AU2007216752A1 - Endogenous and non-endogenous versions of human G protein-coupled receptor hRUP35 - Google Patents

Description

AUSTRALIA

Patents Act 1990 -ig^^ir FB RICE CO Patent and Trade Mark Attorneys COMPLETE SPECIFICATION STANDARD PATENT Applicant: ARENA PHARMACEUTICALS, INC.

Invention Title: Endogenous and non-endogenous versions of the human G protein-coupled receptor The following statement is a full description of this invention including the best method of performing it known to us:- Endogenous and non-endogenous versions of the human G protein-coupled receptor CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of Australian Patent Applications No.

2002219890 under S.79B of the Patents Act 1990, which corresponds to International Application No. PCT/US01/44386 filed on November 26, 2001 in the Australian national phase (published as WO 02/42461 on May 30, 2002), which claims priority from from the following provisional applications, all filed with the United States Patent and Trademark Office on the indicated dates: U.S. Provisional No. 60/253,404, filed Nov. 27, 2000; U.S. Provisional No. 60/255,366, filed Dec. 12, 2000; U.S. Provisional No. 60/270,286 filed Feb. 20, 2001; U.S. Provisional No. 60/282,356, filed Apr. 6, 2001; U.S. Provisional No. 60/270,266, filed Feb. 20, 2001; U.S. Provisional No.

60/282,032, filed Apr. 6, 2001; U.S. Provisional No. 60/282,358, filed Apr. 6, 2001; U.S. Provisional No. 60/282,365, filed Apr. 6, 2001; U.S. Provisional No. 60/290,917, filed May 14, 2001; and U.S. Provisional No. 60/309,208, filed Jul. 31, 2001. The disclosures of each of the foregoing are hereby incorporated in their entirety by reference.

FIELD OF THE INVENTION The present invention relates to transmembrane receptors, in some embodiments to G protein-coupled receptors (GPCRs) and, in some preferred embodiments, to endogenous GPCRs that are altered to establish or enhance constitutive activity of the receptor. In some embodiments, the constitutively activated GPCRs will be used for the direct identification of candidate compounds as receptor agonists or inverse agonists having applicability as therapeutic agents.

BACKGROUND OF THE INVENTION Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR) class.

It is estimated that there are some 30,000-40,000 genes within the human genome, and

NO

of these, approximately 2% are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified, are referred to as "known" receptors, while receptors for which the endogenous ligand has not been identified are referred to as "orphan" receptors.

GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the drugs listed in Table 1 infra (Med Ad News 1999 Data) interact with GPCRs (diseases and/or disorders treated are indicated in parentheses).

TABLE 1 Claritin® (allergies) Paxil® (depression) Cozaar® (hypertension) Propulsid® (reflux disease) Pepcid® (reflux) Effexor® (depression) Allegra® (allergies) Diprivan® (anesthesia) Hytrin® (hypertension) Plavix (MUstroke) Xalatan® (glaucoma) Harnal® (prostatic hyperplasia) Prozac® (depression) Zoloft® (depression) Imitrex® (migraine) Risperdal® (schizophrenia) Gaster® (ulcers) Depakote® (epilepsy) Lupron® (prostate cancer) BuSpar® (anxiety) Wellbutrin® (depression) Toprol-XL® (hypertension) Singulair® (asthma) Vasotec® (hypertension) Zyprexa® (psychotic disorder) Zantac® (reflux) Serevent® (asthma) Atrovent® (bronchospasm) Cardura®(prostatic hypertrophy) Zoladex® (prostate cancer) Ventolin® (bronchospasm) Zyrtec® (rhinitis) Tenormin® (angina) Diovan® (hypertension) GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, transmembrane-1 (TM-1), transmembrane-2 etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and and transmembrane-6 and transmembrane-7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular" side, of the cell membrane (these are referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The "carboxy" terminus of the receptor lies in the intracellular space within the cell, and the "amino" terminus of the receptor lies in the extracellular space outside of the cell.

Generally, when an endogenous ligand binds with the receptor (often referred to as "activation" of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular "Gprotein". It has been reported that GPCRs are "promiscuous" with respect to G proteins, that a GPCR can interact with more than one G protein (Kenakin, Life Sciences 43, p1095 et seq., 1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Although not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.

Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an "active" state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.

A receptor may be stabilized in an active state by a ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent means is termed "constitutive receptor activation." SUMMARY OF THE INVENTION Disclosed herein are endogenous and non-endogenous versions of human GPCRs and uses thereof.

In one example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP28" which comprises the amino acid sequence of SEQ. ID.

NO.: 2, and to non-endogenous, constitutively activated versions of the receptor hRUP28 comprising the substitution H232K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP28 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 1; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 2; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 1; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP28 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 1; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 2; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 1; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP28 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the pancreas or for treatment of a disease or disorder related to the heart, liver, kidney, lung or pancreas. Exemplary diseases/disorders related to the heart include thrombosis, myocardial infarction, atherosclerosis and a cardiomyopathy. Exemplary diseases/disorders related to the liver include cirrhosis, hepatocellular carcinoma optionally associated with HBV or HCV infection, hemochromatosis, hepatitis, hepatic fibrosis, diseases of lipid, free fatty acid or glucose metabolism in the liver diabetes and liver diseases associated with abnormal cytokine production. Exemplary diseases/disorders related to the kidney include renal failure, renal tubular acidosis, renal glyosuria, nephrogenic diabetes insipidus, cystinuria and polycystic kidney disease. Exemplary diseases/disorders related to the lung include chronic obstructive pulmonary disease (COPD), cystic fibrosis, tuberculosis and lung cancer. Exemplary diseases/disorders related to the pancreas 9 include diabetes, pancreatitis, pancreatic cancer, eating disorders, obesity, etc.

d Preferably, such a method comprises performing the method herein of isolating a Cpharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP28 receptor or a variant thereof or fusion protein comprising hRUP28 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

NO

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of CK 10 hRUP28 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP28 receptor function will modulate lipid metabolism and/or free fatty acid levels and/or blood insulin and/or blood glucose and/or blood glucagon in a subject by acting on the receptor in the liver and/or gastrointestinal tract including pancreas and kidney.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP28 receptor or a variant thereof or fusion protein comprising hRUP28 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 2; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 1; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof.

Preferred variants of the hRUP28 receptor comprise a variant of SEQ ID NO: 2 wherein histidine at position 232 is mutated to another amino acid, alanine, arginine or lysine and more preferably, wherein histidine at position 232 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the heart, liver, kidney, lung and pancreas.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP28 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP28 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP28 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 52 and SEQ ID NO: 53 and mixtures thereof.

Preferred vectors comprising hRUP28-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP28 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP28 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP28 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP28 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP28 receptor in the heart, liver, kidney, lung or pancreas. Still more preferably, the agent modulates lipid metabolism and/or free fatty acid levels and/or blood insulin and/or blood glucose and/or blood glucagon in a subject. Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP28 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the heart, liver, kidney, lung or pancreas or for treatment of a disease or disorder related to the heart, liver, kidney, lung or pancreas, or in the preparation of a medicament for modulating a functionality of the pancreas or for treatment of a disease or disorder related to the heart, liver, kidney, lung or pancreas.

In another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP29" which comprises the amino acid sequence of SEQ. ID.

NO.: 4, and to non-endogenous, constitutively activated versions of the receptor hRUP29 comprising the substitution Q224K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP29 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 4; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 4; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 3; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 4; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: 4; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 3; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP29 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 4; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 4; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 3; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 4; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 4; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 3; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP29 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the leukocyte or ovary or for treatment of a disease or disorder related to the leukocyte or ovary. Exemplary diseases/disorders related to the leukocytes include eosinophilia, leukocytosis, leukopenia and leukemia. Exemplary diseases/disorders related to the ovary include ovarian cancer and polycystic ovarian syndrome. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP29 receptor or a variant thereof or fusion protein comprising hRUP29 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP29 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP29 receptor function will modulate cell growth and/or division in the leukocytes or ovaries of a subject, by acting on the receptor in the leukocytes and/or ovary.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP29 receptor or a variant thereof or fusion protein comprising hRUP29 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

I0 S T This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 4; t the amino acid sequence of an endogenous or non-endogenous IN constitutively active variant of C the amino acid sequence of having at least about 80% identity to SEQ O ID NO: 4; N 10 the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 3; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof.

Preferred variants of the hRUP29 receptor comprise a variant of SEQ ID NO: 4 wherein glutamine at position 224 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein glutamine at position 224 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the leukocytes and, if present, the ovary.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP29 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP29 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 3. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP29 receptor or a variant thereof a primer that comprises a sequence selected CsZ from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 54 and SEQ ID NO: 55 and mixtures thereof.

SPreferred vectors comprising hRUP29-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression ltr vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

O To produce recombinant host cells expressing the hRUP29 receptor or variant or fusion C( 10 protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP29 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP29 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP29 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP29 receptor in the leukocytes or ovaries. Still more preferably, the agent modulates cell growth and/or division in a subject. Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP29 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the leukocytes or ovaries or for treatment of a disease or disorder related to the leukocytes or ovaries, or in the preparation of a medicament for modulating a functionality of the leukocytes or ovaries or for treatment of a disease or disorder related to the leukocytes or ovaries.

In another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP30" which comprises the amino acid sequence of SEQ. ID.

NO.: 6, and to non-endogenous, constitutively activated versions of the receptor comprising the substitution T268K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP30 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 5; and c the amino acid sequence of an endogenous or non-endogenous G protein- 2 coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: SEQ ID NO: 56 and SEQ ID NO: 57 and mixtures thereof N Alternatively, the screening method is a method of identifying one or more candidate CN compounds which can inhibit or stimulate a G protein-coupled receptor comprising the 0 steps of: NI 10 contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 6; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: 6; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 5; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 56 and SEQ ID NO: 57 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP30 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 5; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: SEQ ID NO: 56 and SEQ ID NO: 57 and mixtures thereof Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 6; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 6; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 5; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by ri polymerase chain reaction (PCR) using one or more primers comprising a Ssequence selected from the group set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 56 and SEQ ID NO: 57 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate t the G protein-coupled receptor and isolating a compound which inhibits or stimulates IDthe receptor as a pharmaceutical agent.

O For example, the formulation of a pharmaceutical composition for modulating the C1 10 hRUP30 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the pancreas or for treatment of a disease or disorder related to the pancreas, such as diabetes. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP30 receptor or a variant thereof or fusion protein comprising hRUP30 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP30 receptor or a variant thereof. Preferred pharmaceutical agents for modulating receptor function will modulate blood insulin and/or blood glucose and/or blood glucagon in a subject.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP30 receptor or a variant thereof or fusion protein comprising or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 6; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 5; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: SEQ ID NO: 56 and SEQ ID NO: 57 and mixtures thereof.

Preferred variants of the hRUP30 receptor comprise a variant of SEQ ID NO: 6 wherein threonine at position 268 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein threonine at position 268 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the pancreas.

This embodiment clearly extends to any and all nucleic acid encoding the receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP30 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 5. The invention also extends to any and all primers suitable for producing nucleic acid encoding the receptor or a variant thereof a primer that comprises a sequence selected 2 from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 56 and SEQ ID NO: 57.

Preferred vectors comprising hRUP30-encoding nucleic acid or nucleic acid encoding a t variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or C fusion protein is operably linked to a promoter.

O

Ni 10 To produce recombinant host cells expressing the hRUP30 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP30 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP30 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP30 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP30 receptor in the pancreas. Still more preferably, the agent modulates blood insulin and/or blood glucose and/or blood glucagon in a subject.

Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP30 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the pancreas or for treatment of a disease or disorder related to the pancreas diabetes, or in the preparation of a medicament for modulating a functionality of the pancreas or for treatment of a disease or disorder related to the pancreas diabetes.

In a further example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP31" which comprises the amino acid sequence of SEQ. ID.

NO.: 8, and to non-endogenous, constitutively activated versions of the receptor hRUP31 comprising the substitution F263K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP31 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 8; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 8; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 7; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 8; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 8; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 7; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

(For example, the isolation of pharmaceutical agents employing hRUP31 receptor and d variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 8; S(b) the amino acid sequence of an endogenous or non-endogenous INDconstitutively active variant of(a); the amino acid sequence of(b) having at least about 80% identity to SEQ ID NO: 8; S 10 the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 7; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ 1D NO: 8; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 8; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 7; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP31 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the colon, lung, pancreas, thymus, cerebral cortex, hippocampus or fat cells or for treatment of a disease or disorder related to the colon, lung, pancreas, thymus, brain cerebral cortex, hippocampus) or fat cells (adipocytes). Exemplary diseases/disorders related to the colon include colon cancer, ulcerative colitis, diverticulitis, Crohn's disease, irritable bowel syndrome, obesity, etc.

Exemplary diseases/disorders related to the lung include chronic obstructive pulmonary disease (COPD), cystic fibrosis, tuberculosis and lung cancer. Exemplary diseases/disorders related to the pancreas include diabetes, pancreatitis, pancreatic cancer, eating disorders, obesity, etc.Exemplary diseases/disorders related to the thymus include thymoma, endocrine disorders, etc. Exemplary diseases/disorders related to the brain cerebral cortex, hippocampus) include neurodegenerative diseases, movement disorders, ataxia, learning and memory disorders, eating disorders, obesity, cancer, growth disorders, psychosis, migraine, schizophrenia, depression, etc.

Exemplary diseases/disorders related to the adipocytes include obesity, etc. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP31 receptor or a variant thereof or fusion protein comprising hRUP31 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP31 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP31 receptor function will act on the receptor in the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes).

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP31 receptor or a variant thereof or fusion protein comprising hRUP31 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 8; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 8; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 7; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof.

Preferred variants of the hRUP31 receptor comprise a variant of SEQ ID NO: 8 wherein phenylalanine at position 263 is mutated to another amino acid, alanine, Ihistidine, arginine or lysine and more preferably, wherein phenylalanine at position 263 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in C1 the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, O hippocampus) and/or fat cells (adipocytes).

(N This embodiment clearly extends to any and all nucleic acid encoding the hRUP31 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP31 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 7. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP31 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58 and SEQ ID NO: 59 and mixtures thereof.

Preferred vectors comprising hRUP3 -encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP31 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

C Exemplary agents that interact with an isolated or recombinant hRUP31 receptor j polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by tt a method described herein for isolating the hRUP31 receptor or variant. Preferred \D agents will interact with the endogenous receptor or non-endogenous receptor and, CN more preferably, inhibit or stimulate an endogenous or non-endogenous form of the O receptor. Particularly preferred agents are agonists or inverse agonists of the C( 10 endogenous hRUP31 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP31 receptor in the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes).

Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP31 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes), or for treatment of a disease or disorder related to the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes), or in the preparation of a medicament for modulating a functionality of the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes) or for treatment of a disease or disorder related to the colon and/or lung and/or pancreas and/or thymus and/or brain cerebral cortex, hippocampus) and/or fat cells (adipocytes).

In yet another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP32" which comprises the amino acid sequence of SEQ. ID.

NO.: 10, and to non-endogenous, constitutively activated versions of the receptor hRUP32 comprising the substitution A257K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated Scell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify t pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, IND methods of formulating pharmaceutical formulations for modifying functionality of the IC receptor or treatment of conditions associated with the receptor, and to agents that O interact with the receptor and uses of such agents to modulate receptor function.

IC For example, the screening method employing hRUP32 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 9; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: 61 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 9; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: 61 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP32 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 9; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: 61 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 9; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: 61 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP32 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the thymus or for treatment of a disease or disorder related

IN

to the thymus. Exemplary diseases/disorders related to the thymus include thymoma, endocrine disorders, etc. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP32 receptor or a variant thereof or fusion protein comprising hRUP32 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP32 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP32 receptor function will act on the receptor in the thymus.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP32 receptor or a variant thereof or fusion protein comprising hRUP32 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ IDNO: rJ 3 S(d) the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 9; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the s) group set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: CN 61 and mixtures thereof.

C 10 Preferred variants of the hRUP32 receptor comprise a variant of SEQ ID NO: wherein alanine at position 257 is mutated to another amino acid, histidine, arginine or lysine and more preferably, wherein alanine at position 257 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the thymus.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP32 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP32 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 9. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP32 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 60 and SEQ ID NO: 61 and mixtures thereof.

Preferred vectors comprising hRUP32-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP32 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP32 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP32 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP32 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP32 receptor in the thymus. Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP32 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the thymus, or for treatment of a disease or disorder related to the thymus, or in the preparation of a medicament for modulating a functionality of the thymus, or for treatment of a disease or disorder related to the thymus.

In yet another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP33" which comprises the amino acid sequence of SEQ. ID.

NO.: 12, and to non-endogenous, constitutively activated versions of the receptor hRUP33 comprising the substitution V250K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP33 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 11; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 12; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: 12; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 11; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP33 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 11; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 12; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 12; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 11; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP33 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the receptor in the tissue(s) in which it is expressed endogenously or for treatment of a disease or disorder related to said tissue(s).

Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP33 receptor or a variant thereof or fusion protein comprising hRUP33 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP33 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP33 receptor function will act on the receptor in the tissue(s) in which it is expressed endogenously.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP33 receptor or a variant thereof or fusion protein comprising hRUP33 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 12; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 11; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof.

Preferred variants of the hRUP33 receptor comprise a variant of SEQ ID NO: 12 wherein valine at position 250 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein valine at position 250 is mutated to lysine.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP33 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP33 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 11. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP33 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36 and mixtures thereof.

Preferred vectors comprising hRUP33-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP33 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP33 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP33 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP33 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP33 receptor in the tissue(s) in which it is expressed endogenously.

Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP33 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the tissue(s) in which it is expressed endogenously, or for treatment of a disease or disorder related to the tissue(s), or in the preparation of a medicament for modulating a functionality of the tissue(s) or for treatment of a disease or disorder related to the tissue(s).

In yet another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP34" which comprises the amino acid sequence of SEQ. ID.

NO.: 14, and to non-endogenous, constitutively activated versions of the receptor hRUP34 comprising the substitution A256K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP34 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 13; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 14; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: 14; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 13; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP34 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 13; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 14; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 14; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 13; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP34 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of peripheral blood leukocytes (PBLs), prostate or kidney or for treatment of a disease or disorder related to PBLs, prostate or kidney. Exemplary diseases/disorders related to PBLs include cancer, eosinphilia, leukocytosis, inflammation, etc. Exemplary diseases/disorders related to the kidney include renal failure, renal tubular acidosis, renal glyosuria, nephrogenic diabetes insipidus, cystinuria and polycystic kidney disease. Exemplary diseases/disorders related to the prostate include prostate cancer, prostatitis, sexual dyfunction, prostatic hypertrophy, etc. Preferably, such a method comprises performing the method herein of isolating a 2 pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP34 receptor or a variant thereof or fusion protein comprising hRUP34 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

IND It will be apparent from the foregoing that preferred pharmaceutical agents isolated or C1 identified by performing the method of the invention are agonists or inverse agonists of ShRUP34 receptor or a variant thereof. Preferred pharmaceutical agents for modulating C 10 hRUP34 receptor function will act on the receptor in the PBLs and/or prostate and/or kidney.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP34 receptor or a variant thereof or fusion protein comprising hRUP34 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 14; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 13; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof.

Preferred variants of the hRUP34 receptor comprise a variant of SEQ ID NO: 14 wherein alanine at position 256 is mutated to another amino acid, histidine, arginine or lysine and more preferably, wherein alanine at position 256 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in PBLs and/or prostate and/or kidney.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP34 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP34 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 13. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP34 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 62 and SEQ ID NO: 63 and mixtures thereof.

Preferred vectors comprising hRUP34-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP34 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP34 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP34 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP34 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP34 receptor in PBLs and/or prostate and/or kidney. Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP34 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of PBLs and/or prostate and/or kidney, or for treatment of a disease or disorder related to PBLs and/or prostate and/or kidney, or in the preparation of a medicament for modulating a functionality of PBLs and/or prostate and/or kidney or for treatment of a disease or disorder related to PBLs and/or prostate and/or kidney.

In one example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP35" which comprises the amino acid sequence of SEQ. ID.

NO.: 16, and to non-endogenous, constitutively activated versions of the receptor comprising the substitution T227K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid 49 C encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify t pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, IDmethods of formulating pharmaceutical formulations for modifying functionality of the C receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP35 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 16; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP35 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 16; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the thalamus. Exemplary diseases/disorders related to the thalamus include sensorimotor processing and/or arousal. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP35 receptor or a variant thereof or fusion protein comprising or variant and formulating the isolated pharmaceutical agent with a pharmaceuticallyacceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP35 receptor function will act on the receptor in the thalamus.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP35 receptor or a variant thereof or fusion protein comprising hRUP35 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof.

Preferred variants of the hRUP35 receptor comprise a variant of SEQ ID NO: 16 wherein threonine at position 227 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein threonine at position 227 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the thalamus.

This embodiment clearly extends to any and all nucleic acid encoding the receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP35 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 15. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP35 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 64 and SEQ ID NO: 65 and mixtures thereof.

Preferred vectors comprising hRUP35-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP35 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP35 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP35 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP35 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP35 receptor in the thalamus. Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP35 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the thalamus, or for treatment of a disease or disorder related to the thalamus, or in the preparation of a medicament for modulating a functionality of the thalamus or for treatment of a disease or disorder related to the thalamus.

In yet another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP36" which comprises the amino acid sequence of SEQ. ID.

NO.: 18, and to non-endogenous, constitutively activated versions of the receptor hRUP36 comprising the substitution Y215K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP36 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 17; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 18; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 18; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 17; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP36 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 17; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 18; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(i); (iii) the amino acid sequence of(ii) having at least about 80% identity to SEQ ID NO: 18; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 17; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP36 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the receptor in the tissue(s) in which it is expressed endogenously or for treatment of a disease or disorder related to said tissue(s).

Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP36 receptor or a variant thereof or fusion protein comprising hRUP36 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP36 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP36 receptor function will act on the receptor in the tissue(s) in which it is expressed endogenously.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP36 receptor or a variant thereof or fusion protein comprising hRUP36 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of(b) having at least about 80% identity to SEQ ID NO: 18; the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 17; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof.

Preferred variants of the hRUP36 receptor comprise a variant of SEQ ID NO: 18 wherein tyrosine at position 215 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein tyrosine at position 215 is mutated to lysine.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP36 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP36 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 17. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP36 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 43 and SEQ ID NO: 44 and mixtures thereof.

Preferred vectors comprising hRUP36-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP36 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP36 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP36 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP36 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP36 receptor in the tissue(s) in which it is expressed endogenously.

Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP36 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the receptor in the tissue(s) in which it is expressed endogenously or for treatment of a disease or disorder related to said tissue(s), or in the preparation of a medicament for modulating a functionality of said tissue(s) or for treatment of a disease or disorder related to the tissue(s).

In yet another example, the present invention relates to a G protein-coupled receptor designated herein as "hRUP37" which comprises the amino acid sequence of SEQ. ID.

NO.: 20, and to non-endogenous, constitutively activated versions of the receptor hRUP37 comprising the substitution Y215K) and other variants of the receptor having at least about 80% identity thereto or encoded by nucleic acid that hybridizes under stringent conditions to the complement of nucleic acid encoding the receptor or encoded by nucleic acid that is amplifiable using primers that amplify nucleic acid encoding the receptor), fusion proteins comprising the receptor, host cells and isolated cell membranes comprising the receptor or fusion protein, nucleic acid encoding the receptor and vectors expression vectors comprising the nucleic acid, methods of screening candidate compounds using the receptor and non-endogenous constitutively activated versions and other variants thereof or fusion proteins to identify pharmaceutical agents or isolate pharmaceutical agents from candidate compounds, methods of formulating pharmaceutical formulations for modifying functionality of the receptor or treatment of conditions associated with the receptor, and to agents that interact with the receptor and uses of such agents to modulate receptor function.

For example, the screening method employing hRUP37 receptor and variants comprises a method of screening candidate compounds to identify a pharmaceutical agent, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 19; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof.

Alternatively, the screening method is a method of identifying one or more candidate compounds which can inhibit or stimulate a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 19; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof; and measuring the ability of the compound to inhibit or stimulate the G protein-coupled receptor.

For example, the isolation of pharmaceutical agents employing hRUP37 receptor and variants is a method of isolating a pharmaceutical agent from candidate compounds, said method comprising use of a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 19; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof.

Alternatively, the isolation method is method of isolating a pharmaceutical agent from candidate compounds, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 19; and the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof; and measuring the ability of the candidate compounds to inhibit or stimulate the G protein-coupled receptor and isolating a compound which inhibits or stimulates the receptor as a pharmaceutical agent.

For example, the formulation of a pharmaceutical composition for modulating the hRUP37 receptor may be a method for formulating a pharmaceutical composition to modulate a functionality of the testis and/or brain cerebral cortex and/or hippocampus. Exemplary diseases/disorders related to the testis include testicular cancer, impotence, hypogonadism, endocrine diseases and disorders, etc. Exemplary diseases/disorders related to the brain cerebral cortex, hippocampus) include neurodegenerative diseases, movement disorders, ataxia, learning and memory disorders, eating disorders, obesity, cancer, growth disorders, psychosis, migraine, schizophrenia, depression, etc. Preferably, such a method comprises performing the method herein of isolating a pharmaceutical agent from candidate compounds to thereby isolate a pharmaceutical agent that inhibits or stimulates hRUP37 receptor or a variant thereof or fusion protein comprising hRUP37 or variant and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.

It will be apparent from the foregoing that preferred pharmaceutical agents isolated or identified by performing the method of the invention are agonists or inverse agonists of hRUP37 receptor or a variant thereof. Preferred pharmaceutical agents for modulating hRUP37 receptor function will act on the receptor in the testis and/or hippocampus and/or cerebral cortex.

Preferred means for performing the screening and isolation methods herein comprise contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising hRUP37 receptor or a variant thereof or fusion protein comprising hRUP37 or variant and a G protein. The receptor or a constitutively active version thereof or other variant of the receptor, or a fusion protein comprising the receptor or variant and a G protein, may be employed. Preferred eukaryotic host cells are mammalian host cells 293 cells, a 293T cells, or COS-7 cells. Alternatively, the eukaryotic host cell is a yeast host cell. Alternatively, the eukaryotic host cell is a melanophore host cell.

This embodiment clearly provides an isolated or recombinant G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 19; and the amino acid sequence of an endogenous or non-endogenous G proteincoupled receptor encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) using one or more primers comprising a sequence selected from the group set forth in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof.

Preferred variants of the hRUP37 receptor comprise a variant of SEQ ID NO: wherein tyrosine at position 215 is mutated to another amino acid, alanine, histidine, arginine or lysine and more preferably, wherein tyrosine at position 215 is mutated to lysine. Preferably, the endogenous form of the receptor is expressed in the testis and/or cerebral cortex and/or hippocampus.

This embodiment clearly extends to any and all nucleic acid encoding the hRUP37 receptor or variant thereof and fusion proteins comprising the receptor or variants. An isolated polynucleotide encoding the hRUP37 receptor is exemplified by nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 19. The invention also extends to any and all primers suitable for producing nucleic acid encoding the hRUP37 receptor or a variant thereof a primer that comprises a sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 66 and SEQ ID NO: 67 and mixtures thereof.

Preferred vectors comprising hRUP37-encoding nucleic acid or nucleic acid encoding a variant of the receptor or fusion protein comprising the receptor will be expression vectors wherein the polynucleotide encoding the receptor or variant thereof or fusion protein is operably linked to a promoter.

To produce recombinant host cells expressing the hRUP37 receptor or variant or fusion protein, it is preferred to perform a method comprising the steps of: transfecting a vector expressing the receptor or variant or fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or variant thereof or fusion protein from the vector.

Exemplary agents that interact with an isolated or recombinant hRUP37 receptor polypeptide or variant thereof or fusion protein are isolated using the isolated or recombinant receptor or variant thereof or a fusion protein comprising the receptor or variant, or using a recombinant host cell expressing said receptor or variant or fusion protein or using membranes from such cells. Alternatively, such an agent is isolated by a method described herein for isolating the hRUP37 receptor or variant. Preferred agents will interact with the endogenous receptor or non-endogenous receptor and, more preferably, inhibit or stimulate an endogenous or non-endogenous form of the receptor. Particularly preferred agents are agonists or inverse agonists of the endogenous hRUP37 receptor. Still more preferably, the agent is an agonist or inverse agonist of the hRUP37 receptor in the testis and/or hippocampus and/or cerebral cortex.

Accordingly, a suitable agent will be useful for increasing or decreasing activity of the hRUP37 receptor polypeptide when administered to a subject. Such agents are therefore used for modulating a functionality of the testis and/or hippocampus and/or cerebral cortex, or for treatment of a disease or disorder related to the testis and/or hippocampus and/or cerebral cortex, or in the preparation of a medicament for modulating a functionality of the testis and/or hippocampus and/or cerebral cortex or for treatment of a disease or disorder related to the testis and/or hippocampus and/or cerebral cortex.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

The embodiments of the invention described herein with respect to any single embodiment and, in particular, with respect to any GPCR or a use thereof shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein unless the context requires otherwise.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphic representation of activation of RUP32, Gq(del)/Gi, RUP32 cotransfected with Gq(del)/Gi, and CMV (control; expression vector) in a second messenger assay measuring the accumulation of inositol phosphate (IP3) utilizing 293 cells.

Figure 2 provides an illustration of second messenger IP3 production from endogenous version RUP35 and RUP36 as compared with the control DETAILED DESCRIPTION The scientific literature that has evolved around receptors has adopted a number 4 of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document. To the extent that these definitions conflict with other definitions for these terms, the following definitions shall control: AGONISTS shall mean materials ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes. In some embodiments, AGONISTS are those materials not previously known to activate the intracellular response when they bind to the receptor or to enhance GTP binding to membranes.

AMINO ACID ABBREVIATIONS used herein are set out in Table 2 infra.

ANTAGONIST shall mean materials ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists. ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist. In some embodiments, ANTAGONISTS are those materials not previously known to activate the intracellular response when they bind to the receptor or to enhance GTP binding to membranes.

TABLE 2 ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique. Preferably, the phrase "candidate compound" does not include compounds which were publicly known to be compounds selected from the group consisting of inverse agonist, agonist or antagonist to a receptor, as previously determined by an indirect identification process ("indirectly identified compound"); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at

'U

Sleast one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans.

COMPOSITION means a material comprising at least one component; a "pharmaceutical composition" is an example of a composition.

SCOMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality; i.e. the ability to activate/inhibit a signal O transduction pathway, as opposed to receptor binding affinity. Exemplary means of (N 10 detecting compound efficacy are disclosed in the Example section of this patent document.

CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine guanosine cytidine uridine and thymidine coupled to a phosphate group and which encodes an amino acid.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subjected to constitutive receptor activation. A constitutively activated receptor is endogenous or non-endogenous.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.

DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to the phrase "candidate compound", shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound. This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase "indirectly identifying" or "indirectly identified." ENDOGENOUS shall mean a material that a mammal naturally produces.

ENDOGENOUS in reference to, for example and not limitation, the term "receptor," shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. By contrast, the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. For example, and not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a "non-endogenous, constitutively activated receptor." Both terms is utilized to describe both "in vivo" and "in vitro" systems. For example, and not limitation, in a screening approach, the endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in vivo system is viable.

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a nonendogenous protein comprising an endogenous, constitutively activate GPCR or a nonendogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. For example, and not limitation, in an endogenous state, if the G protein "Gsact" is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein based upon the specific GPCR would be a non-endogenous protein comprising the GPCR fused to Gsa; in some circumstances, as will be set forth below, a non-predominant G protein is fused to the GPCR. The G

IL

protein is fused directly to the C-terminus of the constitutively active GPCR or there may be spacers between the two.

(Ni HOST CELL shall mean a cell capable of having a plasmid and/or vector incorporated therein. In the case of a prokaryotic host cell, a plasmid is typically replicated as a t autonomous molecule as the host cell replicates (generally, the plasmid is thereafter IDisolated for introduction into a eukaryotic host cell); in the case of a eukaryotic host cell, a plasmid is integrated into the cellular DNA of the host cell such that when the eukaryotic host cell replicates, the plasmid replicates. In some embodiments the host 10 cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells.

INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the ligandreceptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor.

INHIBIT or INHIBITING, in relationship to the term "response" shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.

INVERSE AGONISTS shall mean materials ligand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists, or decrease GTP binding to membranes.

Preferably, the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% as compared with the baseline response in the absence of the inverse agonist.

KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified.

LIGAND shall mean a molecule specific for a naturally occurring receptor.

MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor. In terms of equivalents to specific sequences, a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if: the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least 80%, at least 85%, at least at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99%. In some embodiments, owing to the fact that some preferred cassettes disclosed herein for achieving constitutive activation include a single amino acid and/or codon change between the endogenous and the nonendogenous forms of the GPCR, it is preferred that the percent sequence homology should be at least 98%.

NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an identified ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway.

ORPHAN RECEPTOR shall mean an endogenous receptor for which the ligand specific for that receptor has not been identified or is not known.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human).

Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

PLASMID shall mean the combination of a vector and cDNA. Generally, a plasmid is introduced into a host cell for the purposes of replication and/or expression of the cDNA as a protein.

SECOND MESSENGER shall mean an intracellular response produced as a result of receptor activation. A second messenger can include, for example, inositol triphosphate

(IP

3 diacycglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP). Second messenger response is measured for a determination of receptor activation. In addition, second messenger response is measured for the direct identification of candidate compounds, including for example, inverse agonists, agonists, and antagonists.

SIGNAL TO NOISE RATIO shall mean the signal generated in response to activation, amplification, or stimulation wherein the signal is above the background noise or the basal level in response to non-activation, non-amplification, or non-stimulation.

SPACER shall mean a translated number of amino acids that are located after the last codon or last amino acid of a gene, for example a GPCR of interest, but before the start codon or beginning regions of the G protein of interest, wherein the translated number amino acids are placed in-frame with the beginnings regions of the G protein of interest. The number of translated amino acids is tailored according to the needs of the skilled artisan and is generally from about one amino acid, preferably two amino acids, more preferably three amino acids, more preferably four amino acids, more preferably five amino acids, more preferably six amino acids, more preferably seven amino acids, more preferably eight amino acids, more preferably nine amino acids, more preferably ten amino acids, more preferably eleven amino acids, and even more preferably twelve amino acids.

STIMULATE or STIMULATING, in relationship to the term "response" shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound.

SUBSTANTIALLY shall refer to a result which is within 40% of a control result, preferably within 35%, more preferably within 30%, more preferably within 25%, more preferably within 20%, more preferably within 1 more preferably within 10%, more preferably within more preferably within and most preferably within 1% of a control result. For example, in the context of receptor functionality, a test receptor may exhibit substantially similar results to a control receptor if the transduced signal, measured using a method taught herein or similar method known to the art-skilled, if within 40% of the signal produced by a control signal.

VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a host cell.

The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to follow.

A. Introduction The traditional study of receptors has typically proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the receptor.

Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been heretofore recognized is that it is the active state of the receptor that is most useful for discovering agonists and inverse agonists of the receptor. For those diseases which result from an overly active receptor or an under-active receptor, what is desired in a therapeutic drug is a compound which acts to diminish the active state of a receptor or enhance the activity of the receptor, respectively, not necessarily a drug which is an antagonist to the endogenous ligand. This is because a compound that reduces or enhances the activity of the active receptor state need not bind at the same site as the endogenous ligand. Thus, as taught by a method of this invention, any search for therapeutic compounds should start by screening compounds against the ligandindependent active state.

B. Identification of Human GPCRs The efforts of the Human Genome project has led to the identification of a plethora of information regarding nucleic acid sequences located within the human genome; it has been the case in this endeavor that genetic sequence information has been made available without an understanding or recognition as to whether or not any particular genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleic acid sequences within the human genome are within the purview of those having ordinary skill in the art. For example, and not limitation, a variety of human GPCRs, disclosed herein, were discovered by reviewing the GenBank T M database. Table 3 infra lists several endogenous GPCRs that we have discovered, along with other GPCRs that are homologous to the disclosed GPCR.

Receptor homology is useful in terms of gaining an appreciation of a role of the receptors within the human body. As the patent document progresses, techniques for mutating these receptors to establish non-endogenous, constitutively activated versions of these receptors will be discussed.

The techniques disclosed herein are also applicable to other human GPCRs known to the art, as will be apparent to those skilled in the art.

TABLE 3 Disclosed GenBank Open reading Reference to Percent Human Orphan Accession No. Frame (bp) homologous homology to GPCRs GPCR designated

GPCR

hRUP28 AC073957 1,002 bp hGPR30 34% hRUP29 AC083865 918 bp hGPR18 27% AC055863 1,125 bp hBRB 27% hRUP31 AL356214 1,086 bp hGALR-I 31% hRUP32 AL513524 1,038 bp hPNR 43% hRUP33 AL513524 1,020 bp GPR57 GPR58 51% hRUP34 AL513524 1,029 bp hPNR AC021089 1,062 bp hK-type 3 opioid 27% hRUP36 AC090099 969 bp GPR90 42% hRUP37 AC090099 969 bp hMRG 41% C. Receptor Screening Screening candidate compounds against a non-endogenous, constitutively activated version of the GPCRs disclosed herein allows for the direct identification of candidate compounds which act at the cell surface receptor, without requiring use of the receptor's endogenous ligand. Using routine and often commercially available techniques, one can determine areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed. The expression location of a receptor in a specific tissue provides a scientist with the ability to assign a physiological functional role of the receptor. It is also possible using these techniques to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document. Furthermore, expression of a receptor in diseased organs can assist one in determining the magnitude of the clinical relevance of the receptor.

Constitutive activation of the GPCRs disclosed herein is based upon the distance from the proline residue at which is presumed to be located within TM6 of the GPCR; this algorithmic technique is disclosed in co-pending and commonly assigned patent document PCT Application Number PCT/US99/23938, published as WO 00/22129 on April 20, 2000 which, along with the other patent documents listed herein, is incorporated herein by reference. The algorithmic technique is not predicated upon traditional sequence "alignment" but rather a specified distance from the aforementioned TM6 proline residue (or, of course, endogenous constitutive substitution for such proline residue). By mutating the amino acid residue located 16 amino acid residues from this residue (presumably located in the 1C3 region of the receptor) to, most preferably, a lysine residue, constitutive activation of the receptor may be obtained. Other amino acid residues may be useful in the mutation at this position to achieve this objective and will be discussed in detail, below.

D. Disease/Disorder Identification and/or Selection As will be set forth in greater detail below, inverse agonists and agonists to the nonendogenous, constitutively activated GPCR is identified by the methodologies of this invention. Such inverse agonists and agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases related to this receptor. Because of the ability to directly identify inverse agonists to the GPCR, thereby allowing for the development of pharmaceutical compositions, a search for diseases and disorders associated with the GPCR is relevant. The expression location of a receptor in a specific tissue provides a scientist with the ability to assign a physiological function to the receptor. For example, scanning both diseased and normal tissue samples for the presence of the GPCR now becomes more than an academic exercise or one which might be pursued along the path of identifying an endogenous ligand to the specific GPCR. Tissue scans is conducted across a broad range of healthy and diseased tissues.

Such tissue scans provide a step in associating a specific receptor with a disease and/or disorder. Furthermore, expression of a receptor in diseased organs can assist one in determining the magnitude of clinical relevance of the receptor.

The DNA sequence of the GPCR is used to make a probe/primer. In some preferred embodiments the DNA sequence is used to make a probe for: dot-blot analysis against tissue-mRNA; and/or RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue, is used to correlate location to function and indicate the receptor's physiological role/function and create a treatment regimen, including but not limited to, a disease associated with that function/role. Receptors are also localized to regions of organs by this technique. Based on the known or assumed roles/functions of the specific tissues to which the receptor is localized, the putative physiological function of the receptor is deduced. For example and not limitation, proteins located/expressed in areas of the thalamus are associated with sensorimotor processing and arousal (In; The Pharmacological Basis of Therapeutics, Goodman Gilman eds., 9 t h Edition, page 465, 1996). Proteins expressed in the hippocampus or in Schwann cells are associated with learning and memory, and myelination of peripheral nerves, respectively (in: Kandel et al., Essentials of Neural Science and Behavior pages 657, 680 and 28, respectively, 1995).

E. Screening of Candidate Compounds 1. Generic GPCR Screening Assay Techniques When a G protein receptor becomes constitutively active, it binds to a G protein (e.g.

Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, 35 S]GTPyS, is used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that 35 S]GTPyS is used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. The use of this assay system is typically for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.

2. Specific GPCR Screening Assay Techniques Once candidate compounds are identified using the "generic" G protein-coupled receptor assay an assay to select compounds that are agonists or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibits adenylyl cyclase. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP as described generally In: "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 r d Nichols, J. G. et al. eds., Sinauer Associates, Inc., 1992. Thus, assays that detect cAMP are utilized to determine if a candidate compound is, an inverse agonist to the receptor such a compound would decrease the levels of cAMP).

Any one or more of a variety of approaches known in the art for measuring cAMP is utilized.

For example, a preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format.

Another type of assay that is utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMPresponsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites (cAMP response elements) and drives the expression of the gene. Reporter systems are constructed which have a promoter containing multiple cAMP response elements before the reporter gene, p-galactosidase or luciferase.

Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and leads to the expression of the reporter protein. The reporter protein such as p-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 releasing two intracellular messengers: diacycloglycerol (DAG) and inositol 1,4,5-triphoisphate (IP 3 Increased accumulation of IP 3 is associated with activation of Gq- and Go-associated receptors. This is described In: "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 rd Ed.) Nichols, J. G. et al. eds., Sinauer Associates, Inc., 1992.

Assays that detect IP 3 accumulation are utilized to determine if a candidate compound is an inverse agonist to a Gq- or Go-associated receptor because such a compound would decrease the levels of IP 3 Gq-associated receptors are also examined using an API reporter assay wherein Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression. Commercially available assays for such detection are available.

3. GPCR Fusion Proteins The use of an endogenous, constitutively activated GPCR or a non-endogenous, constitutively activated GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to differentiate between, the nonendogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist or agonist or have no affect on such a receptor, it is preferred that an approach be utilized that enhances such differentiation. A preferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR.

Coupling of the G protein to the GPCR provides a signaling pathway that is assessed.

In some embodiments it is preferred that screening take place using a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated GPCR will continuously signal. In some embodiments it is preferred that this signal be enhanced such that in the presence of, an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein is preferred for screening with either an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is utilized in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. Important criteria on the construction of such a GPCR Fusion Protein construct include but are not limited to, that the endogenous GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. Other embodiments include constructs wherein the endogenous GPCR sequence and the G protein sequence are not in-frame and/or the "stop" codon is not deleted or replaced. The GPCR is linked directly to the G protein, or there are spacer residues between the two (preferably, no more than about 12, although this number is readily ascertained by one of ordinary skill in the art). Based upon convenience it is preferred to use a spacer. Preferably, the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein a universal G protein construct (see Examples) be available for insertion of an endogenous GPCR sequence therein; this provides for further efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.

As noted above, constitutively activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging the cAMP signal decreases upon activation thus making the direct identification of, inverse agonists (which would further decrease this signal), challenging. As disclosed herein, we have ascertained that for these types of receptors, it is possible to create a GPCR Fusion Protein that is not based upon the GPCR's endogenous G protein, in an effort to establish a viable cyclase-based assay. Thus, for example, an endogenous Gi coupled receptor is fused to a Gs protein--such a fusion construct, upon expression, "drives" or "forces" the endogenous GPCR to couple with, Gs rather than the "natural" Gi protein, such that a cyclase-based assay is established. Thus, for Gi, Gz and Go coupled receptors, in some embodiments it is preferred that when a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).

TABLE 3 G Protein Production upon Accumulation cAMP Accumulation activation of upon activation production upon upon contact GPCR* of GPCR* contact with with inverse inverse agonist agonist Gs Increase N/A Decrease N/A Gi Decrease N/A Increase N/A Gz Decrease N/A Increase N/A Go Decrease Increase Increase Decrease Gq N/A Increase N/A Decrease constitutive activation or agonist binding Equally effective is a G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. In some embodiments a preferred fusion construct is accomplished with a Gq Protein wherein the first six amino acids of the G-protein a-subunit is deleted and the last five amino acids at the C-terminal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest. For example, a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct". This fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylgycerol, is measured in lieu of cAMP production.

4. Co-transfection of a Target Gi Coupled GPCR with a Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays) A Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make assessment of cAMP levels challenging. An effective technique in measuring the decrease in production of cAMP as an indication of constitutive activation of a receptor that predominantly couples Gi upon activation is accomplished by co-transfecting a signal enhancer, a nonendogenous, constitutively activated receptor that predominantly couples with Gs upon activation TSHR-A6231, disclosed below), with the Gi linked GPCR. As is apparent, constitutive activation of a Gs coupled receptor is determined based upon an increase in production of cAMP. Constitutive activation of a Gi coupled receptor leads to a decrease in production cAMP. Thus, the co-transfection approach is intended to advantageously exploit these "opposite" affects. For example, co-transfection of a non-endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with the endogenous Gi coupled receptor (the "target receptor") provides a baseline cAMP signal although the Gi coupled receptor will decrease cAMP levels, this "decrease" will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co-transfecting the signal enhancer with a constitutively activated version of the target receptor, cAMP would be expected to further decrease (relative to base line) due to the increased functional activity of the Gi target which decreases cAMP).

Screening of candidate compounds using a cAMP based assay is then accomplished, with two "changes" relative to the use of the endogenous receptor/G-protein fusion: first, relative to the Gi coupled target receptor, "opposite" effects will result, an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal; second, as would be apparent, candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this is done prior to or after screening against the cotransfected receptors).

F. Medicinal Chemistry Generally, but not always, direct identification of candidate compounds is conducted in conjunction with compounds generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. Generally, the results of such screening will be compounds having unique core structures; thereafter, these compounds may be subjected to additional chemical modification around a preferred core structure(s) to further enhance the medicinal properties thereof.

Such techniques are known to those in the art and will not be addressed in detail in this patent document.

G. Pharmaceutical Compositions Agents identified, selected, or isolated from candidate compounds using the screening assays described herein are selected for further development and formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16 th Edition, 1980, Mack Publishing Co., (Osol et al., eds.).

H. Other Utilities Although a preferred use of the non-endogenous versions of the GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists or agonists (preferably for use as pharmaceutical agents), other uses of these versions of GPCRs exist. For example, in vitro and in vivo systems incorporating GPCRs are utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. In some embodiments it is preferred that the endogenous receptors be "orphan receptors", the endogenous ligand for the receptor has not been identified. In some embodiments, therefore, the modified, non-endogenous GPCRs are used to understand the role of endogenous receptors in the human body before the endogenous ligand therefore is identified. Such receptors can also be used to further elucidate known receptors and the pathways through which they transduce a signal. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.

I. Examples The present invention is further described with reference to the following examples and the accompanying drawings. However, the present invention is not to be limited in scope by the specific examples described herein. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

The examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with the ability to make minor modifications to these sequences while achieving the same or substantially similar results reported below. The traditional approach to application or understanding of sequence cassettes from one sequence to another from rat receptor to human receptor or from human receptor A to human receptor B) is generally predicated upon sequence alignment techniques whereby the sequences are aligned in an effort to determine areas of commonality. The mutational approach disclosed herein does not rely upon this approach but is instead based upon an algorithmic approach and a positional distance from a conserved proline residue located within the TM6 region of human GPCRs. Once this approach is secured, those in the art are credited with the ability to make minor modifications thereto to achieve substantially the same results constitutive activation) disclosed herein. Such modified approaches are considered within the purview of this disclosure.

Example 1 Endogenous Human GPCRs 1. Identification of Human GPCRs The disclosed endogenous human GPCRs were identified based upon a review of the GenBank T M database information. While searching the database, the following cDNA clones were identified as evidenced below (Table 4).

TABLE 4 Disclosed GenBank Open reading Reference to Nucleic acid Amino acid Human Accession No. Frame (bp) homologous SEQ ID NO: SEQ ID NO: Orphan G PCR GPCRs hRUP28 AC073957 1,002 bp hGPR30 1 2 hRUP29 AC083865 918 bp hGPR18 3 4 hRUP3O AC055863 1,125 bp hBRB131 5 6 hRUP31 AL356214 1,086 bp hGALR- 1 7 8 hRUP32 AL513524 1,038 bp hPNR 9 hRUP33 AL513524 1,020 bp GPR57 11 12 GPR58 hRUP34 AL513524 1,029 bp hPNR 13 14 AC021089 1,062 bp hK-type 3 15 16 opioid hRUP36 AC090099 969 bp GPR90 17 18 hRUP37 AC090099 969 bp hMRG 19 2. Full Length Cloning a. hRUP28 (Seq. Id. Nos. I 2) The disclosed human RUP28 was identified based upon the use of GenBank database information. While searching the database, a eDNA clone with Accession Number AC073957 was identified as a human genomic sequence from chromosome 7.

The full length RUP28 was cloned by PCR using the primers: Sense, 5' of initiation codon: 5'-CAGAGCTCTGGTGGCCACCTCTGTCC-3' (SEQ. ID. NO.: 21); and Antisense, 3' of stop codon: 5'-CTGCGTCCACCAGAGTCACGTCTCC-3' (SEQ. ID. NO.: 22),

TM

and human adult liver Marathon-Read y cDNA (Clontech) as template.

Advantage" cDNA polymerase (Clontech) was used for the amplification in a 50 p1 reaction by the following cycle with step 2 to 4 repeated 35 times: 95 0 C for 5 min; 94 0 C for 30 sec; 58 0 C for 30 sec; 72 0 C for 1 min 30 sec; and 72 0 C for 7 min.

A 1.16 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit Biosystems). See, SEQ. ID. NO.: 1 for the nucleic acid sequence and SEQ. ID.

NO.: 2 for the encoded amino acid sequence.

b. hRUP29 (Seq. Id. Nos. 3 4) The disclosed human RUP29 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC0083865 was identified as a human genomic sequence from chromosome 7.

The full length RUP29 was cloned by PCR using primers: Sense, 5' containing the initiation codon (underlined): 5'-GTATGCCTGGCCACAATACCTCCAGG-3' (SEQ ID NO: 23); and Antisense, containing the complement of the stop codon (underlined): 5'-GTTTGTGGCTAACGGCACAAAACACAATTCC-3' (SEQ. ID. NO.: 24), and human genomic DNA as template.

TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a pl reaction by the following cycle with step 2 to 4 repeated 35 times: 94 0 C for min; 94 0 C for 30 sec; 54 0 C for 30 sec; 72'C for 1 min 30 sec; and 72 0 C for 7 min.

A 930 bp PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems).

Rapid amplification of cDNA ends (RACE) was performed using human leukocyte and ovary Marathon-Ready TM cDNA (Clontech) to determine the precise 5'-end of hRUP29 cDNA, using the hRUP29-specific primer having the sequence: 5-GGTACCACAATGACAATCACCAGCGTCC-3' (SEQ. ID. NO.: 25); and an API primer (Clontech) for the first-round PCR reaction; and hRUP29-specific primer having the sequence: 5'-GGAACGTGAGGTACATGTGGATGTGCAGC-3' (SEQ. ID. NO.: 26); and an AP2 primer (Clontech) for the second-round PCR reaction.

The products of the RACE reactions were isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced. See, SEQ. ID. NO.: 3 for the nucleic acid sequence and SEQ. ID. NO.: 4 for the encoded amino acid sequence.

c. hRUP30 (Seq. Id. Nos. 5 6) The disclosed human RUP30 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC055863 was identified as a human genomic sequence from chromosome 17.

The full length RUP30 was cloned by 5'-RACE-PCR using human pancreas Marathon- Ready T cDNA (Clontech) as template and the following primers for the first round

RT-PCR:

Sense, 5' of the initiation codon: 5'-GCAGTGTAGCGGTCAACCGTGAGCAGG-3' (SEQ. ID. NO.: 27); and API primer (Clontech).

For the second round of PCR, the following primers were used: Antisense, 3' of the stop codon: 5'-TGAGCAGGATGGCGATCCAGACTGAGGCGTGG-3'(SEQ. ID. NO.: 28); and AP2 primer (Clontech).

DNA fragments generated by the 5' RACE-PCR were cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene).

Based on the sequence of the 5' RACE products, the full length RUP30 was cloned by RT-PCR, using primers: Sense, containing the intiation codon (underlined): 5'-GAGGTACAGCTGGCGATGCTGACAG-3' (SEQ. ID. NO.: 29); and Antisense, 3' of the stop codon: 5'-GTGGCCATGAGCCACCCTGAGCTCC-3' (SEQ. ID. NO.: and human pancreas Marathon-Ready T M cDNA (Clontech) as template.

Taq DNA polymerase (Stratagene) was used for the amplification in a 50 p reaction by the following cycle with step 2 to 4 repeated 35 times: 94 0 C for 40 seconds; 94 0 C for seconds; 64'C for 20 seconds; 72 0 C for 2 minutes; and 72 0 C for 5 minutes.

A 1.2 Kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRll- TOPO vector (Invitrogen) and several clones were sequenced using the ABI Big Dye Terminator kit Biosystems). See, SEQ. ID. NO.: 5 for the nucleic acid sequence and SEQ. ID. NO.: 6 for the encoded amino acid sequence.

d. hRUP31 (Seq. Id. Nos. 7 8) The disclosed human RUP31 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL356214 was identified as a human genomic sequence from chromosome The full length RUP31 was cloned by RT-PCR using the following primers: Sense, containing the initation codon (underlined): 5'-GGAATGTCCACTGAATGCGCGCGG-3' (SEQ. ID. NO.: 31); and Antisense, 3' of the complement of the stop codon: 5'-AGCTCGCCAGGTGTGAGAAACTCGG-3' (SEQ. ID. NO.: 32), and human mammary gland Marathon-Ready T M cDNA (Clontech) as template.

Advantage T M cDNA polymerase (Clontech) was used for the amplification in a 50 p1 reaction by the following cycle with step 2 to 4 repeated 35 times: 94 0 C for 40 seconds; 94 0 C for 20 seconds; 66 0 C for 20 seconds; 72 0 C for 1 min 30 sec; and 72 0 C for 5 min.

A 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems). See, SEQ. ID. NO.: 7 for the nucleic acid sequence and SEQ. ID. NO.: 8 for the encoded amino acid sequence.

e. hRUP32 (Seq. Id. Nos. 9 The disclosed human RUP32 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL513524 was identified as a human genomic sequence from chromosome 6.

The full length RUP32 was cloned by PCR using the primers: Sense, containing the initiation codon (underlined): 5'-GCGTTATGAGCAGCAATTCATCCCTGCTGG-3' (SEQ. ID. NO.: 33); and Antisense: 5'-GTATCCTGAACTTCGTCTATACAACTGC-3' (SEQ. ID. NO.: 34), and human genomic DNA (Clontech) as template.

TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a 50 pl reaction by the following cycle with step 2 to 4 repeated 35 times: 94 0 C for 3 minutes; 94 0 C for 20 seconds; 58 0 C for 20 seconds; 72 0 C for 1 min 30 sec; and 72°C for 7 minutes.

A 1.06 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit Biosystems). See, SEQ. ID. NO.: 9 for the nucleic acid sequence and SEQ. ID.

NO.: 10 for the encoded amino acid sequence.

f hRUP33 (Seq. Id. Nos. II 12) The disclosed human RUP33 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL513524 was identified as a human genomic sequence from chromosome 6.

The full length RUP33 was cloned by PCR using the primers: Sense, containing the initiation codon (underlined): 5'-CCCTCAGGAATGATGCCCTTTTGCCACAA-3' (SEQ. ID. NO.: 35); and Antisense: 5'-ATCCATGTGGTTGGTGCATGTGGTTCGT-3' (SEQ. ID. NO.: 36), and human genomic DNA (Clontech) as template.

TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a ul reaction by the following cycle with step 2 to 4 repeated 35 times: 94°C for 3 minutes; 94 0 C for 20 seconds; 56 0 C for 20 seconds; 72 0 C for 1 min 30 sec; and 72°C for 7 minutes.

A 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems). See, SEQ. ID. NO.: 11 for the nucleic acid sequence and SEQ. ID. NO.: 12 for the encoded amino acid sequence.

g. hRUP34 (Seq. Id. Nos. 13 14) The disclosed human RUP34 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL513524 was identified as a human genomic sequence from chromosome 6.

The full length RUP34 was cloned by PCR using the primers: Sense, containing the intiation codon (underlined): 5'-AAACAACAAACAGCAGAACCATGACCAGC-3' (SEQ. ID. NO.: 37); and Antisense: 5'-ACATAGAGACAAGTGACATGTGTGAACCAC-3' (SEQ. ID. NO.: 38), and human genomic DNA (Clontech) as template.

TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a pl reaction by the following cycle with step 2 to 4 repeated 35 times: 94°C for 3 minutes; 94 0 C for 20 seconds; 60 0 C for 20 seconds; 72 0 C for 1 min 30 sec; and 72°C for 7 minutes.

A 1.27 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit Biosystems). See, SEQ. ID. NO.: 13 for the nucleic acid sequence and SEQ. ID.

NO.: 14 for the encoded amino acid sequence.

h. hRUP35 (Seq. Id. Nos. 15 16) The disclosed human RUP35 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC021089 was identified as a human genomic sequence from chromosome 16.

The 5' sequence of RUP35 was determined by 5' RACE-PCR with a human fetal brain Marathon-Ready TM cDNA (Clontech) as template and the following primers for the first round RT-PCR: Sense oligonucleotide: 5'-GGTATGAGACCGTGTGGTACTTGAGC-3' (SEQ. ID. NO.: 39); and API primer (Clontech).

For the second round PCR, the following primers were used: Antisense: 5'-GTGGCAGACAGCGATATACCTGTCAATGG-3' (SEQ. ID. NO.: 40); and Sense: AP2 primer (Clontech).

DNA fragments generated by the 5' RACE-PCR were cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene).

Based upon the sequence of the 5' RACE products, the full length RUP35 was cloned by RT-PCR, using the primers: Sense, containing the initation codon (underlined): 5'-GCGCTCATGGAGCACACGCACGCCCAC-3' (SEQ. ID. NO.: 41); and Antisense, 3' of the complement of the stop codon: 5'-GAGGCAGTAGTTGCCACACCTATGG-3' (SEQ. ID. NO.: 42) and human brain Marathon-Ready T M cDNA (Clontech) as template.

Advantage TM cDNA polymerase (Clontech) was used for the amplification in a 100 pl reaction by the following cycle with step 2 to 4 repeated 45 times: 95 0 C for 2 minutes; for 20 seconds; 60 0 C for 20 seconds; 72 0 C for 1 min 30 sec; and 72 0 C for minutes.

A 1.0 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRIl- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems). See, SEQ. ID. NO.: 15 for the nucleic acid sequence and SEQ. ID. NO.: 16 for the encoded amino acid sequence.

i. hRUP36 (Seq. Id. Nos. 17 18) The disclosed human RUP36 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC090099 was identified as a human genomic sequence from chromosome 11.

The full length RUP36 was cloned by PCR using the primers: Sense, 5' of the start codon: 5'-CATCTGGTTTGTGTTCCCAGGGGCACCAG-3' (SEQ. ID. NO.: 43); and Antisense, 3' of the complement of the stop codon: 5'-GACAGTGTTGCTCTCAAAGTCCCGTCTGACTG-3' (SEQ. ID. NO.: 44), and human genomic DNA (Clontech) as template.

TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a pl reaction by the following cycle with step 2 to 4 repeated 30 times: 95 0 C for minutes; 95°C for 30 seconds; 70 0 C for 30 seconds; 72 0 C for 1 min 30 sec; and 72°C for 7 minutes.

A 1.0 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems). See, SEQ. ID. NO.: 17 for the nucleic acid sequence and SEQ. ID. NO.: 18 for the encoded amino acid sequence.

j. hRUP37 (Seq. Id. Nos. 19 The disclosed human RUP37 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC090099 was identified as a human genomic sequence from chromosome 11.

The full length RUP37 was cloned by PCR using the primers: Sense: 5'-CTGTTTCCAGGGTCATCAGACTGGG-3' (SEQ. ID. NO.: 45); and Antisense: 5'-GCAGCATTGCTCTCAAAGTCCTGTCTG-3' (SEQ. ID. NO.: 46), and human genomic DNA (Clontech) as template.

TaqPlus.RTM. Precision DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 95.degree. C. for 5 min; TaqPlus® Precision DNA polymerase (Stratagene) was used for the amplification in a [l reaction by the following cycle with step 2 to 4 repeated 35 times: 95°C for minutes; 95 0 C for 30 seconds; 62 0 C for 30 seconds; 72 0 C for 1 min 30 sec; and 72 0

C

for 7 minutes.

A 969 base pair was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E.

Biosystems). See, SEQ. ID. NO.: 19 for the nucleic acid sequence and SEQ. ID. NO.: for the encoded amino acid sequence.

Example 2 Preparation of Non-Endogenous, Constitutively Activated GPCRs Those skilled in the art are credited with the ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches utilized to create nonendogenous versions of several of the human GPCRs disclosed above. The mutations disclosed below are based upon an algorithmic approach whereby the 16 th amino acid (located in the IC3 region of the GPCR) from a conserved proline (or an endogenous, conservative substitution therefore) residue (located in the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, preferably to an alanine, histidine, arginine or lysine amino acid residue, most preferably to a lysine amino acid residue.

1. Transformer Site-Directed. TM. Mutagenesis Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using inter alia Transformer Site-Directed TM Mutagenesis Kit (Clontech) according to the manufacturer instructions. In some embodiments two mutagenesis primers are used, preferably a lysine mutagenesis oligonucleotide that creates the lysine mutation, and a selection marker oligonucleotide. For convenience, the codon mutation to be incorporated into the human GPCR is also noted, in standard form (Table TABLE Receptor identifier Codon Mutation hRUP28 H232K hRUP29 Q224K T268K hRUP31 F263K hRUP32 A257K hRUP33 V250K hRUP34 A256K T227K hRUP36 Y215K hRUP37 Y215K Example 3 Receptor Expression Although a variety of cells are available to the art-skilled for the expression of proteins, it is preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, utilization of, yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, geneticmechanism and secretary pathways that have evolved for mammalian systems--thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as those obtained using mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized is predicated upon the particular needs of the artisan.

a. Transient Transfection On day one, 6 X 10 6 cells/10 cm dish of 293 cells well were plated out. On day two, two reaction tubes were prepared (the proportions to follow for each tube are per plate): Tube A was prepared by mixing 4 Vg DNA pCMV vector; pCMV vector with C receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); Tube B was prepared dy by mixing 24 pl lipofectamine (Gibco BRL) in 0.5 ml serum free DMEM. Tubes A and B were admixed by inversion (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the "transfection mixture".

Itn Plated 293 cells were washed with 1 x PBS, followed by addition of 5 ml serum free \D DMEM. One ml of the transfection mixture was added to the cells, followed by C1 incubation for 4 hrs at 37 0 C/5% CO 2 The transfection mixture was removed by O aspiration, followed by the addition of 10 ml of DMEM/10% Fetal Bovine Serum.

C 10 Cells were incubated at 37 0 C/5% CO 2 After 48 hr incubation, cells were harvested and utilized for analysis.

b. Stable Cell Lines Approximately 12 x 10 6 293 cells are plated on a 15 cm tissue culture plate, and grown in DME High Glucose Medium containing 10% fetal bovine serum and one percent sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours following plating of 293 cells (to approximately 80% confluency), the cells are transfected using 12 gg of DNA. The 12 Vig of DNA is combined with 60 Jl of lipofectamine and 2 mL of DME High Glucose Medium without serum. The medium is aspirated from the plates and the cells washed once with medium without serum. The DNA, lipofectamine, and medium mixture is added to the plate along with 10 mL of medium without serum. Following incubation at 37 0 C for four to five hours, the medium is aspirated and 25 ml of medium containing serum added. Twenty-four hours following transfection, the medium is aspirated again, and fresh medium with serum added. Forty-eight hours following transfection, the medium is aspirated and medium with serum is added containing geneticin (G418 drug) at a final concentration of 500 utg/ml. The transfected cells then undergo selection for positively transfected cells containing the G418 resistant gene.

The medium is replaced every four to five days as selection occurs. During selection, cells are grown to create stable pools, or split for stable clonal selection.

Example 4 Assays for Determination of Constitutive Activity of Non-Endogenous GPCRs A variety of approaches are available for assessment of constitutive activity of the nonendogenous human GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan.

1. Membrane Binding Assays: sup. 35S]GTP.gamma.S Assay When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog 35 S]GTPyS is utilized to demonstrate enhanced binding of 3 5 S]GTPyS to membranes expressing constitutively activated receptors. Advantages of using 35 S]GTPyS binding to measure constitutive activation include but are not limited to the following: it is generically applicable to all G protein-coupled receptors; it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.

The assay takes advantage of the ability of G protein coupled receptors to stimulate [3S]GTPyS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to constitutively activated G protein-coupled receptors. The assay is generic and has application to drug discovery at all G protein-coupled receptors.

The 35 S]GTPyS assay is incubated in 20 mM HEPES and between 1 and about 20 mM MgCI 2 (this amount is adjusted for optimization of results, although 20 mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM 35 S]GTPyS (this amount is adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 ltg membrane protein 293 cells expressing the Gs Fusion Protein; this amount is adjusted for optimization) and 10 pM GDP (this amount is changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 Wl; Amersham) is then added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500 x g for 5 minutes at room temperature and then counted in a scintillation counter.

2. Adenylyl Cyclase A Flash Plate T M Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays is modified for use with crude plasma membranes. The Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells is quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.

Transfected cells are harvested approximately twenty four hours after transient transfection. Media are carefully aspirated and discarded. Ten ml of PBS is gently added to each dish of cells followed by careful aspiration. One ml of Sigma cell dissociation buffer and 3 ml of PBS is added to each plate. Cells are pipetted off the plate and the cell suspension collected into a 50 ml conical centrifuge tube. Cells are centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet is carefully resuspended into an appropriate volume of PBS (about 3 ml/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (to a final volume of about 50 jpl/well). cAMP standards and Detection Buffer (comprising 1 pCi of tracer [1251 cAMP (50 pl] to 11 ml Detection Buffer) is prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contains 50 ul of Stimulation Buffer, 3 pl of test compound (12 pM final assay concentration) and 50 pl cells. Assay Buffer is stored on ice until utilized. The assay is initiated by addition of 50 pl of cAMP standards to appropriate wells followed by addition of 50 pl of PBSA to wells H-11 and H 12. Fifty pl of Stimulation Buffer is added to all wells. DMSO (or selected candidate compounds) is added to appropriate wells using a pin tool capable of dispensing 3 Ptl of compound solution, with a final assay concentration of 12 jpM test compound and 100 gl total assay volume. The cells are then added to the wells and incubated for 60 min at room temperature. One hundred pl of Detection Mix containing tracer cAMP is then added to the wells. Plates are incubated for an additional 2 hours followed by counting in a Wallac MicroBeta T M scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve contained within each assay plate.

3. Cell-Based cAMPfor Gi Coupled Target GPCRs A Gi-coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging.

TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation.

TSHR is constitutively activated by mutating amino acid residue 623 changing an alanine residue to an isoleucine residue). An effective technique for measuring the decrease in production of cAMP as an indication of constitutive activation of a Gi coupled receptor is accomplished by co-transfecting, most preferably, non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP. Upon creating a non-endogenous version of the Gi coupled receptor, this non-endogenous version of the target GPCR is then co-transfected with the signal enhancer, and it is this material that is used for screening. This approach is utilized to effectively generate a signal when a cAMP assay is used; this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.

On day one, 2 x 10 4 293 cells/well are plated out. On day two, two reaction tubes are prepared (the proportions to follow for each tube are per plate): Tube A is prepared by mixing 2 pg DNA of each receptor transfected into the mammalian cells, for a total of 4 utg DNA pCMV vector; pCMV vector with mutated THSR (TSHR-A6231); TSHR-A6231 and GPCR, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); Tube B is prepared by mixing 120 tl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B are mixed by inversion (several times), followed by incubation at room temperature for 30-45 min. The mixture is referred to as the "transfection mixture". Plated 293 cells are washed with I x PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture is then added to the cells, followed by incubation for 4 hrs at 37 0 C/5% CO 2 The transfection mixture is then removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells are incubated at 37 0 C/5% CO 2 After 24 hr incubation, cells are harvested and utilized for analysis.

A Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) although designed for cell-based assays, is modified for use with crude plasma membranes depending on the need of the skilled artisan. The Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells is quantified by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.

Transfected cells are harvested approximately twenty four hours after transient transfection. Media are carefully aspirated and discarded. Ten ml of PBS are gently added to each dish of cells followed by careful aspiration. One ml of Sigma cell dissociation buffer and 3 ml of PBS are added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 ml conical centrifuge tube. Cells are centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet is carefully resuspended into an appropriate volume of PBS (about 3 ml/plate). The cells are counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (to a final volume of about 50 pl/well). cAMP standards and Detection Buffer (comprising 1 gCi of tracer [1251 cAMP (50 pl] to 11 ml Detection Buffer) is prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer should be prepared fresh for screening and contains 50 al of Stimulation Buffer, 311 of test compound (12 ptM final assay concentration) and 5011 cells. Assay Buffer is stored on ice until utilized. The assay is initiated by addition of 50 gl of cAMP standards to appropriate wells followed by addition of 50 gl of PBSA to wells H-l and HI2. Fifty pl of Stimulation Buffer is added to all wells. Selected compounds TSH) are added to appropriate wells using a pin tool capable of dispensing 3 pl of compound solution, with a final assay concentration of 12 gM test compound and 100 pl total assay volume. The cells are then added to the wells and incubated for 60 min at room temperature. One hundred gl of Detection Mix containing tracer cAMP is added to the wells. Plates are incubated for an additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are then extrapolated from a standard cAMP curve which is contained within each assay plate.

4. Reporter-Based Assays a. CRE-Luc Reporter Assay (Gs-Associated Receptors) 293 and 293T cells are plated-out on 96 well plates at a density of 2 x 10 4 cells per well and transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA/lipid mixture is prepared for each 6-well transfection as follows: 260 ng of plasmid DNA in 100 pl of DMEM are gently mixed with 2 pl of lipid in 100 gl of DMEM (the 260 ng of plasmid DNA consists of 200 ng ofa 8 x CRE-Luc reporter plasmid, 50 ng ofpCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)).

The 8 x CRE-Luc reporter plasmid is prepared as follows: vector SRIF-p-gal is obtained by cloning the rat somatostatin promoter at BgIV-HindIIl site in the ppgal-Basic Vector (Clontech). Eight copies of cAMP response element are obtained by PCR from an adenovirus template AdpCF126CCRE8 (Human Gene Therapy 7, p 18 8 3 et seq., 1996) and cloned into the SRIF-p-gal vector at the Kpn-Bgl site, resulting in the 8 x CRE-p-gal reporter vector. The 8 x CRE-Luc reporter plasmid is then generated by replacing the beta-galactosidase gene in the 8 x CRE-p-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindllI-BamHI site.

Following 30 min. incubation at room temperature, the DNA/lipid mixture is diluted with 400 [tl of DMEM, and 100 p.l of the diluted mixture are added to each well. One hundred 1l of DMEM with 10% FCS are added to each well after a 4 hr incubation in a cell culture incubator. The following day, the transfected cells are changed with 200 gl/well of DMEM with 10% FCS. Eight hours later, the wells are changed to 100 pl/well of DMEM without phenol red, after one wash with PBS. Luciferase activity is measured the next day using the LucLite TM reporter gene assay kit (Packard) following manufacturer's instructions and read on a 1450 MicroBeta T M scintillation and luminescence counter (Wallac).

b. API Reporter Assay (Gq-Associated Receptors) A method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing API elements in their promoter. A Pathdetect T M AP-1 cis-Reporting System (Stratagene, Catalogue #219073) is utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate are 410 ng pAPI-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

c. SRF-Luc Reporter Assay (Gq-Associated Receptors) One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A Pathdetect T M SRF-Luc-Reporting System (Stratagene) is utilized to assay for Gq coupled activity in, COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian Transfection T Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed between 3 wells in a 96-well plate, kept on the cells in a serum free media for 24 hours. The last hours the cells are incubated with 1 pM Angiotensin, where indicated. Cells are then lysed and assayed for luciferase activity using a Luclite T M Kit (Packard, Cat.

#6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. The data are analyzed using GraphPad PriSm T M 2.0a (GraphPad Software Inc.).

d. Intracellular IP3 Accumulation Assay (Gq-Associated Receptors) On day 1, cells comprising the receptors (endogenous and/or non-endogenous) are plated onto 24 well plates, usually 1 x 105 cells/well (although his number is optimized.

On day 2 cells are transfected by firstly mixing 0.25 ug DNA in 50 tl serum free DMEM/well and 2 p.l lipofectamine in 50 pl serum free DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are then washed with 0.5 ml PBS and 400 ptl of serum free media and then mixed with the transfection media and added to the cells. The cells are incubated for 3-4 hrs at 37 0 C/5% CO 2 and then the transfection media is removed and replaced with 1 ml/well of regular growth media. On day 3 the cells are labeled with 3 H-myo-inositol. Briefly, the medium is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media (GIBCO BRL) are added/well with 0.25 pCi of 3 H-myo-inositol/well and the cells incubated for 16-18 hrs overnight at 37 0 C/5% CO 2 On Day 4, the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositolfree/serum free media 10 pM pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50 pl of 10 x ketanserin (ket) to final concentration of 10 p.M. The cells are then incubated for 30 min at 37°C. The cells are then washed with 0.5 ml PBS and 200 tl of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added to each well. The solution is kept on ice for 5-10 min (or until cells are lysed) and then neutralized by 200 pl of fresh/ice cold neutralization solution HCI). The lysate is then transferred into 1.5 ml Eppendorf tubes and 1 ml of chloroform/methanol is added/tube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AGI-X8 T anion exchange resin (100-200 mesh). First, the resin is washed with water at 1:1.25 and 0.9 ml of upper phase is loaded onto the column.

The column is then washed with 10 ml of 5 mM myo-inositol and 10 ml of 5 mM NamM Na-formate. The inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/I M ammonium formate. The columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsed twice with dd H20 and stored at 4 0 C in water.

Reference is made to Figure 1. In Figure 1, 293 cells were transfected with Gq protein containing a six amino acid deletion, "Gq(del)"; Gq protein fused to a Gi protein, "Gq(del)/Gi"; endogenous RUP32; and RUP32 with Gq(del) ("RUP32+Gq(del)/Gi").

The data indicate, based upon measuring IP 3 accumulation of RUP32 co-transfection of Gq(del)/Gi, that RUP32 does not endogenously couple to Gq protein. However when RUP32 was co-transfected with Gq(del)/Gi fusion protein, RUP32 was forced to couple to Gq protein. RUP27+Gq(del)/Gi evidence about a nine fold increase in IP 3 accumulation when compared to endogenous RUP32. These data demonstrate that the Gq(del)/Gi Fusion Construct is co-transfected with a GPCR and used to screen for agonists or inverse agonists.

Reference is made to Figure 2. In Figure 2, 293 cells were transfected with RUP35 and RUP36 receptor and compared to the control, pCMV. The data indicate that both and RUP36 receptor are endogenously, constitutively active. RUP35 evidences about a six fold increase in intracellular inositol phosphate accumulation when compared to pCMV and RUP36 evidences about a four fold increase when compared to pCMV.

Example Fusion Protein Preparation a. GPCR: Gs Fusion Construct The design of the constitutively activated GPCR-G protein fusion construct is I) accomplished as follows: both the 5' and 3' ends of the rat G protein Gsa (long form; SItoh, et al., PNAS (USA) 83, p 3 7 7 6 et seq., 1986) is engineered to include a Hindll NC AAGCTT-3') sequence thereon. Following confirmation of the correct sequence O (including the flanking HindIIIll sequences), the entire sequence is shuttled into C 10 pcDNA3.1(-) (Invitrogen, cat. no. V795-20) by subcloning using the HindIll restriction site of that vector. The correct orientation for the Gsa sequence is determined after subcloning into pcDNA3.1(-). The modified pcDNA3.1(-) containing the rat Gsa gene at HindIIIl sequence is then verified; this vector is then available as a "universal" Gsa protein vector. The pcDNA3.1(-) vector contains a variety of well-known restriction sites upstream of the Hindlll site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach is utilized to create other "universal" G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan is utilized. In some embodiments, the important criteria are that the sequence for the GPCR be upstream and in-frame with that of the G protein.

Spacers in the restriction sites between the G protein and the GPCR are optional. The sense and anti-sense primers included the restriction sites for Xbal and EcoRV, respectively, such that spacers (attributed to the restriction sites) exist between the G protein and the GPCR.

PCR is utilized to secure the respective receptor sequences for fusion within the Gsa universal vector disclosed above, using the following protocol for each: 100 ng cDNA for GPCR are added to separate tubes containing 2 pl of each primer (sense and antisense), 3 Il of 10 mM dNTPs, 10 jl of 10 x TaqPlus TM Precision buffer, 1 il of TaqPlus

TM

Precision polymerase (Stratagene: #600211), and 80 pl of water. Reaction

IUY

NC- temperatures and cycle times for the GPCR are as follows with cycle steps 2 through 4 Swere repeated 35 times: 94 0 C for 1 min; 94 0 C for 30 seconds; 62°C for 20 sec; 72 0 C 1 min 40 sec; and 72 0 C for 5 min. PCR products are run on a 1% agarose gel and then purified. The purified products are digested with Xbal and EcoRV and the desired inserts purified and ligated into the Gs universal vector at the respective restriction Ssites. The positive clones are isolated following transformation and determined by Ds restriction enzyme digestion. Expression using 293 cells is accomplished following the C protocol set forth infra. Each positive clone for GPCR-Gs Fusion Protein is sequenced O to verify correctness.

(N b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct The design of a Gq(del)/Gi fusion construct was accomplished as follows: the Nterminal six amino acids (amino acids 2 through having the sequence of TLESIM (SEQ. ID. NO.: 47) Gaq-subunit was deleted and the C-terminal five amino acids, having the sequence EYNLV (SEQ. ID. NO.: 48) was replaced with the corresponding amino acids of the Gui Protein, having the sequence DCGLF (SEQ. ID.

NO.: 49). This fusion construct was obtained by PCR using the following primers wherein nucleotides in lower caps are included as spacers: 5'-gatcAAGCTTCCATGGCGTGCTGCCTGAGCGAGG-3' (SEQ. ID. NO.: 50); and ATGGTG-3' (SEQ. ID. NO.: 51); and plasmid 63313 which contains the mouse Gaq-wild type version with a hemagglutinin tag as template.

TaqPlus® Precision DNA polymerase (Stratagene) was utilized for the amplification by the following cycles, with steps 2 through 4 repeated 35 times: 95°C for 2 min; for 20 sec; 56 0 C for 20 sec; 72 0 C for 2 min; and 72 0 C for 7 min. The PCR product is cloned into a pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit Biosystems). Inserts from a TOPO clone containing the sequence of the fusion construct are shuttled into the expression vector pcDNA3.1 at the Hindlll/BamHI site by a 2 step cloning process.

Example 6 Tissue Distribution of the Disclosed Human GPCRs: RT-PCR RT-PCR was applied to confirm the expression and to determine the tissue distribution of several novel human GPCRs. Oligonucleotides utilized were GPCR-specific and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) were utilized for the amplification in a 40 pl reaction according to the manufacturer's instructions. Twenty pl of the reaction is loaded on a agarose gel to analyze the RT-PCR products. Table 6, below, lists the receptors, the cycle conditions and the primers utilized, and also lists exemplary diseases/disorders linked to the receptors.

TABLE 6 Receptor Cycle 5'-Primer 3'-Primer DNA Tissue Identifier conditions (SEQ ID NO:) (SEQ ID NO:) Fragment Expression (bp) hRUP28 94 0 C for 5 min; GTCCTCACT CTGCGTCCAC 710 bp heart; kidney; 94 0 C for 30 sec; GGTGGCCAT CAGAGTCAC liver; lung; 58 0 C for 30 sec; GTACTCC (52) GTCTCC (53) pancreas 72°C for 1 min; 72 0 C for 7 min.

hRUP29 94°C for 5 min; CTTGGATGTT GTTTGTGGCT 690 bp leukocyte; ovary 94 0 C for 30 sec; TGGGCTGCCC AACGGCACAA 58°C for 30 sec; TTCTGC (54) AACACAATTC 72 0 C for 1 min; C 72°C for 7 min.

94 0 C for 2 min; CTGCTCACGG GTGGCCATGA 690 bp pancreas 94°C for 15 sec; TTGACCGCTA GCCACCCTGA 58°C for 20 sec; CACTGC (56) GCTCC (57) 72 0 C for I min; 72 0 C for hRUP31 95°C for 4 min; CTTCTTCTCC CCAAATCAGT 516 bp colon; lung; 0 C for I min; GACGTCAAGG GTGCAAATCG pancreas; 52 0 C for 30 sec; (58) (59) thymus; cerebral 72 0 C for I min; cortex; 72 0 C for 7 min. hippocampus; fat cells hRUP32 95 0 C for 4 min; TGAATGGGTC CAACGGTCTG 527 bp thymus 0 C for 1 min; CTGTGTGAAA ACAACCTCCT 52°C for 30 sec; (60) (61) 72 0 C for I min; 72 0 C for 7 min.

hRUP34 95 0 C for 4 min; TTGCTGTGAT CAGGAAGCCC 534 bp peripheral blood 0 C for 1 min; GTGGCATTTT ATAAAGGCAT leukocytes; 52°C for 30 sec; G (62) CAA (63) prostate; kidney 72°C for 1 min; 72°C for 7 min; 95 0 C for 4 min; ACATCACCTG CCAGCATCTT 557 bp thalamus for I min; CTTCCTGACC GATGCAGTGT 52°C for 30 sec; (64) 72°C for I min; 72 0 C for 7 min.

hRUP37 95 0 C for 4 min; CCATCTCCAA GCTGTTAAGA 517 bp testis; cerebral 0 C for I min; AATCCTCAGT GCGGACAGGA cortex; 52°C for 30 sec; C (66) AA (67) hippocampus 72°C for 1 min; 72 0 C for 7 min.

Cycles 2-4 repeated 35 times Diseases and disorders related to receptors located in these tissues or regions include, but are not limited to, cardiac disorders and diseases thrombosis, myocardial infarction; atherosclerosis; cardiomyopathies); kidney disease/disorders renal failure; renal tubular acidosis; renal glycosuria; nephrogenic diabetes insipidus; cystinuria; polycystic kidney disease); eosinophilia; leukocytosis; leukopenia; ovarian cancer; sexual dysfunction; polycystic ovarian syndrome; pancreatitis and pancreatic cancer; irritable bowel syndrome; colon cancer; Crohn's disease; ulcerative colitis; diverticulitis; Chronic Obstructive Pulmonary Disease (COPD); cystic fibrosis; pneumonia; pulmonary hypertension; tuberculosis and lung cancer; Parkinson's disease; movement disorders and ataxias; learning and memory disorders; eating disorders anorexia; bulimia, etc.); obesity; cancers; thymoma; myasthenia gravis; circulatory disorders; prostate cancer; prostatitis; kidney disease/disorders renal failure; renal tubular acidosis; renal glycosuria; nephrogenic diabetes insipidus; cystinuria; polycystic kidney disease); sensorimotor processing and arousal disorders; obsessivecompulsive disorders; testicular cancer; priapism; prostatitis; hernia; endocrine disorders; sexual dysfunction; allergies; depression; psychotic disorders; migraine; reflux; schizophrenia; ulcers; bronchospasm; epilepsy; prostatic hypertrophy; anxiety; rhinitis; angina; and glaucoma.

Accordingly, the methods of the present invention may also be useful in the diagnosis and/or treatment of these and other diseases and disorders.

Example 7 Protocol: Direct Identification of Inverse Agonists and Agonists A. 5 S]GTPyS Assay Although endogenous, constitutively active GPCRs have been used for the direct identification of candidate compounds as, inverse agonists, for reasons that are not altogether understood, intra-assay variation can become exacerbated. In some embodiments a GPCR Fusion Protein, as disclosed above, is also utilized with a nonendogenous, constitutively activated GPCR. When such a protein is used, intra-assay variation appears to be substantially stabilized, whereby an effective signal-to-noise ratio is obtained. This has the beneficial result of allowing for a more robust identification of candidate compounds. Thus, in some embodiments it is preferred that for direct identification, a GPCR Fusion Protein be used and that when utilized, the following assay protocols be utilized.

1. Membrane Preparation Membranes comprising the constitutively active orphan GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agonists or agonists are preferably prepared as follows: a. Materials "Membrane Scrape Buffer" is comprised of 20 mM HEPES and 10 mM EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20 mM HEPES, 100 mM NaCI, and 10 mM MgCI 2 SpH 7.4.

IND

Sb. Procedure O All materials are kept on ice throughout the procedure. Firstly, the media are aspirated 10 from a confluent monolayer of cells, followed by rinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml of Membrane Scrape Buffer are added to scrape cells; this will be followed by transfer of cellular extract into 50 ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4 0 Thereafter, the supernatant is aspirated and the pellet resuspended in 30 ml Membrane Wash Buffer followed by centrifugation at 20,000 rpm for 17 minutes at 4 0 C. The supernatant is aspirated and the pellet resuspended in Binding Buffer. The resuspended pellet is then homogenized using a Brinkman Polytron T M homogenizer (15-20 second bursts until the material is in suspension). This is referred to herein as "Membrane Protein".

2. Bradford Protein Assay Following the homogenization, protein concentration of the membranes is determined, for example, using the Bradford Protein Assay (protein is diluted to about 1.5 mg/ml, aliquoted and frozen for later use; when frozen, protocol for use is as follows: On the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polytron at about 12 x 1,000 rpm for about 5-10 seconds; for multiple preparations, the homogenizer is thoroughly cleaned between homogenization of different preparations).

a. Materials Binding Buffer (as discussed above); Bradford Dye Reagent; Bradford Protein Standard is utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).

b. Procedure Duplicate tubes are prepared, one including the membrane, and one as a control "blank". Each contains 800 l Binding Buffer. Thereafter, 10 pl of Bradford Protein Standard (1 mg/ml) is added to each tube, and 10 pl of membrane Protein are added to just one tube (not the blank). Thereafter, 200 pl of Bradford Dye Reagent are added to each tube, followed by vortexing. After five minutes, the tubes are re-vortexed and the material therein transferred to cuvettes. The cuvettes are read using a CECIL 3041 spectrophotometer, at wavelength 595.

3. Direct Identification Assay a. Materials GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat. no. G- 7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 pM GDP (final concentration of GDP in each well was 0.1 pM GDP); each well comprising a candidate compound, has a final volume of 200 pl consisting of 100 il GDP Buffer (final concentration, 0.1 uM GDP), 50 pl Membrane Protein in Binding Buffer, and pl 3 "S]GTPyS (0.6 nM) in Binding Buffer (2.5 pl ["S]GTPyS per 10 ml Binding Buffer).

b. Procedure Candidate compounds are preferably screened using a 96-well plate format (these are frozen at -80 0 Membrane Protein (or membranes with expression vector excluding the GPCR Fusion Protein, as control), are homogenized briefly until in suspension.

Protein concentration is determined using, for example, the Bradford Protein Assay set forth above. Membrane Protein (and controls) are then diluted to 0.25 mg/ml in Binding Buffer (final assay concentration, 12.5 gg/well). Thereafter, 100 pl GDP Buffer are added to each well of a Wallac Scintistrip T (Wallac). A 5 l1 pin-tool is used to transfer 5 p1 of a candidate compound into such well 5 pl in total assay volume of 200 pl is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 pM). Again, to avoid contamination, after each transfer step the pin tool is rinsed in three reservoirs comprising water (1 ethanol (1 x) and water (2 x)--excess liquid is shaken from the tool after each rinse and the tool is dried with paper and Kim wipes. Thereafter, 50 pl of Membrane Protein is added to each well (a control well comprising membranes without the GPCR Fusion Protein is also utilized), and pre-incubated for 5-10 minutes at room temperature. Thereafter, 50 pl of 3 S]GTPyS (0.6 nM) in Binding Buffer are added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates are covered with foil). The assay is stopped by spinning the plates at 4000 rpm for minutes at 22 0 C. The plates are then aspirated with an 8 channel manifold and sealed with plate covers. The plates are read on a Wallac 1450 using setting "Prot. #37" (as per manufacturer's instructions).

B. Cyclic AMP Assay Another assay approach to directly identify candidate compound is accomplished utilizing a cyclase-based assay. In addition to direct identification, this assay approach is utilized as an independent approach to provide confirmation of the results from the 35 S]GTPyS approach as set forth above.

A modified Flash Plate TM Adenylyl Cyclase kit (New England Nuclear; Cat. No.

SMP004A) is preferably utilized for direct identification of candidate compounds as inverse agonists and agonists to GPCRs in accordance with the following protocol.

Transfected cells are harvested approximately three days after transfection. Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCI 2 Homogenization is performed on ice using a Brinkman Polytron TM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 x g for 15 minutes at 4 0 C. The resulting pellet is resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for seconds, followed by centrifugation at 49,000 x g for 15 minutes at 4 0 C. The resulting pellet is stored at -80 0 C until utilized. On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes are placed on ice until use).

cAMP standards and Detection Buffer (comprising 2 pCi of tracer [1251 cAMP (100 gil)] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contain mM HEPES, pH 7.4, 10 mM MgCI 2 20 mM phosphocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 [M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer is stored on ice until utilized.

Candidate compounds identified as per above (if frozen, thawed at room temperature) are added, preferably, to 96-well plate wells (3 p/well; 12 pM final assay concentration), together with 40 pl Membrane Protein (30 jig/well) and 50 1l of Assay Buffer. This admixture are incubated for 30 minutes at room temperature, with gentle shaking.

Following the incubation, 100 p1 of Detection Buffer are added to each well, followed by incubation for 2-24 hours. Plates are then counted in a Wallac MicroBetaTM plate reader using "Prot. #31" (as per manufacturer instructions).

C. Melanophore Screening Assay A method for identifying candidate agonists or inverse agonists for a GPCR is preformed by introducing tests cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus and expressing an exogenous clone coding for the GCPR. A stimulant, light, sets an initial state of pigment disposition wherein the pigment is aggregated within the test cells if activation of the GPCR induces pigment dispersion. However, stimulating the cell with a stimulant to set an initial state of pigment disposition wherein the pigment is dispersed if activation of the GPCR induces pigment aggregation. The tests cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.

Materials and methods are followed according to the disclosure of U.S. Pat. No.

5,462,856 and U.S. Pat. No. 6,051,386, each of which is incorporated by reference.

Although a variety of expression vectors are available to those in the art, for purposes of utilization for both the endogenous and non-endogenous human GPCRs, in some embodiments it is preferred that the vector utilized be pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

References cited throughout this patent document, including co-pending and related patent applications, unless otherwise indicated, are fully incorporated herein by reference. Modifications and extension of the disclosed inventions that are within the purview of the skilled artisan are encompassed within the above disclosure and the claims that follow.

Claims (27)

1. 2. A method of screening candidate compounds for the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal comprising the steps of: contacting a candidate compound with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor (GPCR) or GPCR fusion protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; (ii) the amino acid sequence of an endogenous or non-endogenous constitutively active variant of (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 16; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of a GPCR fusion protein comprising any one of(i) to (iv) and a G protein; and measuring the ability of the candidate compound to inhibit or stimulate the GPCR or GPCR fusion protein to thereby identify a candidate compound as a compound which can inhibit or stimulate the corresponding endogenous GPCR in the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal.
2. 3. A method of isolating a pharmaceutical agent for the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal, said method comprising using a G protein-coupled receptor (GPCR) or GPCR fusion protein in a method of screening candidate compounds to thereby isolate one or more compounds for the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal by virtue of the ability of said compound(s) to modulate activity of the receptor, said GPCR or GPCR fusion protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G protein- coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of a GPCR fusion protein comprising any one of to and a G protein.
3. 4. A method of isolating a pharmaceutical agent for the treatment of a S e disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal, said method comprising the steps of: contacting candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising a G protein-coupled receptor (GPCR) or GPCR fusion I) protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; CN (ii) the amino acid sequence of an endogenous or non-endogenous 0 constitutively active variant of(i); CN 10 (iii) the amino acid sequence of (ii) having at least about 80% identity to SEQ ID NO: 16; (iv) the amino acid sequence of an endogenous or non-endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: the amino acid sequence of a GPCR fusion protein comprising any one of(i) to (iv) and a G protein; and measuring the ability of the candidate compounds to inhibit or stimulate the GPCR or GPCR fusion protein to thereby identify a candidate compound as a compound which can inhibit or stimulate the corresponding endogenous GPCR in the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal and isolating a compound which inhibits or stimulates the GPCR or GPCR fusion protein as a pharmaceutical agent in the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal.
4. 5. A method of formulating a pharmaceutical composition for the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal, said method comprising performing the method of claim 3 or 4 to thereby isolate a pharmaceutical agent in the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal and formulating the isolated pharmaceutical agent with a pharmaceutically-acceptable carrier.
5. 6. The method according to any one of claims 1 to 5, wherein the GPCR or GPCR fusion protein comprises a sequence selected from the group consisting of: a) the sequence of a GPCR encoded by nucleic acid amplifiable by polymerase chain reaction (PCR) on a human cDNA sample using sequence specific primers selected from SEQ ID NO: 41 and SEQ ID NO: 42; or (ii) SEQ ID NO: 64 and SEQ ID NO: b) the sequence of a GPCR comprising SEQ ID NO: 16 and having a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 16; and the sequence of a GPCR fusion protein comprising or and a G protein.
6. 7. The method of claim 6, wherein the mutated residue has been mutated to an alanine residue, a histidine residue, an arginine residue or a lysine residue.
7. 8. The method according to any one of claims 1 to 7 wherein the GPCR comprises the amino acid sequence set forth in SEQ ID NO: 16.
8. 9. The method according to any one of claims 1 to 8, wherein the GPCR fusion protein comprises Gq. The method according to any one of claims 1 to 4 comprising contacting the one or more candidate compounds with a recombinant eukaryotic host cell or membrane thereof comprising the GPCR or GPCR fusion protein.
9. 11. The method of claim 10, wherein the eukaryotic host cell is a mammalian host cell.
10. 12. The method of claim 11, wherein the mammalian host cell is a 293 host cell, a 293T host cell, or a COS-7 host cell.
11. 13. The method of claim 10, wherein the eukaryotic host cell is a yeast host cell.
12. 14. The method of claim 10, wherein the eukaryotic host cell is a melanophore host cell. The method according to any one of claims 1 to 4 comprising detecting IP 3 and/or measuring an increase or decrease in intracellular IP 3
13. 16. The method according to claim 1 or 2 comprising identifying a candidate compound as an agonist of the GPCR or GPCR fusion protein.
14. 17. The method according to claim 3 or 4 comprising isolating the pharmaceutical agent as an agonist of the GPCR or GPCR fusion protein.
15. 18. The method according to claim 1 or 2 comprising identifying a candidate compound as an inverse agonist of the GPCR or GPCR fusion protein.
16. 19. The method according to claim 3 or 4 comprising isolating the pharmaceutical agent as an inverse agonist of the GPCR or GPCR fusion protein. The method according to any one of claims 16 to 19 further comprising formulating said agonist or inverse agonist as a pharmaceutical composition with a pharmaceutically-acceptable carrier for the treatment of a disease or disorder related to the thalamus and/or sensorimotor processing and/or arousal.
17. 21. The method of claim 20, wherein the pharmaceutical composition is formulated for a disease or disorder related to the thalamus.
18. 22. The method of claim 20, wherein the pharmaceutical composition is formulated for a disorder of sensorimotor processing and/or arousal.
19. 23. An agent that interacts with an isolated or recombinant GPCR or GPCR fusion protein when isolated by the method according to claim 3 or 4 or when isolated by the method according to any one of claims 6 to 22 as appended to claim 3 or 4, wherein said GPCR or GPCR fusion protein comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G protein- coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of a GPCR fusion protein comprising any one of to and a G protein.
20. 24. An agent that interacts with an isolated or recombinant GPCR or GPCR fusion protein when formulated by the method according to claim 5 or when formulated by the method according to any one of claims 6 to 22 as appended to claim 5, wherein said GPCR or GPCR fusion protein comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of(a); the amino acid sequence of having at least about 80% identity to SEQ 1D NO: 16; the amino acid sequence of an endogenous or non-endogenous G protein- coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ 1D NO: 15; and IN the amino acid sequence of a GPCR fusion protein comprising any one of to and a G protein. The agent according to claim 23 or 24, wherein said agent interacts with an isolated or recombinant GPCR or GPCR fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 16.
21. 26. The agent according to any one of claims 23 to 25, wherein said agent inhibits or stimulates an endogenous or non-endogenous GPCR or GPCR fusion protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; the amino acid sequence of an endogenous or non-endogenous G protein- coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement of SEQ ID NO: 15; and the amino acid sequence of a GPCR fusion protein comprising any one of to and a G protein.
22. 27. The agent according to claim 26, wherein said agent is an inverse agonist of the GPCR or GPCR fusion protein.
23. 28. The agent according to claim 26, wherein said agent is an agonist of the GPCR or GPCR fusion protein.
24. 29. The agent according to claim 27 or 28, wherein the agent is an agonist or inverse agonist of the corresponding endogenous GPCR in the thalamus. Use of the agent according to any one of claims 23 to 29 to increase or decrease activity of a GPCR when administered to a subject, wherein said GPCR comprises an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 16; the amino acid sequence of an endogenous constitutively active variant of the amino acid sequence of having at least about 80% identity to SEQ ID NO: 16; and the amino acid sequence of an endogenous G protein-coupled receptor encoded by a nucleic acid that hybridizes under stringent conditions to the complement ofSEQ ID NO:
25. 31. Use of the agent according to any one of claims 23 to 30 for treatment of a disease or disorder related to the thalamus or in the preparation of a medicament for treatment of a disease or disorder related to the thalamus.
26. 32. Use of the agent according to any one of claims 23 to 31 for treatment of sensorimotor processing and/or arousal or in the preparation of a medicament for treatment of sensorimotor processing and/or arousal.
27. 33. The method according to any one of claims 1 to 22 substantially as hereinbefore described with reference to the examples and/or drawings. Dated this TWELFTH day of SEPTEMBER 2007 Arena Pharmaceuticals, Inc. By Patent Attorneys for the Applicant: F B RICE CO