATAAGGAT CCATATGATC

O ■ CCACAGAGGA TCCAGGCACT CCTGATGCTG GTGACATGCA GAAGCCGAAA o I GGGGTGAACC ATTGCCAGGA AACGCACAAC ACTCAGCACG GTCAGGAAAT

I AAATACTGCT GTACATGTTG ACATACAAGG AATAAGACAT AATCCTGCAG

J GCCAGGTCTC CAAATATCCA ATTGGAGCCT CTAAGATAAT AGTCAGCCCT I

GAAGGGAAGC GTGCTTATGA ACAGGAGATC TGAAATGGCC AGATTTAGCA j

¹ GAAAACGTT CACAGATGTG GACTTCTTAT AAGGCTGCAG GAAAACATAT '

ATGGACAACC CATTTCCCAA GACTCCCCAG AAAAATATTA TCAGATATAC

AATTGGGAAA AATTCTCTCT TGAAGTTTTC AATTGTGCAG TTCCTGCTGT _j

¹ TGTTATTGCT GAAGGTGCCA TTTGGTTCCA TTTCTGATAC GGAGATGGAT j

GGTTGCAAGG ACATAAATTT TCTCTC

1 190428 AP000440 [ Gem-muT 426 I AGAGTCATCT GCCTCATTAA TGATGCTTTT TGTCTTAGAA GTCTCTCTTT I ^I U45983 CCR8 chemokine | 1944 1941 1586 1 362 83

, i clone ι i GATTTTATAA ACTGACATGA TTTCTTGCTC TGTTATGAAT GCATGCTGCT ' receptor bp

I LG6894 I CTAGTCCTTT AATAAGCCCA TCACACATTT TTACCATGTC GTCTATAGGC i SEQ ID (CMKBR8) j I ACTTTTTCTG CAGTGTTAAC ATCATCTTCA TTATCATTAT CATCATGATC j NO 32

ATCTTGATTC AGAACCATTT TTGTTACTTC ACCATTGGAC AATGAATGAA I CAACTGGAGC CTCATTATTA CTGTTAAAAA CATCTTTGAT ATCCTCTTCT i I TCCAGCTTAC TGACAGTCTC TGGATGTATA TTTTTTGTAT ATGTAAGGAG

GTCAGACATT ATTTTTTTCT CACTTGACAT ACTGAATCCT TCAAAGTTAC I CATCTTGTTC ATCATCATTA CTGAAC

o co

LS

LS Cluster Cluster

ID: Name and

Database Sequence RepreHomolog

Current Ace No Sequence omolog Name Len. Fr Type Length sentative Ace. No, H om To Aligned Percent

(Original) Sequence

LGNO. (SEQH) NO)

190774 AC007922 Genomic 540 CTTTTTTATA CAAAATATTT TCAAGAAAGC CTTTTGAAAG CGCTTGTGAC Histamme Q9Y5N1 Histamme H3 445 259 " 431 ' 180 29

(190488) clone ACAATGGATA CAAAAGAGGA TTGACAAAGG AATTGAACCA CTGANAGCCA H4 j receptor 1

LG263 AAATGCAATT CTATACCAAA CTGATTTAGG ACCTGTTGCT GAGGAATAAA receptor ' 1 I

ATGAAAGGAC AATTGTGAAC AGAGNAATAT GGAGCCCAGC AAACAGCAAA 1 (SEQ ID

AACCCCHTAA GAGAATGGCC AGTGACTTGG CTAATCTCCT GGCTCTAAGC j NO 37)

AGTTCAACAT GTTCCCTTTG GTGAAGAGCT ACAGAATCTG ATTGGGAGAA 1 i :

GGAACCCATT TTGGAAGCAA TTGTATTGCT ATTCATCTTG GTTCTTGAGG ' 1 ! 1

AAAACATGAG ACTACTCTTT CTCCTCTGTC TCTCTGAATG AAAGGATGCA 1

GGAACTTCTG TCGATGCAGA AAGAGATCTC CTTGAAGATA GTCTACCTCT .

GAATGAGTGT CCACAGATGT TGGAAGAGAC AGCAGTCAGT CCAGGATGGC 1 i

1

TTTGGCACCT ACTGAGATGA TCACGCTTCC ACAGGCTCCA CTTC I

190557 AI806860 Dbest 574 TTTCCAAGAT TTATTTTTAT TATTAATTGA TATTAAATCT ATGACA .Q62851 Pheromone 273 99 269 ' 175 " ³⁶

TGTTGTGTAT TTTTCAGAAT ATTGGTTCTT CTTTTATCTG AAGTGAGGAG receptorVN7, rat , [ ,

CACCAGAGGA CTGATCACGG CATAGACATT GACTACAAGA CTCTGAACTT ¹ 1 i

GCTGGTTGAT TGGGCCATAT GCCCACAACA TTGCTGAGGA GAAGGAGATG ¹

ATGAAATCCA CCCAACAGAG GACCACAGAG AAACTTACCA GCAGCAGTAT ' 1 »

GGTCTGGATG GCCCGTTTCT CAGGGGAAGC TCCTGGGGAA GGACCATTGC ₍

1 TGTGAAGGTG GTGGGATCAC CTCTGAGGCC TGAATAAGAG AGTCACCATG i TATGCAATTA AGAACAGCAG TATTCCTACC AGGAAAGCAT CCCTAAGTGT 1 i 1 TGTCAGAATA AGAAACGTGG CCCTGAGGAT GAAGCTCATG GAGAAAACTG < j AGCAGTACTT ACCTATATTC AGTACATTTG TCTGGCTCAC ACTGGAAGCA [ ' GCTACAGTGT AGAAGATCCT GTTACTACTG AAAGACAAGT TGAGGAACCA 1 TAAGAAGAAG AAACATGGAT AATG 1 ¹ , :ι i^{i ;},

Claims

WHAT IS CLAIMED IS:

1. An isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least about 80%> identical to the sequence set forth in Table 1.

2. The isolated polypeptide of claim 1 , wherein the nucleotide sequence is set forth in Table 1.

3. An isolated nucleic acid molecule, or its complement, encoding the polypeptide of claim 1, wherein said nucleic acid molecule is operably linked to a heterologous promoter.

4. An expression vector comprising a nucleic acid molecule, or its complement, wherein the nucleic acid molecule encodes the polypeptide of claim 1.

5. A host cell comprising the expression vector of claim 4.

6. The host cell of claim 5, wherein the host cell is from a mammal.

7. A nucleic acid probe that specifically hybridizes with a nucleic acid molecule encoding the polypeptide of claim 1.

8. The nucleic acid probe of claim 7, wherein the nucleic acid is a

DNA.

9. The nucleic acid probe of claim 7, wherein the nucleic acid is an

RNA.

10. An expression vector comprising a nucleic acid molecule, or its complement, wherein the nucleic acid molecule selectively hybridizes to a sequence selected from Table 1, wherein the hybridization reaction is incubated overnight at 37°C in a solution comprising 40% formamide, 1 M NaCl and 1% SDS, and washed at 55°C in a solution comprising 0.5x SSC.

11. An antibody that selectively binds to the polypeptide of claim 1.

12. The antibody of claim 11, wherein said antibody is a monoclonal antibody.

13. The antibody of claim 11 , wherein said antibody is a polyclonal antibody.

14. An antisense polynucleotide comprising a sequence capable of specifically hybridizing to a nucleic acid molecule encoding the polypeptide of claim 1.

15. A method for identifying a compound that modulates the expression of a polypeptide in a cell, wherein said polypeptide has at least 80% amino acid sequence identity to a polypeptide encoded by a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1, the method comprising the steps of: (a) culturing said cell in the presence of a modulator to form a first cell culture; (b) contacting RNA or cDNA from the first cell culture with a probe which comprises a polynucleotide sequence encoding said polypeptide; and (c) determining whether the amount of the probe which hybridizes to the RNA or cDNA from the first cell culture is increased or decreased relative to the amount of the probe which hybridizes to RNA or cDNA from a second cell culture grown in the absence of said modulator.

16. A method for identifying a compound that modulates the expression of at least two polypeptides in a cell, wherein each of said polypeptides has at least 80% amino acid sequence identity to a polypeptide encoded by a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1, the method comprising the steps of: (a) culturing said cell in the presence of a modulator to form a first cell culture; (b) contacting RNA or cDNA from the first cell culture with at least two probes, each probe comprising a polynucleotide sequence encoding one of said polypeptides; and (c) determining whether the amount of the probes which hybridizes to the RNA or cDNA from the first cell culture is increased or decreased relative to the amount of the probes which hybridizes to RNA or cDNA from a second cell culture grown in the absence of said modulator.

17. A method for identifying a compound that modulates the activity of a polypeptide, wherein said polypeptide has at least 80% amino acid sequence identity to a polypeptide encoded by a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1, the method comprising the steps of: (a) culturing cells expressing said polypeptide in the presence of a modulator to form a first cell culture; and (b) measuring the activity of said polypeptide or second messenger activity in the first cell culture and determining whether the activity is increased or decreased relative to the activity of said polypeptide or second messenger activity from a second cell culture grown in the absence of said modulator.

18. A method for identifying a compound that modulates the activity of at least two polypeptides, wherein each of said polypeptides has at least 80% amino acid sequence identity to a polypeptide encoded by a nucleotide sequence selected from the group consisting of the sequences set forth in Table 1, the method comprising the steps of: (a) culturing cells expressing said polypeptides in the presence of a modulator to form a first cell culture; and (b) measuring the activity of said polypeptides or second messenger activity in the first cell culture and determining whether the activity is increased or decreased relative to the activity of said polypeptides or second messenger activity from a second cell culture grown in the absence of said modulator.