AU2007202241A1

AU2007202241A1 - Human Orphan G protein-coupled receptor hRUP3

Info

Publication number: AU2007202241A1
Application number: AU2007202241A
Authority: AU
Inventors: Ruoping Chen; James Leonard
Original assignee: Arena Pharmaceuticals Inc
Current assignee: Arena Pharmaceuticals Inc
Priority date: 1998-11-20
Filing date: 2007-05-11
Publication date: 2007-06-07
Also published as: AU2007202155A1; AU2009202228A1; AU2009202228B2; AU2007202139A1; AU2007202155B2; AU2007202139B8; AU2007202121A1; AU2007202139B2

Description

AUSTRALIA

Patents Act 1990 ARENA PHARMACEUTICALS, INC.

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Human Orphan G protein-Coupled receptor hR UP3 The following statement is a full description of this invention including the best method of performing it known to us:- 2 C HUMAN ORPHAN G PROTEIN-COUPLED RECEPTOR HRUP3 RELATED APPLICATION DATA This application is a divisional application of Australian Patent Application No.

_2004202147 filed May 12, 2004 which is a divisional application of Australian Patent Application No. 37904/00 (Acceptance No. 770056) the contents of which are incorporated herein in their entirety by reference. Australian Patent Application No.

S37904/00 (Acceptance No. 770056) claims priority benefit of each of the following applications, all filed with the United States Patent and Trademark Office via U. S.

Express Mail on the indicated filing dates: U. S. Provisional Number 60/121,852, filed February 26, 1999; U. S. Provisional Number 60/109,213, filed November 20,1998; U.

S. Provisional Number 60/120,416, filed February 16, 1999; U. S. Provisional Number 60/123,946, filed March 12,1999; U. S. Provisional Number 60/123,949, filed March 12, 1999; U. S. Provisional Number 60/136,436, filed May 28, 1999; U. S. Provisional Number 60/136,439, filed May 28,1999; U. S. Provisional Number 60/136,567, filed May 28, 1999; U. S. Provisional Number 60/137,127, filed May 28, 1999; U. S.

Provisional Number 60/137,131, filed May 28, 1999; U. S. Provisional Number 60/141,448, filed June 29, 1999; U. S. Provisional Number 60/136,437, filed May 28, 1999; U. S. Provisional Number 60/156,653, filed September 29, 1999; U. S.

Provisional Number 60/156, 633, filed September 29, 1999; U. S. Provisional Number 60/156,555, filed September 29, 1999; U. S. Provisional Number 60/156,634, filed September 29, 1999; U. S. Provisional Number 60/157,280, filed October 1, 1999; U.S.

Provisional Number 60/157,281, filed October 1,1999; U. S. Provisional Number 60/157,282, filed October 1, 1999; U. S. Provisional Number 60/157,293, filed October 1, 1999; U. S. Provisional Number 60/157,294, filed October 1,1999; U.S.

Serial Number 09/417,044 filed October 12, 1999; and U.S. Serial Number 09/416,760 filed October 12, 1999. This patent document is related to U. S. Serial Number 09/170,496 filed October 13,1998, and U. S. Serial Number 09/416,760 (Woodcock Washburn Kurtz Mackiewicz Norris, LLP docket number AREN-0054) filed on October 12,1999 (via U. S. Express Mail) both being incorporated herein by reference.

This patent document also is related to U. S. Serial No. 09/364,425; filed July 30,1999, which is incorporated by reference in its entirety. Each of the foregoing applications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to endogenous, orphan, human G protein-coupled receptors ("GPCRs").

BACKGROUND OF THE INVENTION General This specification contains nucleotide and amino acid sequence information prepared using PatentIn Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

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.

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.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e.

one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.

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.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only.

Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151; Nucleic Acid Hybridization: A Practical Approach D. Hames S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Animal Cell Culture: Practical Approach, Third Edition (John R.W. Masters, ed., 2000), ISBN 0199637970, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; 9 Perbal, A Practical Guide to Molecular Cloning (1984); and Methods In Enzymology Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

It is intended that each of the patents, applications, and printed publications mentioned in this patent document be hereby incorporated by reference in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention and the claims that follow.

Description of the related art 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 or GPCRs) class. It is estimated that there are some 100, 000 genes within the human genome, and of these, approximately 2% or 2,000 genes, 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, 60% of all prescription pharmaceuticals have been developed. This distinction is not merely semantic, particularly in the case of GPCRs. Thus, the orphan GPCRs are to the pharmaceutical industry what gold was to California in the late 19th century-an opportunity to drive growth, expansion, enhancement and development.

GPCRs share a common structural motif. All these receptors have 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 10 transmembrane-2 etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, 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 15 by strands of amino acids between transmembrane-I and transmembrane-2, transmembrane-3 and transmembrane-4, and and transmembrane-6 on the interior, or "intracellular" side, of the ri 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 c "activation" of the receptor), there is a change in the conformation of the intracellular O 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. See, Kenakin, 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. 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 produce 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 an endogenous ligand or a compound such as a drug.

SUMMARY OF THE INVENTION Disclosed herein are recombinant human endogenous orphan G protein-coupled receptors and nucleic acid encoding same.

In one embodiment, the presently claimed invention relates to a recombinant human G protein-coupled receptor (GPCR) that is preferably expressed in accordance with data provided herein and more preferably expressed selectively in accordance with the data provided in TABLE C and/or TABLE D, and isolated nucleic acid encoding same. The present invention further extends to nucleic acids encoding constitutively-activated variants of the exemplified GPCR polypeptides, fusion proteins comprising the GPCR polypeptides "GPCR fusion proteins") and a G protein, and nucleic acid encoding the constitutively-activated variants and GPCR fusion proteins.

A further embodiment of the present invention provides an isolated polynucleotide encoding a GPCR fusion protein, wherein said polynucleotide comprises a nucleotide sequence encoding the GPCR and nucleic acid encoding a G protein. In the construction of such fusions, the G protein may be a cognate or non-cognate G protein with respect to the GPCR, however it may be preferred to use a cognate G protein.

A further embodiment of the present invention provides a vector comprising a polynucleotide encoding a human GPCR or constitutively-activated variant thereof or GPCR fusion protein comprising said human GPCR or said variant. Preferably, the vector is an expression vector wherein the polynucleotide encoding the human GPCR or variant or GPCR fusion protein is operably linked to a promoter.

A further embodiment of the present invention provides a recombinant host cell comprising the vector described in the preceding paragraph.

A further embodiment of the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises a recombinant human GPCR polypeptide or constitutively-activated variant thereof or GPCR fusion protein comprising said human GPCR or said variant.

A further embodiment of the present invention provides a method of producing a human GPCR or constitutively-activated variant thereof or a GPCR fusion protein comprising the steps of: transfecting an expression vector comprising nucleic acid encoding the GPCR or variant or GPCR fusion protein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human GPCR or variant or GPCR fusion protein from the expression vector.

Preferably, the method of producing a human GPCR or GPCR fusion protein further comprises obtaining the transfected host cell. In one embodiment, the host cell is a melanophore. In another embodiment, the host cell is a mammalian cell, a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell.

A further embodiment of the present invention provides an isolated or recombinant human G protein-coupled receptor (GPCR) polypeptide or a constitutively-activated variant thereof comprising a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of said GPCR.

A further embodiment of the invention provides an isolatfed or recombinant GPCR fusion protein comprising an amino acid sequence of a human GPCR or constitutivelyactivated variant thereof, preferably further comprising a G protein. Preferably, the G protein is coupled to a C-terminal portion of the GPCR or a constitutively-activated variant thereof. The GPCR or variant and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the human GPCR or a constitutively-activated variant thereof or an isolated membrane comprising said human GPCR polypeptide or said variant; and measuring the ability of the compound to modulate functionality of the G protein coupled receptor polypeptide or variant wherein modulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Unless specifically stated otherwise, or the context requires otherwise, the term "functionality" of a GPCR shall be taken to mean expression and/or activity of the GPCR or a constitutively-activated variant thereof. By "expression" is meant the level or rate of transcription and/or translation of mRNA encoding the GPCR to produce an active protein. By "activity" is meant any activity of a GPCR protein or a constitutively-activated variant thereof, including a function mediated by binding of GPCR to a ligand such as, for example, its cognate ligand, and any signalling effected thereby.

Preferably, the method according to this embodiment further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector comprising nucleic acid encoding the human GPCR or constitutively-activated variant thereof.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor (GPCR) comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses a GPCR fusion protein comprising the human GPCR or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to modulate functionality of the GPCR polypeptide or variant portion of said GPCR fusion protein wherein modulation of said functionality indicates that the candidate compound is a modulator of the GPCR polypeptide.

Preferably, the method of identifying a GPCR modulator further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR fusion protein. In a particularly preferred embodiment, the GPCR fusion protein comprises the amino acid sequence of the GPCR or variant thereof and the amino acid sequence of a G protein.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR fusion protein or an isolated membrane comprising said GPCR fusion protein; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate functionality of the G protein-coupled receptor polypeptide portion of said GPCR fusion protein wherein modulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

The screening methods supra are particularly useful for identifying compounds that modulate signal transduction mediated by the GPCR in a cell, such as, for example, in the diagnosis or treatment of a disease or disorder associated with the tissue(s) in which the GPCR is expressed. In one embodiment, expression of the GPCR may be aberrant or dysfunctional compared to expression in the same or corresponding tissue in a healthy or normal subject a subject not having the disease or disorder.

Accordingly, the terms "expression" and "activity" of a stated GPCR of the present I invention in the context of a disorder or disease shall be taken to include aberrant or N, dysfunctional expression and/or activity of the GPCR relative to a non-diseased tissue or cell or a subject not having the disease or disorder, but not mean exclusively such Saberrant or dysfunctional expression.

A further embodiment of the present invention provides a process for identifying a compound that modulates signal transduction mediated by a G-protein coupled receptor in one or more tissues in which the GPCR is expressed, said process comprising performing a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR fusion protein or an isolated membrane comprising said GPCR fusion protein; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide portion of said GPCR fusion protein wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

In a particularly preferred embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of a GPCR of the present invention; optionally, determining the structure of the compound; and providing the compound or modulator or the name or structure of the compound.

In an alternative embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of a GPCR of the present invention; optionally, determining.the structure of the compound; optionally, providing the name or structure of the compound; and producing or synthesizing the compound.

In a related embodiment, the present invention provides a process for modulating the activity and/or expression of a GPCR of the present invention said process comprising performing the screening method supra to thereby identify a modulator of the GPCR and then contacting a GPCR with the identified modulator or administering the identified modulator to a subject under conditions sufficient to modulate the activity and/or expression of the GPCR.

Preferred cells for use in the screening assays over express a GPCR of the present invention by virtue of having been stably transformed or transiently transfected with a nucleic acid comprising a GPCR-encoding gene of the invention. For example, 293 cells that have been stably transformed or transiently transfected with nucleic acid comprising said GPCR-encoding gene are particularly suitable for this purpose. This embodiment of the present invention clearly encompasses a preferred form comprising obtaining or producing the transformed or transfected cell.

Any class of candidate modulator described herein can be tested in the inventive screening assay.

In one embodiment, the test compound is a small molecule, such as, for example, a modulator of a receptor that shares some structural homology with a receptor of the present invention.

In an alternative embodiment, the test compound is an antibody that binds to a GPCR protein of the present invention.

Without limiting the invention, antibodies and small molecules are typically capable of modulating the activity of a target protein.

In a further embodiment, the test compound comprises siRNA or shRNA comprising a nucleotide sequence derived from the nucleotide sequence of a GPCR-encoding gene of the present invention according to standard procedures.

In an alternative embodiment, the test compound comprises antisense RNA.

As will be known to the skilled artisan, siRNA, shRNA and antisense RNA are generally capable of modulating expression of a target gene.

In an alternative embodiment, the test compound comprises a peptide of at least about 4-10 amino acids in length derived from the amino acid sequence of the GPCR protein of the present invention.

Preferably, the signal transduction mediated by a G-protein coupled receptor in the tissues(s) modulates a downstream physiological or biochemical parameter in a subject such as involved in the development or aetiology of disease or a medical condition. Accordingly, the present invention clearly contemplates prophylactic and/or therapeutic treatments wherein the level of GPCR expression and/or activity is modulated in a subject in need thereof.

A further embodiment of the present invention provides a method of treating a disorder or disease associated with expression and/or activity of a GPCR of the present invention said method comprising administering a modulator of said GPCR to a subject in need thereof for a time and under conditions sufficient to modulate GPCR expression and/or activity in said subject.

Preferably, the method comprises administering an amount of an agent that reduces GPCR activity or expression to the subject sufficient to reduce the level or activity of a GPCR protein or mRNA encoding a GPCR protein in a cell of the subject.

Accordingly, the antagonist may comprise nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, or nucleic acid that forms a triple helical structure, capable of reducing GPCR expression in a cell of the individual. Antibodies, peptides or small molecules that bind a GPCR and inhibit its activity are also useful in this context. As with the other embodiments described herein, it is particularly preferred to use an agent that reduces GPCR expression comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecule, or alternatively, an antibody that binds to a GPCR protein thereby inhibiting its activity. Other antagonists, dominant negative mutants, small molecules etc are not to be excluded.

Alternatively, the method comprises administering an amount of an agent that enhances or agonizes GPCR activity or expression to the subject sufficient to enhance the level or activity of a GPCR protein or mRNA encoding a GPCR protein in a cell of the subject. Accordingly, the agonist may comprise a peptide or small molecule agonist.

Such agents are able to be administered by injection or preferably, by non-invasive routes such as by oral ingestion, intranasal inhalation, intra-aural formulation, or suppository.

A further embodiment of the present invention provides for the use of a composition that modulates GPCR expression and/or activity in the preparation of one or more medicaments for treating a disease or disorder associated with expression and/or activity of a GPCR in a subject.

A further embodiment of the present invention provides a commercial package comprising: a composition that modulates GPCR expression and/or activity in a subject; and instructions for use of the composition and agent for treating a disease or disorder associated with expression and/or activity of the GPCR in a subject.

Particularly preferred embodiments of the present invention in accordance with the exemplified subject matter are described in the following paragraphs: hRUP3 In one embodiment, the presently claimed invention relates to a recombinant human G protein-coupled receptor (GPCR) designated hRUP3 that is selectively expressed in pancreatic tissue, and isolated nucleic acid encoding same. The present invention further extends to encoding ligand-independent versions of the hRUP3 polypeptide (also referred to herein as ligand-independent active versions of the hRUP3 polypeptide), GPCR fusion proteins comprising the hRUP3 polypeptide and a G protein, Gs, a cognate G protein that couples with RUP3) and nucleic acid encoding the ligand-independent versions and GPCR fusion proteins.

For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 7 is the sequence of a hRUP3-encoding cDNA. The sequence set forth in SEQ ID NO: 8 is the sequence of a hRUP3 polypeptide.

Accordingly, one embodiment of the present invention provides an isolated polynucleotide encoding a GPCR polypeptide designated hRUP3, wherein said polynucleotide comprises a nucleic acid that is amplifiable by polymerase chain reaction (PCR) on a human sample using sequence specific primers SEQ ID NO: 61 and SEQ ID NO: 62. Alternatively, or in addition, the present invention provides an isolated polynucleotide encoding a GPCR polypeptide designated hRUP3, wherein said polynucleotide comprises the nucleotide sequence of a polynucleotide that is amplifiable by PCR using one or more primers selected from primers comprising SEQ ID NO: 61 and/or SEQ ID NO: 62. Preferably, the isolated polynucleotide encodes a ligand-independent version of a human GPCR polypeptide comprising SEQ ID NO: 8.

in one example, the polynucleotide encodes a non-endogenous GPCR polypeptide. In one example, the polynucleotide encodes a ligand-independent version of a human GPCR other than a human GPCR consisting of SEQ ID NO: 8.

In one embodiment, the present invention provides a polynucleotide comprising a sequence selected from the group consisting of: a sequence encoding a ligand-independent version of a human GPCR polypeptide that comprises substantially the same sequence as SEQ ID NO: 8 and having a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 8; and a sequence encoding a variant of SEQ ID NO: 8 and comprising a mutation positioned at a codon encoding an amino acid 16 amino acid residues N-terminal from the codon encoding the conserved proline residue within the TM6 domain of SEQ ID NO: 8.

Preferably, the present invention provides a polynucleotide encoding a GPCR that comprises a mutation that introduces a lysine residue into the sequence of the encoded human G protein-receptor polypeptide, wherein said mutation is positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 8. For example, the mutation results in the substitution of leucine at position 224 of SEQ ID NO: 8 for lysine.

It will be apparent from the description herein that the isolated polynucleotide encoding the human GPCR designated hRUP3 is preferably obtained by a process comprising performing PCR on a sample of human genomic DNA or by a process comprising performing RT-PCR on a sample of mRNA obtained from human pancreas tissue. As shown in Figure 3, the isolated polynucleotide encoding the human GPCR designated hRUP3 is expressed endogenously by human pancreas, as determined using a process that comprises performing RT-PCR. Based on the foregoing, the present invention also provides an isolated polynucleotide encoding the GPCR designated hRUP3 when amplified by PCR, using one or more primers selected from primers comprising SEQ ID NO: 61 and/or SEQ ID NO: 62. For example, the isolated polynucleotide is isolated from pancreas tissue, human pancreas tissue.

A further embodiment of the present invention provides an isolated polynucleotide encoding a GPCR fusion protein, wherein said polynucleotide comprises a nucleotide sequence encoding the human GPCR designated hRUP3 or a ligand-independent version thereof. For example, the polynucleotide encoding the GPCR fusion protein further comprises a nucleic acid encoding a G protein. Preferably, the G protein is coupled to a C-terminal portion of hRUP3 or a ligand-independent version thereof. The hRUP3 or ligand-independent version thereof and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

A further embodiment of the present invention provides a vector comprising a polynucleotide encoding the human GPCR designated hRUP3 or a ligand-independent version thereof. Preferably, the vector is an expression vector, wherein the polynucleotide encoding the human GPCR designated hRUP3 or a ligand-independent version thereof is operably linked to a promoter.

A further embodiment of the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises a human GPCR polypeptide designated hRUP3 or a ligand-independent version thereof.

A further embodiment of the present invention provides a method of producing a GPCR, or a GPCR fusion protein comprising the steps of: transfecting an expression vector comprising nucleic acid encoding hRUP3 or a ligand-independent version thereof or GPCR fusion protein comprising said hRUP3 or said ligand-independent version thereof into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human GPCR designated hRUP3 or a ligand-independent version thereof or a GPCR fusion protein from the expression vector.

The present invention also provides a method of producing a G protein-coupled receptor or GPCR fusion protein comprising: performing PCR on human RNA or DNA using one or more primers selected from primers comprising SEQ ID NO: 61 and/or SEQ ID NO: 62; isolating DNA amplified using said primers; and expressing said DNA to produce a G protein-coupled receptor polypeptide or GPCR fusion protein comprising substantially the same sequence as SEQ ID NO: 8.

Suitable sources of RNA or DNA will be apparent to the skilled artisan. For example, the DNA is genomic DNA or the RNA is from pancreas tissue.

Preferably, the PCR is performed using sequence specific primers SEQ ID NO: 61 and SEQ ID NO: 62.

In one embodiment, the step of expressing the DNA to produce a GPCR polypeptide or GPCR fusion protein comprises introducing the DNA into a host cell for a time and under conditions sufficient for expression to occur.

Preferably, the method of producing a human GPCR or GPCR fusion protein described supra further comprises obtaining the transfected host cell. In one embodiment, the host cell is a eukaryotic cell, a mammalian cell. For example, the host cell is a melanophore. In another embodiment, the host cell is a pancreatic cell. In another embodiment, the host cell is a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell. In another embodiment, the host cell is a yeast cell.

Preferably, the method of producing a human GPCR or GPCR fusion protein described supra further comprises modifying the GPCR to thereby produce a non-endogenous ligand-independent version of the GPCR. For example, the method comprises introducing a mutation at a position 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of the GPCR to a residue other than leucine, or introducing a mutation at an equivalent position in the DNA encoding the GPCR so as to encode a ligand-independent version of SEQ ID NO: 8 wherein the amino acid positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of the GPCR is other than leucine. For example, the mutation is the substitution of leucine at position 224 of SEQ ID NO: 8 for lysine.

A further embodiment of the present invention provides an isolated or recombinant human GPCR designated hRUP3, wherein said GPCR comprises an amino acid sequence encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample using sequence specific primers SEQ ID NO: 61 and SEQ ID NO: 62. Alternatively, or in addition, the present invention provides an isolated or recombinant GPCR polypeptide designated hRUP3, wherein said polypeptide comprises an amino acid sequence encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using one or more primers selected from primers comprising SEQ ID NO: 61 and/or SEQ ID NO: 62. In one example, the present invention provides an isolated or recombinant ligand-independent version of a human GPCR polypeptide designated hRUP3. Preferably, the isolated or recombinant ligand-independent version of a human GPCR polypeptide is a non-endogenous receptor polypeptide. For example, the polypeptide comprising SEQ ID NO: 8. In one example, the human GPCR is a ligand independent version of a human GPCR other than a GPCR comprising SEQ ID NO: 8.

In one embodiment, the isolated or recombinant ligand-independent version of a human G protein-coupled receptor polypeptide comprises a mutation at a position 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 8 to a residue other than leucine. Preferably, the mutation is the substitution of leucine at position 224 of SEQ ID NO: 8 for lysine.

In one embodiment, the isolated or recombinant ligand-independent version of a human GPCR designated hRUP3 polypeptide is the non-endogenous version of a receptor in the pancreas that modulates the concentration of blood glucose and/or glucagon and/or insulin in the blood.

A further embodiment of the invention provides an isolated or recombinant GPCR fusion protein comprising an amino acid sequence of the human GPCR designated hRUP3 or a ligand-independent version thereof, preferably further comprising a G protein, Gs. Preferably, the G protein coupled to a C-terminal portion of hRUP3 or a constitutively-activated variant thereof. The hRUP3 or ligand-independent version thereof and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

The present invention also provides a method of identifying a modulator of a GPCR comprising the steps of: contacting a candidate compound with a recombinant host cell or an isolated membrane comprising a GPCR which is an endogenous or nonendogenous version of a GPCR comprising or having SEQ ID NO: 8; and measuring the ability of the compound to inhibit or stimulate functionality of the GPCR, wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of a G protein-coupled receptor.

A further embodiment of the present invention provides a method of identifying a modulator of a GPCR comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the human GPCR polypeptide designated hRUP3 or a ligand-independent version thereof or an isolated membrane comprising said human GPCR polypeptide or said ligand-independent version thereof; and measuring the ability of the compound to inhibit or stimulate functionality of the GPCR polypeptide or ligand-independent version thereof wherein inhibition or stimulation of functionality indicates that the candidate compound is a modulator of a GPCR polypeptide.

Preferably, the cell expresses a ligand-independent version of the human GPCR designated hRUP3 or the membrane comprises a ligand-independent version of the human GPCR designated hRUP3.

In one embodiment, the candidate compound is a candidate small molecule compound.

Lzu Preferably, the method according to this embodiment further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector comprising nucleic acid encoding the human GPCR designated hRUP3 or a ligandindependent version thereof.

A further embodiment of the present invention provides a method of identifying a C modulator of a GPCR comprising the steps of: N contacting a candidate compound with a recombinant host cell that expresses a GPCR fusion protein comprising the human GPCR designated hRUP3 or a ligand-independent version thereof or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to inhibit or stimulate functionality of the GPCR polypeptide portion of said GPCR fusion protein wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the GPCR polypeptide.

Preferably, the candidate compound is a candidate small molecule compound.

Preferably, the method of identifying a GPCR modulator further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR fusion protein. In one embodiment, the host cell is a eukaryotic cell, a mammalian cell. For example, the host cell is a melanophore.

In another embodiment, the host cell is a pancreatic cell. In another embodiment, the host cell is a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell. In another embodiment, the host cell is a yeast cell.

In one embodiment, a method for identifying a modulator of a GPCR as described herein according to any embodiment additionally comprises isolating a candidate compound that modulates functionality of the GPCR. In one example, the candidate compound is an agonist or a partial agonist of a ligand-independent version of a human GPCR designated hRUP7 or a GPCR fusion protein comprising same. In another example, the candidate compound is an inverse-agonist of a ligand-independent version of a human GPCR designated hRUP7 or a GPCR fusion protein comprising same.

Preferably, a method for identifying a modulator of a GPCR as described herein according to any embodiment additionally comprises formulating a candidate compound that modulates functionality of a human GPCR designated hRUP7 or a ligand-independent version thereof or a GPCR fusion protein comprising same as a pharmaceutical in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a process comprising: performing a method as described herein according to any embodiment to thereby identify or isolate a candidate compound that modulates functionality of a human GPCR polypeptide or GPCR fusion protein; optionally, determining the structure of the compound; and providing the compound or modulator or the name or structure of the compound.

The present invention also provides a process for producing or synthesizing a compound that modulates a GPCR comprising: performing a method as described herein according to any embodiment to thereby identify or isolate a candidate compound that modulates functionality of a human GPCR polypeptide or GPCR fusion protein; optionally, determining the structure of the compound; optionally, providing the name or structure of the compound; and producing or synthesizing the compound.

In a related embodiment, the present invention provides a process for modulating the functionality of hRUP3 said process comprising performing a screening method supra to thereby identify, isolate, formulate, produce or synthesize a modulator of a GPCR and then contacting the GPCR with the modulator or administering the modulator to a subject under conditions sufficient to modulate the functionality of the GPCR.

The present invention also provides a process for identifying, isolating, formulating, producing or synthesizing a compound that modulates signal transduction mediated by a GPCR designated hRUP3, a GPCR having substantially the sequence of SEQ ID NO: 8 in the pancreas, said process comprising performing the method or process described herein according to any embodiment to thereby identify, isolate, formulate, produce or synthesize the compound. Preferably, the signal transduction mediated by a GPCR in the pancreas modulates blood concentration of glucose and/or glucagon and/or insulin.

Any class of candidate modulator described herein can be tested in the inventive screening assay. In a preferred embodiment, the candidate compound is a candidate small molecule. For example, a small molecule compound that shares some structural homology with hRUP3. In a further embodiment, the candidate compound is an antibody that binds to hRUP3. In an alternative embodiment, the test compound comprises a peptide of at least about 4-10 amino acids in length derived from the amino acid sequence of the hRUP3 protein of the present invention. In a further embodiment, the test compound comprises siRNA or shRNA comprising a nucleotide sequence derived from the nucleotide sequence encoding hRUP3. In an alternative embodiment, the test compound comprises antisense RNA that targets expression of hRUP3 in a cell.

The screening methods supra are particularly useful for identifying compounds that modulate hRUP3-mediated signal transduction in a cell, such as, for example, in the diagnosis or treatment of a disease or disorder associated with pancreas. Accordingly, a further embodiment of the present invention provides a process for identifying a compound for the treatment of diabetes or a condition associated with diabetes said process comprising performing a method or process for identifying, isolating, formulating, producing or synthesizing a compound as described herein according to any embodiment. Preferably, the condition associated with diabetes is a defect in glucagon regulation and/or insulin regulation.

The present invention also provides a compound for the treatment of a disorder of the pancreas in a subject. Preferably, the present invention provides a compound for the treatment of a disorder of the pancreas in a subject wherein said compound interacts with an isolated or recombinant GPCR polypeptide comprising the sequence set forth in SEQ ID NO: 8 or a ligand-independent version thereof and wherein said compound is isolated using one or more of: an isolated or recombinant G protein-coupled receptor comprising an amino acid sequence selected from the group consisting of: a sequence encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using one or more primers selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 71 and SEQ ID NO: 72; and (ii) the sequence of a ligand-independent version of SEQ ID NO: 8; and/or a fusion protein comprising the G protein-coupled receptor of(a); and/or a recombinant host cell expressing or and/or a membrane comprising or Preferably, the compound for the treatment of a disorder of the pancreas in a subject wherein said compound interacts with an isolated or recombinant G protein-coupled receptor polypeptide comprising the sequence set forth in SEQ ID NO: 8 or a ligandindependent version thereof and wherein said compound is isolated using a method or process for isolating a compound that modulates a GPCR as described herein according to any one or more embodiment(s). For example, the compound is a small molecule compound In one example of the invention, the compound interacts with an isolated or recombinant GPCR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or a ligand-independent version of SEQ ID NO: 8. For example, the compound interacts with a ligand-independent version of SEQ ID NO: 8 comprising substantially the same sequence as set forth in SEQ ID NO: 8 and having a mutation at a position 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 8. For example, the mutation is to a residue other than leucine, the mutation is the substitution of leucine at position 224 of SEQ ID NO: 8 for lysine.

Preferably, the compound as described herein according to any one or more embodiments inhibits or stimulates an endogenous version of a GPCR designated hRUP7. In one example, the compound is an inverse agonist of a GPCR designated hRUP7 or a ligand-independent version thereof. In another example, the compound is an agonist or a partial agonist of a GPCR designated hRUP7 or a ligand-independent version thereof. For example, the compound is an agonist, partial agonist or inverse agonist of an endogenous GPCR polypeptide in pancreas.

The present invention also provides a compound as described herein according to any one or more embodiment(s) when used to modulate activity of a GPCR designated hRUP7 or a ligand-independent version thereof when administered to a subject in the treatment of a disease or disorder caused by inappropriate activity of said endogenous version of a G protein-coupled receptor.

In one embodiment, a disease or disorder caused by inappropriate activity of said endogenous version of a GPCR or a disorder of the pancreas affects blood concentration of glucose and/or glucagon and/or insulin. For example, the disease or disorder is diabetes or a condition associated with diabetes.

The present invention clearly contemplates prophylactic and/or therapeutic treatments wherein the level of hRUP3 expression and/or activity is modulated in a subject in need thereof.

Accordingly, a further embodiment of the present invention provides a method of treating a disorder or disease associated with pancreas, said method comprising administering a modulator of hRUP3 to a subject in need thereof for a time and under conditions sufficient to modulate hRUP3 activity in said subject. The present invention also the use of an antagonist of a GPCR designated hRUP3 or a ligand-independent version thereof in the manufacture of a medicament of a disease, disorder or abnormality alleviated by decreasing the activity of the GPCR or ligand-independent version thereof.. Conversely, the present invention provides the use of an agonist of a GPCR designated hRUP3 or a ligand-independent version thereof in the manufacture of a medicament of a disease, disorder or abnormality alleviated by increasing the activity of the GPCR or ligand-independent version thereof. For example, the disease, disorder or abnormality is associated with and/or affects blood concentration of glucose and/or glucagon and/or insulin, the disease, disorder or abnormality is diabetes or a condition associated with diabetes.

By "hRUP3 activity" is meant any activity of hRUP3 including a function mediated by binding of hRUP3 to a ligand such as, for example, its cognate ligand, and any signalling effected thereby.

SIn certain embodiments, the method comprises administering an amount of an agent that reduces hRUP3 activity or expression to the subject sufficient to reduce the level or activity of a hRUP3 protein or mRNA encoding hRUP3 in a cell of the subject.

SAccordingly, the antagonist may comprise nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, or nucleic acid that forms a triple helical structure, capable of reducing hRUP3 expression in a cell of the individual. Antibodies, peptides or small molecules that bind hRUP3 and inhibit its activity are also useful in this context. As C with the other embodiments described herein, it is particularly preferred to use an agent that reduces hRUP3 expression comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecule, or alternatively, an antibody that binds to a hRUP3 protein thereby inhibiting its activity. Other antagonists, dominant negative mutants, small molecules etc are not to be excluded.

In certain embodiments, the method comprises administering an amount of an agent that enhances or agonizes hRUP3 activity or expression to the subject sufficient to enhance the level or activity of a hRUP3 protein or mRNA encoding hRUP3 in a cell of the subject. Accordingly, the agonist may comprise a peptide or small molecule agonist.

hRUP 7 In another embodiment, the presently claimed invention relates to a recombinant human G protein-coupled receptor (GPCR) designated hRUP7, that is expressed highly in peripheral blood leukocytes, especially eosinophils and in spleen or thymus tissue, and isolated nucleic acid encoding same. The present invention further extends to encoding a constitutively-activated variants of the hRUP7 polypeptide, GPCR fusion proteins comprising the hRUP7 polypeptide and a G protein Gi, the predominate G protein that couples with RUP7) and nucleic acid encoding the constitutivelyactivated variants and GPCR fusion proteins. Coupling to non-cognate G proteins is also encompassed by the invention.

For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 13 is the sequence of a hRUP7-encoding cDNA. The sequence set forth in SEQ ID NO: 14 is the sequence of a hRUP7 polypeptide.

Accordingly, one embodiment of the present invention provides an isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated hRUP7, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 14; the sequence set forth in SEQ ID NO: 13; the sequence of a polynucleotide that hybridizes under stringent conditions to (a) or or a complementary sequence thereto; the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 59 and/or SEQ ID NO: 60 and/or SEQ ID NO: 75 and/or SEQ ID NO: 76; a sequence encoding a constitutively-activated variant of SEQ ID NO: 14 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 14; and a variant of SEQ ID NO: 13 comprising a mutation positioned at a codon encoding an amino acid 16 amino acid residues N-terminal from the codon encoding the conserved proline residue within the TM6 domain of SEQ ID NO: 14.

In a particularly preferred embodiment, the isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated hRUP7 comprises a nucleotide sequence selected from the group consisting of: a sequence encoding the amino acid sequence set forth in SEQ ID NO: 14; and the nucleotide sequence set forth in SEQ ID NO: 13.

It will be apparent from the description herein that the isolated polynucleotide encoding the human GPCR designated hRUP7 is preferably obtained from human peripheral blood leukocytes, spleen or thymus tissue eg., by a process comprising performing RT- PCR on a sample of human mRNA obtained from such tissue.

A further embodiment of the present invention provides an isolated polynucleotide encoding a GPCR fusion protein, wherein said polynucleotide comprises a nucleotide sequence encoding the human GPCR designated hRUP7 or a constitutively activated variant thereof and nucleic acid encoding a G protein, Gi.

A further embodiment of the present invention provides a vector comprising a polynucleotide encoding the human GPCR designated hRUP7 or a constitutively activated variant thereof. Preferably, the vector is an expression vector, wherein the polynucleotide encoding the human GPCR designated hRUP7 or a constitutively activated variant thereof is operably linked to a promoter.

A further embodiment of the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises a recombinant human GPCR polypeptide designated hRUP7 or a constitutively activated variant thereof.

A further embodiment of the present invention provides a method of producing a human GPCR or a GPCR fusion protein comprising the steps of: transfecting an expression vector comprising nucleic acid encoding hRUP7 or a constitutively activated variant thereof or GPCR fusion protein comprising said hRUP3 or said variant or a GPCR fusion protein comprising same into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human GPCR designated hRUP7 or a constitutively activated variant thereof or a GPCR fusion protein from the expression vector.

Preferably, The method of producing a human GPCR or GPCR fusion protein further comprises obtaining the transfected host cell. In one embodiment, the host cell is a melanophore. In another embodiment, the host cell is a leukocyte, such as an eosinophil. In another embodiment, the host cell is a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell.

28 A further embodiment of the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated hRUP7 comprising an amino acid sequence selected from the group consisting of: a sequence comprising the amino acid sequence set forth in SEQ ID NO: 14; a sequence encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 13 or a complementary sequence thereto; a sequence encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 59 and/or SEQ ID NO: 60 and/or SEQ ID NO: 75 and/or SEQ ID NO: 76; and the sequence of a constitutively-activated variant of SEQ ID NO: 14 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 14.

Preferably, the primers are used in pairs selected from SEQ ID NO: 59 and SEQ ID NO: 60 and (ii) SEQ ID NO: 75 and SEQ ID NO: 76.

In a particularly preferred embodiment, the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated hRUP7 comprising the amino acid sequence set forth in SEQ ID NO: 14.

A further embodiment of the invention provides an isolated or recombinant GPCR fusion protein comprising an amino acid sequence of the human GPCR designated hRUP7 or a constitutively-activated variant thereof, preferably further comprising a G protein, preferably Gi. Preferably, the G protein is coupled to a C-terminal portion of hRUP7 or a constitutively-activated variant thereof. The hRUP7 or variant and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the human GPCR designated hRUP7 or a constitutively-activated variant thereof or an isolated membrane comprising said human GPCR polypeptide or said variant; and

C

29 measuring the ability of the compound to modulate expression and/or activity of the G protein coupled receptor polypeptide or variant wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Preferably, the method according to this embodiment further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector comprising nucleic acid encoding the human GPCR designated hRUP7 or a constitutively-activated variant thereof. In a particularly preferred embodiment, the human GPCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses a GPCR fusion protein comprising the human GPCR designated hRUP7 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to modulate expression and/or activity of the GPCR polypeptide or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the GPCR polypeptide.

Preferably, the method of identifying a GPCR modulator further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR fusion protein. In a particularly preferred embodiment, the GPCR fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 14 and the amino acid sequence of a G protein.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 14 or a constitutively-activated variant thereof and a G protein or an isolated membrane comprising said GPCR fusion protein;

.JU

S(b) contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide portion or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

SIn a particularly preferred embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that Smodulates activity and/or expression of hRUP7; optionally, determining the structure of the compound; and providing the compound or modulator or the name or structure of the compound.

In an alternative embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of hRUP7; optionally, determining the structure of the compound; optionally, providing the name or structure of the compound; and producing or synthesizing the compound.

In a related embodiment, the present invention provides a process for modulating the functionality of hRUP7 said process comprising performing a screening method supra to thereby identify a modulator of hRUP7 and then contacting hRUP7 with the identified modulator or administering the identified modulator to a subject under conditions sufficient to modulate the functionality of hRUP7.

Any class of candidate modulator described herein can be tested in the inventive screening assay. In one embodiment, the test compound is a small molecule, such as, for example, a modulator of a receptor that shares some structural homology with hRUP7. In a further embodiment, the test compound is an antibody that binds to hRUP7. In an alternative embodiment, the test compound comprises a peptide of at least about 4-10 amino acids in length derived from the amino acid sequence of the hRUP7 protein of the present invention. In a further embodiment, the test compound comprises siRNA or shRNA comprising a nucleotide sequence derived from the nucleotide sequence encoding hRUP7. In an alternative embodiment, the test compound comprises antisense RNA that targets expression of hRUP7 in a cell.

Preferably, the candidate compound for screening is a known modulator of a histamine receptor, such as, for example, a histamine 3 receptor (hH3R). Such molecules are known in the art and described, for example, by Leurs et al., Trends Pharmacol Sci.

19(5):177-83 (1998) which is incorporated herein by reference. In one particularly preferred embodiment, the candidate compound is an agonist selective for the H3 receptor, such as, for example, R-alpha-methyl histamine, imetit, immepip, or a derivative compound thereof or structurally-related compound thereto, which may mimic the effect of histamine. In an alternative preferred embodiment, the candidate compound is an antagonist selective for the H3 receptor, such as, for example, clobenpropit, thioperamide, thioperanide, or iodoproxyfan or a derivative compound thereof or structurally-related compound thereto.

The screening methods supra are particularly useful for identifying compounds that modulate hRUP7-mediated signal transduction in a cell, such as, for example, in the diagnosis or treatment of a disease or disorder associated with a cell or tissue selected from the group consisting of peripheral blood leukocytes, spleen and thymus.

Accordingly, a further embodiment of the present invention provides a process for identifying a compound that modulates signal transduction mediated by a G-protein coupled receptor in a cell or tissue selected from the group consisting of peripheral blood leukocytes, spleen or thymus, said process comprising performing a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR polypeptide or variant thereof; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide or variant wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Preferably, the signal transduction mediated by the RUP7 GPCR in a cell or tissue selected from the group consisting of peripheral blood leukocytes, spleen and thymus modulates inflammation, an inflammatory response, histamine secretion, histamine release or gastric acid secretion in an animal.

Accordingly, the present invention clearly contemplates prophylactic and/or therapeutic treatments wherein the level of hRUP7 expression and/or activity is modulated in a subject in need thereof.

Accordingly, a further embodiment of the present invention provides a method of treating a disorder or disease associated with peropheral blood leukocytes, spleen, or thymus, said method comprising administering a modulator of hRUP7 to a subject in need thereof for a time and under conditions sufficient to modulate hRUP7 activity in said subject.

By "hRUP7 activity" is meant any activity of hRUP7 including a function mediated by binding of hRUP7 to a ligand such as, for example, its cognate ligand, and any signalling effected thereby.

In certain embodimens, the method comprises administering an amount of an agent that reduces hRUP7 activity or expression to the subject sufficient to reduce the level or activity of a hRUP7 protein or mRNA encoding hRUP7 in a cell of the subject.

Accordingly, the antagonist may comprise nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, or nucleic acid that forms a triple helical structure, capable of reducing hRUP7 expression in a cell of the individual. Antibodies, peptides or small molecules that bind hRUP7 and inhibit its activity are also useful in this context. As with the other embodiments described herein, it is particularly preferred to use an agent that reduces hRUP7 expression comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecule, or alternatively, an antibody that binds to a hRUP7 protein thereby inhibiting its activity. Other antagonists, dominant negative mutants, small molecules etc are not to be excluded.

In certain embodiments, the method comprises administering an amount of an agent that enhances or agonizes hRUP7 activity or expression to the subject sufficient to enhance the level or activity of a hRUP7 protein or mRNA encoding hRUP7 in a cell of the subject. Accordingly, the agonist may comprise a peptide or small molecule agonist.

A further embodiment of the present invention provides for the use of a composition that modulates hRUP7 expression and/or activity in the preparation of one or more medicaments for treating a disease or disorder associated with peripheral blood leukocytes, spleen, or thymus in a subject, such as for treating inflammation, an inflammatory response, asthma, histamine secretion, histamine release or gastric acid secretion.

hARE-2 In another embodiment, the presently claimed invention relates to a recombinant human G protein-coupled receptor (GPCR) designated hARE-2, that is expressed in human brain tissue, preferably in brain tissue selected from the group consisting of cerebellum, and substantia nigra, and isolated nucleic acid encoding same. The present invention further extends to encoding a constitutively-activated variants of the hARE-2 polypeptide, GPCR fusion proteins comprising the hARE-2 polypeptide and a G protein, and nucleic acid encoding the constitutively-activated variants and GPCR fusion proteins.

For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 19 is the sequence of a hARE-2-encoding cDNA. The sequence set forth in SEQ ID NO: is the sequence of hARE-2 polypeptide. The amino acid sequence of the encoded hARE-2 polypeptide shares sequence homology with another polypeptide of the present invention designated hGPCR27 the amino acid sequence of which is also set forth in SEQ ID NO: 16.

Accordingly, one embodiment of the present invention provides an isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated hARE-2, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: the sequence set forth in SEQ ID NO: 19; the sequence of a polynucleotide that hybridizes under stringent conditions to (a) or or a complementary sequence thereto; a sequence encoding a constitutively-activated variant of SEQ ID NO: wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 20; and a variant of SEQ ID NO: 19 comprising a mutation positioned at a codon encoding an amino acid 16 amino acid residues N-terminal from the codon encoding the conserved proline residue within the TM6 domain of SEQ ID NO: In a particularly preferred embodiment, the isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated hARE-2 comprises a nucleotide sequence selected from the group consisting of: a sequence encoding the amino acid sequence set forth in SEQ ID NO: 20; and the nucleotide sequence set forth in SEQ ID NO: 19.

It will be apparent from the description herein that the isolated polynucleotide encoding the human GPCR designated hARE-2 is preferably obtained, by a process comprises performing RT-PCR on a sample of mRNA obtained from human brain, such as from cerebellum or substantia nigra.

A further embodiment of the present invention provides an isolated polynucleotide encoding a GPCR fusion protein, wherein said polynucleotide comprises a nucleotide sequence encoding the human GPCR designated hARE-2 or a constitutively activated variant thereof and nucleic acid encoding a G protein.

A further embodiment of the present invention provides a vector comprising a polynucleotide encoding the human GPCR designated hARE-2 or a constitutively activated variant thereof. Preferably, the vector is an expression vector, wherein the polynucleotide encoding the human GPCR designated hARE-2 or a constitutively activated variant thereof is operably linked to a promoter.

A further embodiment of the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises a recombinant human GPCR polypeptide designated hARE-2 or a constitutively activated variant thereof.

A further embodiment of the present invention provides a method of producing a human GPCR, or constitutive activated variant thereof, or a GPCR fusion protein comprising the steps of: transfecting an expression vector comprising nucleic acid encoding hARE-2 or a constitutively activated variant thereof or GPCR fusion protein comprising said hARE-2 or said variant or a GPCR fusion protein comprising same into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human GPCR designated hARE-2 or a constitutively activated variant thereof or a GPCR fusion protein from the expression vector.

Preferably, The method of producing a human GPCR or GPCR fusion protein further comprises obtaining the transfected host cell. In one embodiment, the host cell is a melanophore. In another embodiment, the host cell is a a brain cell or a neuronal cell or neuronal cell line NT2, hNT, GTl-trk). In another embodiment, the host cell is a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell.

A further embodiment of the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated hARE-2 comprising an amino acid sequence selected from the group consisting of: a sequence comprising the amino acid sequence set forth in SEQ ID NO: a sequence encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 19 or a complementary sequence thereto; and the sequence of a constitutively-activated variant of SEQ ID NO: 20 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: In a particularly preferred embodiment, the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated hARE-2 comprising the amino acid sequence set forth in SEQ ID NO: A further embodiment of the invention provides an isolated or recombinant GPCR fusion protein comprising an amino acid sequence of the human GPCR designated hARE-2 or a constitutively-activated variant thereof, preferably further comprising a G protein. Preferably, the G protein is coupled to a C-terminal portion of the GPCR or a constitutively-activated variant thereof. The GPCR or variant and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the human GPCR designated hARE-2 or a constitutively-activated variant thereof or an isolated membrane comprising said human GPCR polypeptide or said variant; and measuring the ability of the compound to modulate expression and/or activity of the G protein coupled receptor polypeptide or variant wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Preferably, the method according to this embodiment further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector comprising nucleic acid encoding the human GPCR designated hARE-2 or a constitutively-activated variant thereof. In a particularly preferred embodiment, the human GPCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses a GPCR fusion protein comprising the human GPCR designated hARE-2 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to modulate expression and/or activity of the GPCR polypeptide or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the GPCR polypeptide.

Preferably, the method of identifying a GPCR modulator further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR fusion protein. In a particularly preferred embodiment, the GPCR fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 20 and the amino acid sequence ofa G protein.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 20 or a constitutively-activated variant thereof and a G protein or an isolated membrane comprising said GPCR fusion protein; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide portion or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

In a particularly preferred embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of hARE-2; optionally, determining the structure of the compound; and providing the compound or modulator or the name or structure of the compound.

In an alternative embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a modulates activity and/or expression of hARE-2; optionally, determining the structure of the compound; optionally, providing the name or structure of the compound; and compound that S(d) producing or synthesizing the compound.

In a related embodiment, the present invention provides a process for modulating the Sfunctionality of hARE-2 said process comprising performing a screening method supra to thereby identify a modulator of hARE-2 and then contacting hARE-2 with the _identified modulator or administering the identified modulator to a subject under Sconditions sufficient to modulate the functionality of hARE-2.

SAny class of candidate modulator described herein can be tested in the inventive screening assay. In one embodiment, the test compound is a small molecule, such as, Sfor example, a modulator of a receptor that shares some structural homology with hARE-2. In a further embodiment, the test compound is an antibody that binds to hARE-2. In an alternative embodiment, the test compound comprises a peptide of at least about 4-10 amino acids in length derived from the amino acid sequence of the hARE-2 protein of the present invention. In a further embodiment, the test compound comprises siRNA or shRNA comprising a nucleotide sequence derived from the nucleotide sequence encoding hARE-2. In an alternative embodiment, the test compound comprises antisense RNA that targets expression of hARE-2 in a cell.

The screening methods supra are particularly useful for identifying compounds that modulate hARE-2-mediated signal transduction in a cell, such as, for example, in the diagnosis or treatment of a disease or disorder associated with the brain, especially the cerebellum or substantia nigra. Accordingly, a further embodiment of the present invention provides a process for identifying a compound that modulates signal transduction mediated by a G-protein coupled receptor in the brain, such as cerebellum or substantia nigra, said process comprising performing a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR polypeptide or variant thereof; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide or vriant thereof wherein modulation (NI of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Preferably, the signal transduction mediated by a G-protein coupled receptor in the cerebellum, basal ganglia substantia nigra and/or caudate nucleus) modulates Smotor function and neurotransmitter dopamine, hydroxy tryptamine etc) N release associated therewith such as, for example, in Huntington's Chorea, Parkinson's disease, striatonigral degeneration when parkinsonism predominates, multiple system atrophy (associated with a combination of parkinsonism, cerebellar and pyramidal signs, and autonomic dysfunction), olivopontocerebellar atrophy (OPCA) when cerebellar signs predominate, progressive supranuclear palsy or Shy-Drager syndrome.

Accordingly, the present invention clearly contemplates prophylactic and/or therapeutic treatments wherein the level of hARE-2 expression and/or activity is modulated in a subject in need thereof.

Accordingly, a further embodiment of the present invention provides a method of treating a disorder or disease associated with the brain, such as with cerebellum or substantia nigra, said method comprising administering a modulator of hARE-2 to a subject in need thereof for a time and under conditions sufficient to modulate hARE-2 activity in said subject.

By "hARE-2 activity" is meant any activity of hARE-2 including a function mediated by binding of hARE-2 to a ligand such as, for example, its cognate ligand, and any signalling effected thereby.

In certain embodiments, the method comprises administering an amount of an agent that reduces hARE-2 activity or expression to the subject sufficient to reduce the level or activity of a hARE-2 protein or mRNA encoding hARE-2 in a cell of the subject.

Accordingly, the antagonist may comprise nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, or nucleic acid that forms a triple helical structure, capable of reducing hARE-2 expression in a cell of the individual. Antibodies, peptides or small molecules that bind hARE-2 and inhibit its activity are also useful in this context. As with the other embodiments described herein, it is particularly preferred to use an agent 04 c that reduces hARE-2 expression comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecule, or alternatively, an antibody that binds to a hARE-2 protein thereby inhibiting its activity. Other antagonists, dominant negative mutants, small molecules etc are not to be excluded.

In certain embodiments, the method comprises administering an amount of an agent that enhances or agonizes hARE-2 activity or expression to the subject sufficient to C enhance the level or activity of a hARE-2 protein or mRNA encoding hARE-2 in a cell of the subject. Accordingly, the agonist may comprise a peptide or small molecule agonist.

A further embodiment of the present invention provides for the use of a composition that modulates hARE-2 expression and/or activity in the preparation of one or more medicaments for treating a disease or disorder associated with the brain, in a subject, such as for treating Huntington's Chorea, Parkinson's disease, striatonigral degeneration when parkinsonism predominates, multiple system atrophy (associated with a combination of parkinsonism, cerebellar and pyramidal signs, and autonomic dysfunction), olivopontocerebellar atrophy (OPCA) when cerebellar signs predominate, progressive supranuclear palsy or Shy-Drager syndrome.

CHN4 In another embodiment, the presently claimed invention relates to a recombinant human G protein-coupled receptor (GPCR) designated CHN4 and isolated nucleic acid encoding same. The present invention further extends to encoding a constitutivelyactivated variants of the CHN4 polypeptide, GPCR fusion proteins comprising the CHN4 polypeptide and a G protein, and nucleic acid encoding the constitutivelyactivated variants and GPCR fusion proteins.

(N *For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 27 ¢c is the sequence of a CHN4-encoding cDNA. The sequence set forth in SEQ ID NO: 28 is the sequence of CHN4 polypeptide.

Accordingly, one embodiment of the present invention provides an isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated CHN4, wherein said polynucleotide comprises a nucleotide sequence selected from the C group consisting of: C a sequence encoding a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 28; the sequence set forth in SEQ ID NO: 27; the sequence of a polynucleotide that hybridizes under stringent conditions to (a) or or a complementary sequence thereto; a sequence encoding a constitutively-activated variant of SEQ ID NO: 28 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 28; and a variant of SEQ ID NO: 27 comprising a mutation positioned at a codon encoding an amino acid 16 amino acid residues N-terminal from the codon encoding the conserved proline residue within the TM6 domain of SEQ ID NO: 28.

In a particularly preferred embodiment, the isolated polynucleotide encoding a human G protein-coupled receptor polypeptide designated CHN4 comprises a nucleotide sequence selected from the group consisting of: a sequence encoding the amino acid sequence set forth in SEQ ID NO: 28; and the nucleotide sequence set forth in SEQ ID NO: 27.

It will be apparent from the description herein that the isolated polynucleotide encoding the human GPCR designated CHN4 is preferably obtained, by a process comprising performing RT-PCR on a sample of mRNA obtained from leukocytes including T-cells (including CD8' T-cells and CD4 T-cells), B-cells (including CD19+ B-cells), and neutrophils, or from spleen cells.

A further embodiment of the present invention provides an isolated polynucleotide encoding a GPCR fusion protein or a constitutively activated variant thereof, wherein 42 c said polynucleotide comprises a nucleotide sequence encoding the human GPCR designated CHN4 or a constitutively activated variant thereof and nucleic acid encoding a G protein.

A further embodiment of the present invention provides a vector comprising a polynucleotide encoding the human GPCR designated CHN4 or a constitutively activated variant thereof. Preferably, the vector is an expression vector, wherein the Ci polynucleotide encoding the human GPCR designated CHN4 or a constitutively activated variant thereof is operably linked to a promoter.

t OA further embodiment of the present invention provides a recombinant host cell comprising the vector described in the preceding paragraph.

A further embodiment of the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises a recombinant human GPCR polypeptide designated CHN4 or a constitutively activated variant thereof.

A further embodiment of the present invention provides a method of producing a human GPCR, or constitutively activated variant thereof, or a GPCR fusion protein comprising the steps of: transfecting an expression vector comprising nucleic acid encoding CHN4 or a constitutively activated variant thereof or GPCR fusion protein comprising said CHN4 or said variant thereof or a GPCR fusion protein comprising same into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human GPCR designated CHN4 or a constitutively activated variant thereof or a GPCR fusion protein from the expression vector.

Preferably, The method of producing a human GPCR or GPCR fusion protein further comprises obtaining the transfected host cell. In one embodiment, the host cell is a melanophore. In another embodiment, the host cell is a leukocyte such as a B cell or B cell line, a T-cell or T-cell line, a neutrophil or neutrophil cell line, or a spleen cell. In another embodiment, the host cell is a cell selected from the group consisting of a CHO cell, a COS cell COS-7 cell), a 293 cell, and a 293T cell. Human B cell lines, including lymphoma cell lines are particularly preferred.

A further embodiment of the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated CHN4 comprising an amino acid sequence selected from the group consisting of: a sequence comprising the amino acid sequence set forth in SEQ ID NO: 28; a sequence encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 27 or a complementary sequence thereto; and the sequence of a constitutively-activated variant of SEQ ID NO: 28 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 28.

In a particularly preferred embodiment, the present invention provides an isolated or recombinant human G protein-coupled receptor polypeptide designated CHN4 comprising the amino acid sequence set forth in SEQ ID NO: 28.

A further embodiment of the invention provides an isolated or recombinant GPCR fusion protein comprising an amino acid sequence of the human GPCR designated CHN4 or a constitutively-activated variant thereof, preferably further comprising a G protein. Preferably, the G protein is coupled to a C-terminal portion of the GPCR or a constitutively-activated variant thereof. The GPCR or variant and the G protein may be juxtaposed or separated by an intervening spacer such as, for example a spacer of about up to about 6 amino acids in length.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the human GPCR designated CHN4 or a constitutively-activated variant thereof or an isolated membrane comprising said human GPCR polypeptide or said variant; and measuring the ability of the compound to modulate expression and/or activity of the G protein coupled receptor polypeptide or variant wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Preferably, the method according to this embodiment further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector comprising nucleic acid encoding the human GPCR designated CHN4 or a constitutively-activated variant thereof. In a particularly preferred embodiment, the human GPCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses a GPCR fusion protein comprising the human GPCR designated CHN4 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to modulate expression and/or activity of the GPCR polypeptide or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the GPCR polypeptide.

Preferably, the method of identifying a GPCR modulator further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR fusion protein. In a particularly preferred embodiment, the GPCR fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 28 and the amino acid sequence of a G protein.

A further embodiment of the present invention provides a method of identifying a modulator of a G protein-coupled receptor comprising the steps of: providing a recombinant host cell that expresses a GPCR fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 28 or a constitutively-activated variant thereof and a G protein or an isolated membrane comprising said GPCR fusion protein; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide portion or variant portion of said GPCR fusion protein wherein modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

In a particularly preferred embodiment, the screening assay comprises: performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of hCHN4; optionally, determining the structure of the compound; and providing the compound or modulator or the name or structure of the compound.

In an alternative embodiment, the screening assay comprises: S(a) performing a method supra to thereby identify or determine a compound that modulates activity and/or expression of hCHN4; optionally, determining the structure of the compound; optionally, providing the name or structure of the compound; and producing or synthesizing the compound.

In a related embodiment, the present invention provides a process for modulating the functionality of hCHN4 said process comprising performing a screening method supra to thereby identify a modulator of hCHN4 and then contacting hCHN4 with the identified modulator or administering the identified modulator to a subject under conditions sufficient to modulate the functionality of hCHN4.

The screening methods supra are particularly useful for identifying compounds that modulate hCHIN4-mediated signal transduction in a cell, such as, for example, in the diagnosis or treatment of a disease or disorder associated with leukocytes, especially Bcells or T-cells or neutrophils, or the spleen. Accordingly, a further embodiment of the present invention provides a process for identifying a compound that modulates signal transduction mediated by a G-protein coupled receptor in leukocytes or spleen cells, said process comprising performing a method of identifying a modulator of a G protein-coupled receptor (GPCR) comprising the steps of: providing a recombinant host cell that expresses a GPCR polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28 or a constitutively-activated variant thereof or an isolated membrane comprising said GPCR polypeptide or variant thereof; contacting a candidate compound with the recombinant host cell or isolated membrane; and measuring the ability of the compound to modulate expression and/or activity of the G protein-coupled receptor polypeptide or variant thereof wherein 4b c modulation of expression and/or activity indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

Any class of candidate modulator described herein can be tested in the inventive screening assay. In one embodiment, the test compound is a small molecule, such as, for example, a modulator of a receptor that shares some structural homology with hCHN4. In a further embodiment, the test compound is an antibody that binds to C hCHN4. In an alternative embodiment, the test compound comprises a peptide of at CI least about 4-10 amino acids in length derived from the amino acid sequence of the hCHN4 protein of the present invention. In a further embodiment, the test compound Scomprises siRNA or shRNA comprising a nucleotide sequence derived from the nucleotide sequence encoding hCHN4. In an alternative embodiment, the test compound comprises antisense RNA that targets expression of hCHN4 in a cell.

Accordingly, the present invention clearly contemplates prophylactic and/or therapeutic treatments wherein the level of hCHN4 expression and/or activity is modulated in a subject in need thereof.

In certain embodiments, the candidate compound for screening is a known modulator of a thrombin receptor, such as, for example, PAR-1. In one particularly preferred embodiment, the candidate compound is an agonist selective for the PAR-1 receptor, such as, for example, the hexapeptide analogue 3-Mercapto-propionyl-Phe-Cha-Cha- Arg-Lys-Pro-Asn-Asp-Lys-amide or a derivative compound thereof or structurallyrelated compound thereto Mercapto-propionyl-Phe-Cha-Cha-Leu-Arg-Asn-Proamide or Mercapto-propionyl-Phe-Cha-Cha-Leu-Arg-Asn-Pro-Ala-amide.

The screening methods supra are particularly useful for identifying compounds that modulate CHN4-mediated signal transduction in a cell, such as, for example, in the diagnosis or treatment of a disorder or disease associated with leukocytes, especially Bcells, T-cells, or neutrophils, or the spleen. Preferably, the signal transduction modulated by hCHN4 modulates an inflammatory or autoimmune response in a subject. Accordingly, the identified compounds are particularly useful for treating inflammatory or autoimmune diseases or disorders such as, for example, sepsis, endotoxemia, systemic lupus erythematosis (SLE), rheumatoid arthritis, myasthenia gravis, Crohn's Disease, Crohn's disease in which fistulas form, rheumatoid arthritis, juvenile rheumatoid arthritis, active ankylosing spondylitis, psoriasis, psoriatic arthritis, reactive arthritis, inflammatory bowel disease arthritis, colitis, neuronal inflammation or non-neuronal inflammation.

Accordingly, a further embodiment of the present invention provides a method of treating a disorder or disease associated with leukocytes, espcially B-cells, T-cells, or neutrophils, or the spleen, said method comprising administering a modulator of hCHN4 to a subject in need thereof for a time and under conditions sufficient to modulate hCHN4 activity in said subject.

By "hCHN4 activity" is meant any activity of hCHN4 including a function mediated by binding of hCHN4 to a ligand such as, for example, its cognate ligand, and any signalling effected thereby.

In certain embodiments, the method comprises administering an amount of an agent that reduces hCHN4 activity or expression to the subject sufficient to reduce the level or activity of a hCHN4 protein or mRNA encoding hCHN4 in a cell of the subject.

Accordingly, the antagonist may comprise nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, or nucleic acid that forms a triple helical structure, capable of reducing hCHN4 expression in a cell of the individual. Antibodies, peptides or small molecules that bind hCHN4 and inhibit its activity are also useful in this context. As with the other embodiments described herein, it is particularly preferred to use an agent that reduces hCHN4 expression comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecule, or alternatively, an antibody that binds to a hCHN4 protein thereby inhibiting its activity. Other antagonists, dominant negative mutants, small molecules etc are not to be excluded.

In certain embodiments, the method comprises administering an amount of an agent that enhances or agonizes hCHN4 activity or expression to the subject sufficient to enhance the level or activity of a hCHN4 protein or mRNA encoding hCHN4 in a cell of the subject. Accordingly, the agonist may comprise a peptide or small molecule agonist.

A further embodiment of the present invention provides for the use of a composition that modulates hCHN4 expression and/or activity in the preparation of one or more medicaments for treating a disease or disorder associated with leukocytes, especially Bcells, T-cells, or neutrophils, or the spleen in a subject, such as for treating neuroinflammation, such as in Alzheimer's disease, and such as for treating sepsis, endotoxemia, systemic lupus erythromatosis (SLE), rheumatoid arthritis, myasthenia gravis, Crohn's Disease, Crohn's disease in which fistulas form, rheumatoid arthritis, juvenile rheumatoid arthritis, active ankylosing spondylitis, psoriasis, psoriatic arthritis, reactive arthritis, inflammatory bowel disease or colitis.

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and 1B provide reference "grids" for certain dot-blots provided herein (see also, Figure 2A and 2B, respectively).

Figures 2A and 2B provide reproductions of the results of certain dot-blot analyses resulting from hCHN3 and hCHN8, respectively (see also, Figures 1A and 1B, respectively).

Figure 3 provides a reproduction of the results of RT-PCR analysis of hRUP3. These data support the conclusion that hRUP3 is selectively expressed in the pancreas.

Figure 4 provides a reproduction of the results of RT-PCR analysis of hRUP4.

Figure 5 provides a reproduction of the results of RT-PCR analysis of hRUP6.

Figure 6 provides a graphical representation showing expression of hRUP7 in human tissues and cell types as shown on the x-axis. Highest expression was observed in eosinophils.

Figure 7 provides a graphical representation showing expression of hCHN4 in human tissues and cell types as shown on the x-axis. Highest expression was observed in Tcells (including CD8* T-cells and CD4 T-cells), B-cells (including CD19 B-cells), neutrophils and spleen.

Figure 8 provides an alignment between the amino acid sequence of the novel human GPCR designated hRUP7 (SEQ ID NO: 14) and the reported amino acid sequence of human histamine H3 receptor (hH3R). The sequence identity between hRUP7 and hH3R is about 43% as determined by this alignment. Conserved amino acid residues between the two sequences are shown in bold type above each block of residues in the alignment. The asterisk above the consensus sequence indicates the position of the alanine residue in hRUP7 that is mutated to produce a constitutively-activated variant of the receptor the position of hRUP7 A302).

Figure 9 provides an alignment between the amino acid sequence of the novel human ARE-2 (SEQ ID NO: 20) and the amino acid sequence of human GPCR27 (SEQ ID NO: 16). The sequence identity between hARE-2 and hGPCR27 is about 53% as determined by this alignment. Conserved amino acid residues between the two sequences are shown in bold type above each block of residues in the alignment. The asterisk above the consensus sequence indicates the position of the glycine residue in hARE-2 that is mutated to produce a constitutively-activated variant of the receptor the position of hARE-2 G285).

Figure 10 provides an alignment between the amino acid sequence of the novel human CHN4 (SEQ ID NO: 28) and the amino acid sequence of human thrombin receptor PAR-1, a seven-transmembrane receptor (GenBank Accession No. NM_001992). The sequence identity between hCHN4 and PAR-1 is about 32% as determined by this alignment. Conserved amino acid residues between the two sequences are shown in bold type above each block of residues in the alignment. The asterisk above the consensus sequence indicates the position of the valine residue in hCHN4 that is mutated to produce a constitutively-activated variant of the receptor the position of hCHN4 V236).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions The scientific literature that has evolved around receptors has adopted a number 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: AGONIST shall mean an agent ligand, candidate compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response or GTP binding to membrane mediated by the GPCR.

AMINO ACID ABBREVIATIONS used herein are set out in Table 1: TABLE 1 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 ANTAGONIST shall mean an agent ligand, candidate compound) that binds, and preferably binds competitively, to a GPCR at about the same site as an agonist or partial agonist but which does not activate an intracellular response initiated by the active form of the GPCR, and can thereby inhibit the intracellular response by agonist or partial agonist. An antagonist typically does not diminish the baseline intracellular response in the absence of an agonist or partial agonist.

CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) which is amenable to a screening technique.

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

COMPOSITION means a material comprising at least one component.

COMPOUND EFFICACY or EFFICACY shall mean the ability of a compound to inhibit or stimulate one or more GPCR functions directly or indirectly, by measurement of GTP (via [35S]GTPyS) or cAMP level in the presence or absence of a candidate compound.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject to constitutive receptor activation. A constitutively activated receptor can be 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 endogenous ligand or a chemical equivalent thereof.

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

ENDOGENOUS shall mean a material that a mammal naturally produces.

ENDOGENOUS in reference to, for example and not limited to the term "GPCR" or "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.

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, shall mean a nonendogenous protein comprising a GPCR such as an endogenous or non-endogenous, constitutively activated orphan GPCR preferably fused to at least one G protein, most preferably, the alpha subunit of such G protein that binds GTP, with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. In the preferred form, the G protein can be fused directly to the C-terminus of the GPCR or there may be spacers positioned between the two.

HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein. In the present context, the Plasmid or vector will typically contain nucleic acid encoding a GPCR or GPCR fusion protein in operable connection with a suitable promoter sequence to permit expression of the GPCR or GPCR fusion protein to occur.

In the preferred embodiment, the host cell is a eukaryotic cell, such as a melanophore, or a mammalian cell a leukocyte, neuronal cell, pancreatic cell, spleen cell, brain cell, 293 cell, 293T cell, CHO cell or COS cell.

INHIBIT or INHIBITING, in relation 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 AGONIST shall mean an agent ligand, candidate compound) which binds to either the endogenous form of a GPCR or to the constitutively activated form of a GPCR and which inhibits 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 an agonist or partial agonist, or decreases GTP binding to membranes.

Preferably, the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.

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

LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.

MODULATE shall mean a variation in activity and/or expression of a GPCR such as, for example, a variation in activity and/or expression produced by a modulator.

MODULATOR shall mean and antagonist, agonist, inverse agonist or partial agonist as hereinbefore defined.

MUTANT or MUTATION in reference to an endogenous receptor's amino acid sequence shall mean one or more amino acid deletions, insertions or substitutions that produce a modified sequence of similar functionality including a constitutively activated form of an endogenous non-constitutively activated 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 the receptor is substantially the same as that evidenced by the first mutation of the receptor. In any event, it is preferred for the percent amino acid sequence identity between a mutated form of a receptor and the non-mutated receptor is at least about 80%, more preferably at least about 90% and more preferably at least 95%. Ideally, and owing to the fact that the most preferred mutation disclosed herein for achieving constitutive activation includes a single amino acid and/or codon change between the endogenous and the nonendogenous forms of the GPCR, it is preferred for the percent sequence amino acid sequence identity with the non-mutated sequence should be at least about 98%. As will be known to the skilled artisan, a mutation in the receptor per se may be readily accomplished, for example, by introducing a mutation into the coding region of the nucleic acid encoding the receptor.

MUTANT or MUTATION in reference to nucleic acid encoding an endogenous receptor shall be taken to include any GPCR-encoding nucleic acid of the invention modified to comprise a nucleotide deletion, insertion or substitution and encoding a modified form of a receptor of the invention, including a constitutively activated form of an endogenous non-constitutively activated receptor. The term MUTANT or MUTATION shall also be taken to include any modified nucleic acid relative to a nucleotide sequence of the invention as disclosed herein that, by virtue of the degeneracy of the genetic code, encodes a receptor of the invention as disclosed herein.

It is preferred for the percent nucleotide sequence identity between a mutated form of a receptor and the non-mutated receptor is at least about 60%, preferably, at least about more preferably at least about 80%, even more preferably at least about 90% and still more preferably at least about 95% or 98%.

PARTIAL AGONIST shall mean an agent ligand, candidate compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response or GTP binding to a membrane mediated by the GPCR, albeit to a lesser extent or degree than that observed for an agonist as hereinbefore defined.

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 limited to 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 a circular DNA molecule of bacteria and yeasts that is capable of autonomous replication independent of chromosomal DNA, and which typically carries one or more genes encoding antibiotic resistance proteins. A preferred PLASMID is amenable to insertion of nucleic acid encoding a GPCR or GPCR fusion protein and is capable of being introduced into a Host Cell for the purposes of replication and/or expression of the inserted nucleic acid.

Jo SMALL MOLECULE shall be taken to mean a compound having a molecular weight of less than about 10,000 grams per mole, including a peptide, peptidomimetic, amino acid, amino acid analogue, polynucleotide, polynucleotide analogue, nucleotide, nucleotide analogue, organic compound or inorganic compound including a heteroorganic compound or organometallic compound), and salts, esters and other pharmaceutically acceptable forms thereof. In certain preferred embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than O about 5,000 grams per mole. In certain preferred embodiments, small molecules are organic or inorganic compounds having molecular weight of less than about 1,000 10 grams per mole. In certain preferred embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 500 grams per mole.

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

TRAVERSE or TRAVERS1NG, in reference to either a defined nucleotide sequence or a defined amino acid sequence, shall mean that the sequence comprises or is contiguous with more than one region or domain of a protein. For example, in an amino acid sequence of a GPCR comprising 10 amino acids in length wherein 3 amino acid residues of said sequence are within the TM6 region of the GPCR and the remaining 7 residues are within the IC3 region of the GPCR, the 10-residue amino acid sequence is considered to traverse the TM6 and IC3 regions of the GPCR.

VECTOR shall mean a circular DNA capable of having at least one heterologous nucleic acid a cDNA or other nucleic acid encoding a GPCR or a variant thereof or a GPCR fusion protein) inserted therein and which is capable of incorporation into a host cell. The vector may be an expression vector.

57 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.

Identification of Human GPCRs The efforts of the Human Genome project have led to the identification of a plethora of information regarding nucleotide sequences located within the human genome; it has 0 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 N, 10 genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleotide 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 GPCRs, disclosed herein, were discovered by reviewing the GenBankTM database, while other GPCRs were discovered by utilizing a nucleotide sequence of a GPCR, previously sequenced, to conduct a BLAST T M search of the EST database. Table A, below, lists the disclosed endogenous orphan GPCRs along with a GPCR's respective homologous GPCR: Homologies between orphan receptors, and between orphan receptors and known receptors, and patterns of receptor expression, are used to determine one or more roles and associated diseases or disorders for the receptors within the human body.

Additionally, such homology can provide insight as to possible endogenous ligand (s) that may be natural activators for orphan GPCRs and disorders associated therewith.

For example, the nucleotide sequence of the novel human receptor designated RUP7 (SEQ ID NO: 13) isolated from human leukocytes, is similar with three unordered segments of AC007922, suggesting that the RUP7 gene comprises 3 exons. An alignment of the encoded amino acid sequence set forth in SEQ ID NO: 14 and the reported amino acid sequence for the human histamine receptor 3 Lovenberg et. al., Molecular Pharmacology,55: 1101 (1999); Figure 9) indicates there is about a 43% sequence identity between these two receptors, suggesting RUP7 has homology to a histamine receptor and may function as a histamine receptor. Those skilled in the art will be aware that histamine is a neurotransmitter found in the central and peripheral nervous system and is proven to play a role in the inflammatory "triple response" and in gastric acid secretion (Lovenberg et al., supra).

Similarly, as shown in Figure 10 there is approximate 53% identity between hARE-2 (SEQ ID NO: 20) and hGPCR27 (SEQ ID NO: 16). The similar patterns of expression of these receptor proteins especially in the basal ganglia (TABLE C and TABLE D), substantia nigra in the case of hARE-2, suggests that they may have similar roles or be involved in similar transduction pathways in the brain, such as, for example, the modulation of motor function including that which is compromised in Parkinson's disease and/or neurotransmitter dopamine, hydroxy tryptamine etc) release associated therewith.

TABLE A Disclosed Accession Open Reading Per Cent Reference to Human Number Frame Homology Homologous Orphan Identified (base Pairs) To Designated GPCR GPCRs GPCR (Accession No.) hARE-3 AL033379 1,260 bp 52.3% LPA-R U92642 hARE-4 AC006087 1,119 bp 36% P2Y5 AF000546 AC006255 1,104 bp 32% Oryzias D43633 latipes hGPR27 AA775870 1,128 bp hARE-I A1090920 999 bp 43% D13626 KIAAOOO1I hARE-2 AA359504 1,122 bp 53% GPR27 hPPRI H67224 1,053 bp 39% EB11 L31581 hG2A AA754702 1,113 bp 3 1% GPR4 L36148 hRUP3 AL035423 1,005 bp 30% 2133653 Drosophila melanogaster hRUP4 A1307658 1,296 bp 32% pNPGPR NP_-004876 28% and 29% AAC4 1276 Zebrafish Ya and and Yb, AAB94616 respectively AC005849 1,413 bp 25% DEZ Q99788 23% FMLPR P21462 hRUP6 AC005871 1,245 bp 48% GPR66 NP_-006047 hRUP7 AC007922 1,173 bp 43% H3R AF140538 hCH-N3 EST 36581 1,113 bp 53% GPR27 hCHN4 AA804531 1,077 bp 32% PAR-I 4503637 hCHN6 EST 2134670 1,503 bp 3 6% edg-1I NP_001391 KUMN EST 764455 1,029 bp 47% D13626 KlAAOOO I hCHN9 EST 1541536 1,077 bp 4 1% LTB34R NM_-000752 hCHN 10 EST 1365839 1,055bp 35% P2Y NM_002563 Furthermore, there is also about 32% identity between the novel receptor designated hCHN4 and human thrombin receptor PAR-1 (Vu et al., Cell 64. 1057-1068 (1991); GenBank Accession No. NM_001992; Figure 11), suggesting that these seventransmembrane receptor proteins may have similar roles or be involved in similar transduction pathways, e.g. in regulating an inflammatory response. On proteolytic activation by thrombin, PAR-1 exerts modulatory function in inflammation and fibrosis and plays a critical role in homeostasis. Based upon the expression profile for hCHN4, modulators of hCHN4 may be useful for treating inflammatory conditions, such as, for example, sepsis, endotoxemia, systemci lupus erythematosus, rheumatoid arthritis, myasthenia gravis, Crohn's Disease, Crohn's disease in which fistulas form, rheumatoid arthritis, juvenile rheumatoid arthritis, active ankylosing spondylitis, psoriasis, psoriatic arthritis, reactive arthritis, inflammatory bowel disease arthritis, colitis, neuronal inflammation or non-neuronal inflammation.

Receptor Screening Techniques have become more readily available over the past few years for endogenous-ligand identification (this, primarily, for the purpose of providing a means of conducting receptor-binding assays that require a receptor's endogenous ligand) because the traditional study of receptors has always 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, partial 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 ligand-independent active state.

As is known in the art, GPCRs can be "active" in their endogenous state even without the binding of the receptor's endogenous ligand thereto. Such naturally-active receptors can be screened for the direct identification without the need for the receptor's endogenous ligand) of, in particular, inverse agonists. Alternatively, the receptor can be "activated" via, e. mutation of the receptor to establish a non-endogenous version of the receptor that is active in the absence of the receptor's endogenous ligand.

Screening candidate compounds against an endogenous, non-constitutively activated human orphan GPCR disclosed herein can also provide for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand.

By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed, it is possible 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.

With respect to creation of a mutation that may evidence constitutive activation of human orphan GPCRs disclosed herein is based upon the distance from the proline residue at which is presumed to be located within TM6 of the GPCR typically nears the TM6/1C3 interface (such proline residue appears to be quite conserved). By mutating the amino acid residue located 16 amino acid residues from this residue (presumably located in the IC3 region of the receptor) to, most preferably, a lysine residue, such activation may be obtained. Other amino acid residues may be useful in the mutation at this position to achieve this objective.

For example, by mutating the leucine residue located 16 amino acid residues from the proline residue at position 240 of hRUP 3 (SEQ ID NO: preferably to a lysine residue hRUP3 L224K), such activation is obtainable. Other amino acid residues than lysine may be substituted at this position to achieve constitutive activation.

For example, by mutating the alanine residue located 16 amino acid residues from the proline residue at position 318 of hRUP 7 (SEQ ID NO: 14; Figure preferably to a lysine residue hRUP7 A302K), such activation is obtainable. Other amino acid residues than lysine may be substituted at this position to achieve constitutive activation.

Similarly, the glycine residue at position 285 of hARE-2 (SEQ ID NO: 20; Figure is in the IC3 region of that receptor and positioned 16 amino acid residues from the proline residue at position 301. By mutating the glycine residue located 16 amino acid residues from the proline residue at position 301 of hARE-2 (SEQ ID NO: preferably to a lysine residue hARE-2 G285K), such activation is obtainable.

Other amino acid residues than lysine may be substituted at this position to achieve constitutive activation.

Similarly, the valine residue at position 236 of hCHN4 (SEQ ID NO: 28; Figure 11) is in the IC3 region of that receptor and positioned 16 amino acid residues from the proline residue at position 252 within the TM6 domain. By mutating the valine residue located 16 amino acid residues from the proline residue at position 252 of hCHN4 (SEQ ID NO: 28), preferably to a lysine residue hCHN4 V236K), such activation is obtainable. Other amino acid residues than lysine may be substituted at this position to achieve constitutive activation.

Disease/Disorder Identification and/or Selection Preferably, the DNA sequence of the human orphan GPCR can be 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, can be preferably cN utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease. Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the functional role of the receptor can be deduced.

C As the data herein indicate, hRUP3 is expressed within the human pancreas, suggesting O that RUP3 may play a role in insulin regulation and/or glucagon regulation.

Accordingly, candidate compounds identified using a constitutively activated form of RUP3 may be useful for understanding the role of RUP3 in diabetes and/or as therapeutics for diabetes.

Similarly, hRUP7 has been determined to be specifically expressed in the peripheral blood leukocytes especially in eosinophils, and in the spleen and thymus. This distribution of RUP7 and the high similarity of this receptor with human H3 receptor suggests that RUP7 mediates histamine actions on immuno-active cells. Thus, by way of example only, compounds that modulate RUP7 are intended to be used as potential anti-inflammatory drugs or in the prophylactic or therapeutic treatment of asthma.

Additionally, hARE-2 has been determined to be expressed preferentially in adult and fetal brain and in particular, left and right cerebella and substantia nigra. This tissue distribution and similarity with hGPCR27 which is expressed in similar tissues suggest a role for these receptors in modulating motor function, that compromised in Parkinson's disease, and/or neurotransmitter release.

Additionally, hCHN4 has been determined to be expressed preferentially in leukocytes, including neutrophils, B-cells and T-cells, and in the spleen. The distribution of hCHN4 and the similarity of this receptor with human PAR-1 suggests that CHN4 mediates inflammatory and/or autoimmune responses. By way of example, compounds that modulate hCHN4 are intended to be used in the treatment of inflammation or autoimmune diseases/disorders.

Screening of Candidate Compounds 1. Generic GPCR screening assay techniques When a G protein receptor becomes constitutively active active in the absence of endogenous ligand binding thereto), it binds to a G protein Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly 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, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that 35 S]GTPyS can be 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 preferred use of this assay system is 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, partial 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.

2a. Gs and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the other hand, inhibit this enzyme. 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 c the Gi (or Go) protein are associated with decreased cellular levels of cAMP. See, generally, "Indirect Mechanisms of Synaptic Transmission,"Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays _that detect cAMP can be utilized to determine if a candidate compound is, e. an inverse agonist to the receptor such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; C a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISAbased format. Another type of assay that can be 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 cAMP-responsive DNA binding protein or transcription factor (CREB) which then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e. p-galactosidase or luciferase. Thus, a constitutively activated Gslinked receptor causes the accumulation of cAMP that then activates the gene and 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).

2b. 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-triphosphate (IP 3 Increased accumulation of IP 3 is associated with activation of Gq-and Go-associated receptors. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP 3 accumulation can be utilized to determine if a candidate compound is, e. an inverse agonist to a Gq-or Go associated receptor such a compound would decrease the levels of IP 3 Gq-associated receptors can also been examine using an API reporter assay in that 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 Protein The use of an endogenous, constitutively activate orphan GPCR, or a non-endogenous, constitutively activated orphan GPCR, for screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provides a unique challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, it is often useful that an approach be utilized that can enhance the signal obtained by the activated receptor. A preferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a GPCR is constitutively active or 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 can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the constitutively activated orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e. 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 GPCR. The GPCR Fusion Protein is preferred for screening with a nonendogenous. constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques, although the GPCR Fusion Protein can also be (and preferably is) used with an endogenous, constitutively activated GPCR. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is import 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 variety of approaches that can fit the particular needs of an investigator.

A variety of expression vectors are available to those in the art, for purposes of utilization for both endogenous and non-endogenous human GPCRs and fusion proteins comprising same. In one embodiment, the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 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. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

The criteria of importance for such a GPCR Fusion Protein construct is that the GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the GPCR is upstream of the G protein sequence) and that the "stop" codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). We have a preference (based upon convenience) of use of a spacer in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the 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) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.

Other Utility Although a preferred use of the human orphan GPCRs disclosed herein may be for the identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these GPCRs can also be utilized in research settings. For example, in vitro and in vivo systems incorporating GPCRs can be 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. The value in human orphan GPCRs is that its utility as a research tool is enhanced in that by determining the location(s) of such receptors within the body, the GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified.

Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.

Examples The following 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. Unless otherwise indicated below, all nucleotide sequences for the disclosed endogenous orphan human GPCRs have been sequenced and verified. For purposes of equivalent receptors, those of ordinary skill in the art will readily appreciate that conservative substitutions can be made to the disclosed sequences to obtain a functionally equivalent receptor.

Example 1 Endogenous human GPCRs 69

O

C1 1. Identification of Human GPCRs Several of the disclosed endogenous human GPCRs were identified based upon a Sreview of the GenBank database information. While searching the database, the following cDNA clones were identified as evidenced below in Table B.

TABLE B Disclosed Accession Complete DNA Open Reading Nucleic Amino Human Number Sequence Frame Acid Acid Orphan (Base Pairs) (Base Pairs) SEQ ID SEQ ID GPCRs No. No.

hARE-3 AL033379 111,389 bp 1,260 bp 1 2 hARE-4 AC006087 226,925 bp 1, 119 bp 3 4 AC006255 127,605 bp 1,104 bp 5 6 hRUP3 AL035423 140,094 bp 1,005 bp 7 8 AC005849 169,144 bp 1,413 bp 9 hRUP6 AC005871 218,807 bp 1,245 bp 11 12 hRUP7 AC007922 158,858 bp 1, 173 bp 13 14 Other disclosed endogenous human GPCRs were identified by conducting a BLAST(TM) search of EST database (dbest) using the following EST clones as query sequences. The following EST clones (TABLE C) identified were then used as a probe to screen a human genomic library.

TABLE C Disclosed Query EST Clone/ Open Reading Nucleic Amino Human (Sequence) Accession No. Frame Acid Acid Orphan Identified (Base Pairs) SEQ ID SEQ ID GPCRs No. No.

hGPCR27 Mouse AA775870 1,125 bp 15 16 GPCR27 hARE-I TDAG 1689643 999 bp 17 18 A1090920 hARE-2 GPCR27 68530 1,122 bp 19 AA359504 hPPRI Bovine 238667 1,053 bp 21 22 PPRI H67224 hG2A Mouse See Example 1,113 bp 23 24 1179426 below hCHN3 N.A. EST 36581 1,113 bp 25 26 (full length) hCHN4 TDAG 1184934 1,077 bp 27 28 AA804531 hCHN6 N.A. EST 2134670 1,503 bp 29 (full length) hCHN8 KIAA0001 EST 764455 1,029 bp 31 32 hCHN9 1365839 EST 1541536 1,077 bp 33 34 hCHNIO Mouse EST Human 1365839 1,005 bp 35 36 1365839 hRUP4 N.A. A1307658 1,296 bp 37 38 S N.A. "not applicable".

2. Full Length Cloning 2a. hG2A (Seq.Id. Nos. 23 24) Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid hG2A coding sequences. The 5' end of this coding sequence was obtained by using 5' RACE

TM

and the template for PCR was Clontech's Human Spleen Marathon-Ready T M cDNA. The disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ ID NO: 39 and SEQ ID NO: 40 as follows: 5'-CTGTGTACAGCAGTTCGCAGAGTG-3' (SEQ ID NO: 39; 1st round PCR) 5'-GAGTGCCAGGCAGAGCAGGTAGAC-3' (SEQ ID NO: 40; second round PCR).

PCR was performed using Advantage M GC Polymerase Kit (Clontech; manufacturing instructions is followed), at 94 0 C. for 30 sec followed by 5 cycles of 94°C. for 5 sec and 72 0 C. for 4 min; and 30 cycles of 94°C. for 5 sec and 70° for 4 min. An approximate 1.3 Kb PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen). The N cloned-insert was sequenced using the T7 Sequenase TM kit (USB Amersham; Smanufacturer instructions is followed) and the sequence was compared with the 0presented sequence. Expression of the human G2A is detected by probing an RNA dot c blot (Clontech; manufacturer instructions is followed) with the P 32 -labeled fragment.

The hG2A open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

b. hCHN9 (Seq.Id. Nos. 33 34) Sequencing of the EST clone 1541536 indicated that hCHN9 is a partial cDNA clone having only an initiation codon; the termination codon was missing. When hCHN9 was used to "blast" against data base the 3' sequence of hCHN9 was 100% homologous to the 5' untranslated region of the leukotriene B4 receptor cDNA, which contained a termination codon in the frame with hCHN9 coding sequence. To determine whether the 5' untranslated region of LTB4R cDNA was the 3' sequence of hCHN9, PCR was performed using primers based upon the 5' sequence flanking the initiation codon found in hCHN9 and the 3' sequence around the termination codon found in the LTB4R 5' untranslated region. The 5' primer sequence utilized was as follows: 5'-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3' (SEQ ID NO: 41; sense) and 5'-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3' (SEQ ID NO: 42; antisense).

PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 M of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94°C. for 1 min, 65 0 C. for 1 min and 72 0 C. for 1 min and 10 sec. A 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV for expression as described according to the general procedure described in Example 2, and for sequence analysis (see, SEQ ID NO: 33).

c. hRUP4 (Seq. Id. Nos. 37 38) The full length RUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates: 5'-TCACAATGCTAGGTGTGGTC-3' (SEQ ID NO: 43; sense) and 5'-TGCATAGACAATGGGATTACAG-3' (SEQ ID NO: 44; antisense).

PCR was performed using TaqPlus T M Precision T M polymerase (Stratagene; manufacturing instructions followed) by the following cycles: 94°C for 2 min; 94 0 C. sec; 55 0 C. for 30 sec, 72°C. for 45 sec, and 72 0 C. for 10 min. Cycles 2 through 4 were repeated 30 times.

The PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the T7 DNA Sequenase T M kit (Amsham) and the SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCR fragment was indeed an alternatively spliced form of AI307658 having a continuous open reading frame with similarity to other GPCRs. The completed sequence of this PCR fragment was as follows:

GGCACGTGCAACAACTTGAGATCAAATATGACTTCCTATATGAAAAGGAACACAT

CTGCTGCTTAAGAGTGGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATC

CTTGTCATCCTCTTCCTCCTGCCTCTTATGGTGATGCTTATTCTGTACGTAAAATTGG

TTATGAACFFI'GGATAAAGAAAAGAGTTGGGGATGGTTCAGTGCTTCGAACTATTC

ATGGAAAAGAAATGTCCAAAATAGCCAGGAAGAAGAAACGAGCTGTCATTATGAT

GGTGACAGTGGTGGCTCTCTTTGCTGTGCTGGGCACCATTCCATGTTGTCCATATGA

TGATTGAATACAGTAATTF[GAAAAGGAATATGATGATGTCACAATCAAGATGATT

TTTGCTATCGTGCAAATFAITGGAYFF-FCCAACTCCATCTGTAATCCCATTFGTCTAT

GCA-3' (SEQ ID NO: Based on the above sequence, two sense oligonucleotide primer sets: 5'-CTGCTTAGAAGAGTGGACCAG-3' (SEQ.ID.NO: 46; oligo 1), 5'-CTGTGCACCAGAAGATCTACAC-3' (SEQ.ID NO: 47; oligo 2) and two antisense oligonucleotide primer sets: 5'-CAAGGATGAAGGTGGTGTAGA-3' (SEQ ID NO: 48; oligo 3) 5'-GTGTAGATGTTCTGGTGCAGAGG-3' (SEQ ID NO: 49; oligo 4) were used for and 5'-RACE PCR with a human brain Marathon-Ready TM cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions. DNA fragments generated by the RACE PCR were cloned into the pCRII-TOpOT vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers. The 3' RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon. The 5' RACE product contained an incomplete end; the ATG initiation codon was not present.

Based on the new 5' sequence, oligo 3 and the following primer: (SEQ ID NO: 50; oligo were used for the second round of 5' race PCR and the PCR products were analyzed as above.

A third round of 5' race PCR was carried out utilizing antisense primers: 5'-TGGAGCATGGTGACGGGAATGCAGAAG-3' (SEQ ID NO: 5 1; oligo 6) and 5'-GTGATGAGCAGGTCACTGAGCGCCAAG-3' (SEQ ID NO: 52; oligo7).

The sequence of the 5' RACE PCR products revealed the presence of the initiation codon ATG, and further round of 5' race PCR did not generate any more 5' sequence.

The completed 5' sequence was confirmed by RT-PCR using sense primer 5'-GCAATGCAGGCGCTTAACATTAC-3' (SEQ ID NO: 53; oligo 8) and oligo 4 as primers and sequence analysis of the 650 bp PCR product generated from human brain and heart cDNA templates (Clontech, Cat# 7404-1). The completed 3' sequence was confirmed by RT-PCR using oligo 2 and the following antisense primer: 5'-TTGGGTTACAATCTGAAGGGCA-3' (SEQ ID NO: 54; oligo 9) and sequence analysis of the 670 bp PCR product generated from human brain and heart cDNA templates. (Clontech, Cat# 7404-1).

The hRUP4 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

d. hRUPS (Seq. Id. Nos. 38 39) The full length hRUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ ID NO: 55), and an antisense primer containing TCA as the stop codon (SEQ ID NO: 56), which had the following sequences: 5'-ACTCCGTGTCCAGCAGGACTCTG-3' (SEQ ID NO: 5'-TGCGTGTTCCTGGACCCTCACGTG-3' (SEQ ID NO: 56) and human peripheral leukocyte cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50ul reaction by the following cycle with step 2 through step 4 repeated 30 times: 94°C. for 30 sec; 94° for sec; 69° for 40 sec; 72 0 C. for 3 min; and 72 0 C. fro 6 min. A 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPO T M vector (Invitrogen) and completely sequenced using the T7 DNA Sequenase T M kit (Amsham). See, SEQ ID NO: 9.

The hRUP5 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

e. hRUP6 (Seq. Id. Nos. 11 12) The full length hRUP6 was cloned by RT-PCR using primers: 5'-CAGGCCTTGGATTTTAATGTCAGGGATGG-3' (SEQ ID NO: 57) and 5'-GGAGAGTCAGCTCTGAAAGAATTCAGG-3' (SEQ ID NO: 58); and human thymus Marathon-Ready TM cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech, according to manufacturer's instructions) was used for the amplification in a 50ul reaction by the following cycle: 94 0 C for 30 sec; 94 0 C for sec; 66 0 C for 40 sec; 72 0 C for 2.5 sec and 72 0 C. for 7 min. Cycles 2 through 4 were repeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-

TOPO

T M vector (Invitrogen) and completely sequenced (see, SEQ ID NO: 11) using the ABI Big Dye TerminatorTM kit Biosystem).

The hRUP6 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

f RUP7 (Seq. Id. Nos. 13 14) The full length RUP7 was cloned by RT-PCR using primers: 5'-TGATGTGATGCCAGATACTAATAGCAC-3' (SEQ ID NO: 59; sense) and 5'-CCTGATTCATTTAGGTGAGATTGAGAC-3' (SEQ ID NO: 60; antisense) and human peripheral leukocyte cDNA (Clontech) as a template. Advantage TM cDNA polymerase (Clontech) was used for the amplification in a 50ul reaction by the following cycle with step 2 to step 4 repeated 30 times: 94 0 C for 2 minutes; 94°C for seconds; 60 0 C for 20 seconds; 72 0 C for 2 minutes; 72 0 C for 10 minutes. A 1.25 Kb PCR fragment was isolated and cloned into the pCRII-TOPO TM vector (Invitrogen) and completely sequenced using the ABI Big Dye TerminatorTM kit Biosystem). See, SEQ ID NO: 13.

The hRUP7 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

g. hARE-5 (Seq. Id. Nos. 5 6) The full length hARE-5 was cloned by PCR using the hARE5 specific primers: 5'-CAGCGCAGGGTGAAGCCTGAGAGC-3' (SEQ ID NO: 69; sense), 5' of initiation codon ATG, and 5'-GGCACCTGCTGTGACCTGTGCAGG-3' (SEQ ID NO: 70; antisense), 3' of stop codon TGA and human genomic DNA as template. TaqPlus Precision T DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 96 0 C, 2 minutes; 96 0 C, 20 seconds; 58 0 C, 30 seconds; 72 0 C, 2 minutes; and 72 0 C, 10 minutes.

A 1.1 Kb PCR fragment of predicated size was isolated and cloned into the pCRII- TOPO'M vector (Invitrogen) and completely sequenced (SEQ ID NO: 5) using the T7 DNA Sequenase T M kit (Amsham).

The hARE-5 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

h. hARE-4 (Seq. Id. Nos.: 3 4) The full length hARE-4 was cloned by PCR using the hARE-4 specific primers: 78

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CTGGTGTGCTCCATGGCATCCC-3' (SEQ ID NO: 67; sense), 5' of initiation codon ATG, and 5'-GTAAGCCTCCCAGAACGAGAGG-3' (SEQ ID NO: 68; antisense), 3' of stop codon TGA, and human genomic DNA as template. Taq DNA polymerase (Stratagene) and

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DMSO was used for the amplification by the following cycle with step 2 to step 3 Srepeated 35 times: 94 0 C, 3 minutes; 94°C, 30 seconds; 59 0 C, 2 minutes; 72 0 C, 0minutes.

A 1.12 Kb PCR fragment of predicated size was isolated and cloned into the pCRII- TOPOTM vector (Invitrogen) and completely sequenced (SEQ ID NO: 3) using the T7 DNA SequenaseT kit (Amsham).

The hARE-4 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

i. hARE-3 (Seq. Id. Nos.: 1 2) The full length hARE-3 was cloned by PCR using the hARE-3 specific primers: 5'-gatcaagcttCCATCCTACTGAAACCATGGTC-3' (SEQ ID NO: 65; sense), wherein lower case nucleotides represent HindlIl overhang and ATG is the translation initiation codon, and 5'-gatcagatctCAGTTCCAATATTCACACCACCGTC-3' (SEQ ID NO: 66; antisense), wherein lower case nucleotides represent XbaI overhang and trinucleotide TCA is the complement of a translation stop codon in the gene, and human genomic DNA as template. TaqPlusPrecision DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94°C, 3 minutes; 94 0 C, 1 minute; 55 0 C, 1 minute; 72 0 C, 2 minutes; 72 0 C, 10 minutes.

A 1.3 Kb PCR fragment of predicated size was isolated and digested with Hind III and Xba I, cloned into the pRC/CMV2 vector (Invitrogen) at the Hind III and Xba I sites and completely sequenced (SEQ ID NO: 1) using the T7 DNA Sequenase kit (Amsham).

The hARE-3 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

j. hRUP3 (Seq. Id. Nos.: 7 8) The full length hRUP3 was cloned by PCR using the hRUP3 specific primers: 5'-GTCCTGCCACTTCGAGACATGG-3' (SEQ ID NO: 71; sense), wherein ATG is the traslation initiation codon, and 5'-GAAACTTCTCTGCCCTTACCGTC-3' (SEQ ID NO: 72; antisense) 3' of stop codon TAA, and human genomic DNA as template. TaqPlusPrecision T M DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94°C, 3 minutes; 94 0 C, 1 minute; 58 0 C, 1 minute; 72 0 C, 2 minutes; 72 0 C, 10 minutes.

A 1.0 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-

TOPO'

M vector (Invitrogen) and completely sequenced (SEQ ID NO: 7) using the T7 DNA sequenase kit (Amsham).

The hRUP3 open reading frame is also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

k. hARE-2 (Seq. Id. Nos.: 19 The disclosed human ARE-2 gene was identified based upon the use of EST database information. The nucleotide sequence of human GPCR27 (SEQ ID NO: 15) was used to conduct a BLAST search of the EST database ("dbest" search). EST clone 68530 (Genbank Accession Number AA359504) was identified from this search and then used c as a probe to screen a human genomic library (Stratagene, #942503), following the manufacturer instructions. This screening resulted in the identification and purification Sof a genomic clone which was mapped using restriction enzymes and Southern NI hybridization techniques. A fragment of the genomic clone containing a coding sequence was thus localized, subcloned into pBluScript (Stratagene), and the nucleotide sequence determined. The nucleotide sequence of the open reading frame is set forth in SEQ ID NO: 19. The encoded amino acid sequence for ARE-2 is set forth in SEQ ID NO: The hARE-2 open reading frame was also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

1. hCHN4 (Seq. Id. Nos.: 27 28) The disclosed human CHN4 gene was identified based upon the use of EST database information. The nucleotide sequence of human TDAG receptor was used to conduct a BLAST search of the EST database ("dbest" search). EST clone 1184934 (Genbank Accession Number AA804531) was identified from this search and then used as a probe to screen a human genomic library (Stratagene, #942503), following the manufacturer instructions. This screening resulted in the identification and purification of a genomic clone which was mapped using restriction enzymes and Southern hybridization techniques. A fragment of the genomic clone containing a coding sequence was thus localized, subcloned into pBluScript (Stratagene), and the nucleotide sequence determined. The nucleotide sequence of the open reading frame is set forth in SEQ ID NO: 27. The encoded amino acid sequence for CHN4 is set forth in SEQ ID NO: 28.

The hCHN4 open reading frame was also sub-cloned into pCMV for expression as described according to the general procedure described in Example 2.

Example 2 Receptor expression A variety of cells are available to the art for the expression of proteins, however the use of eukaryotic cells is preferred for the expression of GPCRs or constitutively activated variants thereof or GPCR fusion proteins.

In one embodiment, melanocytes are used to express a GPCR or constitutively activated variants thereof or GPCR fusion protein. Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomes.

Melanophores are able to redistribute these melanosomes along a microtubule network upon G-protein coupled receptor (GPCR) activation. The result of this pigment movement is an apparent lightening or darkening of the cells. In melanophores, the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAMP levels are then raised, following activation of a Gscoupled receptor, the melanosomes are re-dispersed and the cells appear dark again.

The increased levels of diacylglycerol that result from activation of Gq-coupled receptors can also induce this re-dispersion. In addition, the technology is also suited to the study of certain receptor tyrosine kinases. The response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change.

The response can be easily detected using a conventional absorbance microplate reader or a modest video imaging system. Unlike other skin cells, the melanophores derive from the neural crest and appear to express a full complement of signaling proteins. In particular, the cells express an extremely wide range of G-proteins and so are able to functionally express almost all GPCRs.

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I Melanophores are utilized to identify compounds, including natural ligands, against GPCRs. This method can be conducted by introducing test cells of a pigment cell line 0capable of dispersing or aggregating their pigment in response to a specific stimulus N and expressing an exogenous clone coding for the GCPR. A stimulant, melatonin, 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 test 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, including but not limited to a ligand, coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.

The use of melanophores to screen for modulators of GPCR activity and/or expression is in accordance with the materials and methods described in U.S. Patent Number 5,462,856 and U.S. Patent Number 6,051,386. These patent disclosures are hereby incorporated by reference in their entirety.

In particular, the melanophores are plated in e.g. 96-well plates (one receptor per plate).

48 hours post-transfection, half of the cells on each plate are treated with melatonin. Melatonin activates an endogenous Gi-coupled receptor in the melanophores and causes them to aggregate their pigment. The remaining half of the cells are transferred to serum-free medium 0.7X L-15 (Gibco). After one hour, the cells in serum-free media remain in a pigment-dispersed state while the melatonintreated cells are in a pigment-aggregated state. At this point, the cells are treated with a dose response of a test/candidate compound. If the plated GPCRs bind to the test/candidate compound, the melanophores would be expected to undergo a color change in response to the compound. If the receptor were either a Gs or Gq coupled receptor, then the melatonin-aggregated melanophores would undergo pigment dispersion. In contrast, if the receptor was a Gi-coupled receptor, then the pigmentdispersed cells would be expected to undergo a dose-dependent pigment aggregation.

In other embodiments of the invention, mammalian cells are utilized. Preferred mammalian cells include, but are not limited to CHO cells, COS-7 cells, 293 cells and 293T cells, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan and the tissue in which the GPCR is expressed in nature.

For the purposes of exemplification, the general procedure for expression of the disclosed GPCRs in 293T cells is as follows.

On day one, 1X10 7 293T cells per 150mm plate 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 20p.g DNA pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2ml serum free DMEM (Irvine Scientific, Irvine, CA); tube B is prepared by mixing 120pl lipofectamine (Gibco BRL) in 1.2mI serum free DMEM.

Tubes A and B are admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the "transfection mixture". Plated 293T cells are washed with IX PBS, followed by addition of serum free DMEM. 2.4ml 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 84 removed by aspiration, followed by the addition of 25ml of DMEM/10% Fetal Bovine Serum. Cells are incubated at 37 0 C/5% CO 2 After 72hr incubation, cells are harvested and utilized for analysis.

Example 3 N Tissue distribution of human GPCRs C. Several approaches are used for determination of the tissue distribution of the GPCRs Sdisclosed herein.

(N 1. Dot-Blot Analysis Using a commercially available human-tissue dot-blot format, endogenous orphan GPCRs were probed for a determination of the areas where such receptors are localized. cDNA fragments from the GPCRs of Example 1 (radiolabeled) were (or can be) used as the probe: radiolabeled probe was (or can be) generated using the complete receptor cDNA (excised from the vector) using a Prime-It IITM Random Primer Labeling Kit (Stratagene, #300385), according to manufacturer's instructions. A human RNA Master BlotTM (Clontech, #7770-1) was hybridized with the endogenous human GPCR radiolabeled probe and washed under stringent conditions according manufacturer's instructions. The blot was exposed to Kodak BioMaxTM Autoradiography film overnight at-80 0 C. Results are summarized for several receptors in Table D and E (see Figures 1A and 1B for a grid identifying the various tissues and their locations, respectively). Exemplary dot-blots are provided in Figure 2A and 2B for results derived using hCHN3 and hCHN8, respectively.

TABLE D Orphan GPCR Tissue Distribution (highest levels, relative to other tissues in the dot-blot) hGPCR27 hARE-1 hPPRI hRUP3 hCHN3 hCHN9 hCHNIO Fetal brain, Putamen, Pituitary gland, Caudate nucleus Spleen, Peripheral blood leukocytes, Fetal spleen Pituitary gland, Heart, salivary gland, Small intestine, Testis Pancreas Fetal brain, Putamen, Occipital cortex Pancreas, Small intestine, Liver Kidney, Thyroid TABLE E Orphan GPCR hARE-3 hGPCR3 hARE-2 hCHN8 Tissue Distribution (highest levels, relative to other tissues in the dot-blot) Cerebellum left, Cerebellum right, Testis, Accumbens Corpus callosum, Caudate nucleus, Liver, Heart, Inter- Ventricular Septum Cerebellum left, Cerebellum right, Substantia Nigra, Fetal Brain Cerebellum left, Cerebellum right, Kidney, Lung 2. GeneChip analyses a. hRUP7 The amino acid sequence of hRUP7 (SEQ ID NO: 14) was submitted to Affymetrix for the design and manufacture of a microarray containing oligonucleotides specific for the GPCR, to monitor its expression using their GeneChip® Technology. Also present on the microarray were probes for characterized human brain tissues from Harvard Brain Band or obtained from commercially available sources. RNA samples were amplified, labeled, hybridized to the microarray, and data analyzed according to manufacturer's instructions.

As shown in Figure 6, expression of hRUP7 was highest in eosinophils.

b. hCHN4 The amino acid sequence of hCHN4 (SEQ ID NO: 28) was submitted to Affymetrix for the design and manufacture of a microarray containing oligonucleotides specific for the GPCR, to monitor its expression using their GeneChip® Technology. Also present on the microarray were probes for characterized human brain tissues from Harvard Brain Band or obtained from commercially available sources. RNA samples were amplified, labeled, hybridized to the microarray, and data analyzed according to manufacturer's instructions.

As shown in Figure 7, hCHN4 expression was detected strongly in leukocytes, including neutrophils, B-cells and T-cells, and in spleen tissue.

3. RT-PCR a. hRUP3 To ascertain the tissue distribution of hRUP3 mRNA, RT-PCR was performed using hRUP3-specific primers and human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was utilized for the PCR reaction, using the following reaction cycles in a 40ul reaction: 94 0 C for 2 min; 94°C for 15 sec; for 30 sec; 72 0 C for 1 min; 72 0 C, for 10 min. Primers were as follows: 5'-GACAGGTACCTTGCCATCAAG-3' (SEQ ID NO: 61; sense) 5-CTGCACAATGCCAGTGATAAGG-3' (SEQ ID NO: 62; antisense).

of the reaction was loaded onto a 1% agarose gel; results are set forth in Figure 3.

As is supported by the data of Figure 3, of the 16 human tissues in the cDNA panel utilized (brain, colon, heart, kidney, lung, ovary, pancreas, placenta, prostate, skeleton, small intestine, spleen, testis, thymus leukocyte, and liver) a single hRUP3 band is evident only from the pancreas. Additional comparative analysis of the protein sequence of hRUP3 with other GPCRs suggest that hRUP3 is related to GPCRs having small molecule endogenous ligand such that it is predicted that the endogenous ligand for hRUP3 is a small molecule.

b. hRUP4 RT-PCR was performed using hRUP4 oligo's 8 and 4 as primers and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was used for the amplification in a 40ul reaction by the following cycles: 94°C for 30 seconds, 94 0 C for 10 seconds, 55°C for 30 seconds, 72 0 C for 2 minutes, and 72 0 C for 5 minutes with cycles 2 through 4 repeated 30 times.

20 pl of the reaction were loaded on a 1 agarose gel to analyze the RT-PCR products, and hRUP4 mRNA was found expressed in many human tissues, with the strongest expression in heart and kidney. (see, Figure To confirm the authenticity of the PCR fragments, a 300 bp fragment derived from the 5' end of hRUP4 was used as a probe for the Southern Blot analysis. The probe was labeled with 32 P-dCTP using the Prime-It II M Random Primer Labeling Kit (Stratagene) and purified using the ProbeQuant T M G-50 microcolumns (Amersham). Hybridization was done overnight at 42 0 C following a 12 hr pre-hybridization. The blot was finally washed at 65 0 C with 0.1 x SSC. The Southern blot did confirm the PCR fragments as hRUP4.

r c. RT-PCR was performed using the following hRUP5 specific primers: 5'-CTGACTTCTTGTTCCTGGCAGCAGCGG-3' (SEQ ID NO: 63; sense) S'-AGACCAGCCAGGGCACGCTGAAGAGTG-3' (SEQ ID NO: 64; antisense) and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was used for the amplification in a 40ul reaction by the following cycles: 94 0 C for 30 sec, 94 0 C for 10 sec, 62°C for 1.5 min, 72 0 C for 5 min, and with cycles 2 through 3 repeated 30 times. 20 pl of the reaction were loaded on a 1.5% agarose gel to analyze the RT-PCR products, and hRUP5 mRNA was found expressed only in the peripheral blood leukocytes (data not shown).

d. hRUP6 RT-PCR was applied to confirm the expression and to determine the tissue distribution of hRUP6. Oligonucleotides used, based on an alignment of AC005871 and GPR66 segments, had the following sequences: 5'-CCAACACCAGCATCCATGGCATCAAG-3' (SEQ ID NO: 73; sense), 5'-GGAGAGTCAGCTCTGAAAGAATTCAGG-3' (SEQ ID NO: 74; antisense) and the human multiple tissue cDNA panels (MTC, Clontech) were used as templates.

PCR was performed using TaqPlus Precision T M polymerase (Stratagene; manufacturing instructions will be followed) in a 40ul reaction by the following cycles: 94 0 C for sec; 94°C 5 sec; 66 0 C for 40 sec, 72 0 C for 2.5 min, and 72 0 C for 7 min. Cycles 2 through 4 were repeated 30 times.

20 ul of the reaction were loaded on a 1.2% agarose gel to analyze the RT-PCR products, and a specific 760bp DNA fragment representing hRUP6 was expressed predominantly in the thymus and with less expression in the heart, kidney, lung, prostate small intestine and testis. (see, Figure e. hRUP7 RT-PCR was applied to confirm the expression and to determine the tissue distribution of RUP7. Oligonucleotides, based on an alignment of AC007922 and human H3 receptor segments, had the following sequences: c, 5'-CTGTGGACACTCATTCAGAGGTAGAC-3' (SEQ ID NO: 75; sense) and

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CI 5'-GCCAGTGACTTGGCTAATCTCCTGGC-3' (SEQ ID NO: 76; antisense) and the human multiple tissue DNA panels (MTC, Clontech) were used as templates.

Taq DNA polymerase (Stratagene) was used for the amplification in a 40 ul reaction by the following cycle with step 2 to step 4 repeated 30 times: 94 0 C for 2 minutes; 94°C for 15 seconds; 60 0 C for 20 seconds; 72 0 C for 40 seconds; 72 0 C for 10 minutes. 20 ul of the reaction was loaded on a 1.5% agarose gel to analyze the RT-PCR products, and a specific 310 bp DNA fragments representing RUP7 was detected strongly in peripheral blood leukocytes, spleen, thymus, and weakly in kidney and small intestine.

0 u Example 4 Preparation of Non-Endogenous, Constitutively Activated GPCRs 1. hRUP3 Preparation of the non-endogenous human RUP3 receptor that may evidence constitutive activation of the receptor disclosed herein is accomplished by creating a c mutation at position 224L, most preferably an L224K mutation. Mutagenesis is preferably performed using a Transformer Site-Directed TM Mutagenesis Kit (Clontech) Saccording to manufacturer's instructions. The two mutagenesis primers are to be utilized, a lysine mutagenesis oligonucleotide that creates the lysine mutation at amino acid position 224L changing CTC to AAA at nucleotides 670-672) and a selection marker oligonucleotide.

2. hRUP7 Preparation of the non-endogenous human RUP7 receptor that evidences constitutive activation of the hRUP7 receptor disclosed herein is achieved by creating a mutation at position 302A (Figure most preferably by substituting alanine for lysine RUP7 A302K). Mutagenesis is preferably performed using a Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to manufacturer's instructions. Two mutagenesis primers are utilized, a lysine mutagenesis oligonucleotide that creates the lysine mutation at amino acid position 302A changing GCC to AAA at nucleotides 904- 906 of SEQ ID NO: 13) and a selection marker oligonucleotide.

3. hARE-2 Preparation of the non-endogenous human ARE-2 receptor that evidences constitutive activation of the hARE-2 receptor disclosed herein is achieved by creating a mutation at position 285G (Figure most preferably by substituting glycine for lysine at this position hARE-2 G285K). Mutagenesis is preferably performed using a Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to manufacturer's instructions. Two mutagenesis primers are utilized, a lysine mutagenesis oligonucleotide that creates the lysine mutation at amino acid position 285G changing GGC to AAA at nucleotides 853-855 of SEQ ID NO: 19) and a selection marker oligonucleotide.

4. hCHN4 Preparation of the non-endogenous human CHN4 receptor that evidences constitutive activation of the CHN4 receptor disclosed herein is achieved by creating a mutation at position 236V (Figure 10) most preferably by substituting valine for lysine at this position hCHN4 V236K). Mutagenesis is preferably performed using a Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to manufacturer's instructions. Two mutagenesis primers are utilized, a lysine mutagenesis oligonucleotide that creates the lysine mutation at amino acid position 236V changing GTG to AAA at nucleotides 706-708 of SEQ ID NO: 27) and a selection marker oligonucleotide.

Claims

1. An isolated polynucleotide encoding a G protein-coupled receptor polypeptide, wherein said polynucleotide comprises a nucleic acid that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample using sequence specific primers SEQ ID NO: 61 and SEQ ID NO: 62.

2. An isolated or recombinant G protein-coupled receptor, wherein said G protein- coupled receptor comprises an amino acid sequence encoded by a nucleic acid that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample using sequence specific primers SEQ ID NO: 61 and SEQ ID NO: 62.

3. A method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell or an isolated membrane comprising a G protein-coupled receptor which is an endogenous or non-endogenous version of a G protein-coupled receptor having SEQ ID NO: 8; and measuring the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor, wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of a G protein-coupled receptor.

4. A compound for the treatment of a disorder of the pancreas in a subject. Dated this ELEVENTH day of MAY 2007 Arena Pharmaceuticals, Inc. Patent Attorneys for the Applicant: F B RICE CO