AU2010201829B2 - Endogenous and non-endogenous versions of human G protein-coupled receptors - Google Patents

Abstract

The invention disclosed in this patent document relates to transmembrane receptor polypeptides, more particularly to orphan G protein-coupled receptor (GPCR) polypeptides designated hRUP8, hRUP9, hRUP10, hRUP11, hRUP12, hRUP13, hRUP14, hRUP15, hRUP16, hRUP17, hRUP18, hRUP19, hRUP20, hRUP21, hRUP22, hRUP23, hRUP24, hRUP25, hRUP26 and hRUP27 and to non-endogenous variants thereof, especially ligand-independent active versions of the endogenous GPCR polypeptides and GPCR fusion protein comprising said GPCR polypeptides. The present invention also relates to isolated nucleic acid encoding the endogenous and non-endogenous GPCR polypeptides and GPCR fusion proteins. The present invention also relates to the use of the endogenous and non-endogenous GPCR polypeptides and GPCR fusion proteins and nucleic acids encoding same to identify and isolate modulatory compounds and formulate those modulatory compounds as pharmaceutical compositions for therapy.

Description

AUSTRALIA Patents Act 1990 FB RICE & CO Patent and Trade Mark Attorneys ARENA PHARMACEUTICALS, INC. COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Endogenous and non-endogenous versions of human G protein-coupled receptors The following statement is a full description of this invention including the best method of performing it known to us: 1 ENDOGENOUS AND NON-ENDOGENOUS VERSIONS OF HUMAN G PROTEIN-COUPLED RECEPTORS CROSS REFERENCE TO RELATED APPLICATIONS 5 This application is a divisional application under Section 79B of the Patents Act 1990 of Patent Application No. 2005244540 filed December 15, 2005 as a divisional application under Section 79B of the Patents Act 1990 of Patent Application No. 200117696 which corresponds to International Patent Application No. PCT/US2000/031509 filed on November 16, 2000 and which claims the benefit of 10 priority from USSN 60/166088 filed November 17, 1999 and USSN 60/166099 filed November 17, 1999 and USSN 60/166369 filed November 17, 1999 and USSN 60/171900 filed December 23, 1999 and USSN 60/171901 filed December 23, 1999 and USSN 60/171902 filed December 23, 1999 and USSN 60/181749 filed February 11, 2000 and USSN 60/189258 filed March 14, 2000 and USSN 60/189259 filed March 14, 15 2000 and USSN 60/195898 filed April 10, 2000 and USSN 60/195899 filed April 10, 2000 and USSN 60/196078 filed April 10, 2000 and USSN 60/200419 filed April 28, 2000 and USSN 60/203630 filed May 12, 2000 and USSN 60/210741 filed June 12, 2000 and USSN 60/210982 filed June 12, 2000 and USSN 60/226760 filed August 21, 2000 and USSN 60/235418 filed September 26, 2000 and USSN 60/235779 September 20 26, 2000 and USSN 60/242332 filed October 20, 2000 and USSN 60/242343 filed October 20, 2000 and USSN 60/243019 filed October 24, 2000. The contents of each foregoing application is incorporated herein in its entirety by way of reference. 2 FIELD OF THE INVENTION The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to human G protein-coupled receptors, and specifically to endogenous human GPCRs with particular emphasis on non- endogenous versions of 5 the GPCRs that have been altered to establish or enhance constitutive activity of the receptor. The invention also relates to the use of the GPCRs for the identification of receptor agonists, inverse agonists or partial agonists of the receptors and their therapeutic utility. 10 BACKGROUND OF THE INVENTION Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the GPCR polypeptide (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 15 GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as "known" receptors, while receptors for which the endogenous ligand has not been identified are referred to as "orphan" receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription 20 pharmaceuticals have been developed. 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, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices 25 are joined by strands of amino acids between transmembrane-2 and transmembrane-3, 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 by strands of amino acids between 5 transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular" side, of the cell membrane (these are referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and IC- 3), respectively). The "carboxy" ("C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" ("N') terminus of the receptor lies in 10 the extracellular space outside of the cell. Generally, when an endogenous ligand binds with the receptor (often referred to as "activation" of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular "G-protein". It has been reported that GPCRs are "promiscuous" with 15 respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz 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 20 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 25 transduction pathway to produce a biological response. Changing the receptor 4 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. Recent discoveries, including but not exclusively limited to 5 modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed "constitutive receptor activation". 10 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 15 of this application. SUMMARY OF THE INVENTION Disclosed herein are endogenous and non-endogenous versions of G protein coupled receptors (GPCRs), nucleic acids or polynucleotides encoding same, and uses thereof 20 In one example, the present invention provides endogenous and non endogenous versions of novel GPCR polypeptides e.g., designated herein as hRUP8, hRUP9, hRUP10, hRUP11, hRUP12, hRUP13, hRUP14, hRUP15, hRUP16, hRUP17, hRUP18, hRUP19, hRUP20, hRUP21, hRUP22, hRUP23, hRUP24, hRUP25, hRUP26 and hRUP27 and variants thereof of non-human origin. 5 It will be understood from the disclosure herein that a non-endogenous version of a GPCR of the present invention will generally, but not necessarily exclusively, comprise a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of an endogenous GPCR from which it is 5 derived. Exemplary non-endogenous forms of the human GCPRs designated herein as hRUP8, hRUP9, hRUP10, hRUPl1, hRUPl2, hRUP13, hRUP14, hRUP15, hRUP16, hRUP17, hRUP18, hRUP19, hRUP20, hRUP21, hRUP22, hRUP23, hRUP24, hRUP25, hRUP26 and hRUP27 are disclosed in Table D and Table E hereof. Such forms may be ligand-independent active versions of a human GPCR or they may be ligand 10 independent active variants of a human GPCR. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 15 In another example, an endogenous or non-endogenous GPCR of the present invention is encoded by a polynucleotide that is amplifiable using one or more oligonucleotide primers based on the sequence of the receptor sequence or the sequence of a polynucleotide encoding the receptor. In another example, the present invention extends to any polymorphic forms of 20 the exemplified endogenous and non-endogenous GPCRs and any other recepotrs that comprise an amino acid sequence that is at least about 80% identical or at least about 90% identical or at least about 95% identical to the disclosed amino acid sequences, or alternatively or additionally, are encoded by polynucleotides having at least about 80% identity or at least about 90% identity or at least about 95% identity to the disclosed 6 nucleotide sequences or able to hybridize under stringent conditions to a complement of a disclosed nucleotide sequence. For the purposes of nomenclature, the amino acid sequences of endogenous and non-endogenous versions of GPCRs of the present invention are disclosed in the 5 Sequence Listing e.g., selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96. In a further example, the endogenous or non-endogenous version of a GPCR of the invention is a constitutively activated (i.e., ligand-independent active) version of a human GPCR polypeptide (GPCR), such as a ligand-independent active version of an 10 endogenous GPCR set forth in any one of Table B or C hereof or the accompanying examples or a ligand-independent active version of non-endogenous GPCR set forth in any one of Tables D or E or F hereof or the accompanying examples. Ligand independent forms of a GPCR are particularly useful where the receptor ligand is unknown and the endogenous form of the GPCR is either ligand-dependent or active 15 albeit to a lesser degree than a ligand-independent active version of the receptor. Accordingly, to enhance the ability to identify and/or isolate agonists, partial agonist or inverse agonists of a GPCR, the present inventors employed a mutation approach to produce non-endogenous constitutively activated i.e., ligand-independent versions of GPCRs disclosed herein, thereby permitting screening of candidate compounds against 20 the non-endogenous, constitutively activated i.e., ligand-independent versions of the receptors. Specific sequences of ligand-independent versions of the disclosed receptors will be apparent from the disclosure herein and are presented in the Sequence Listing. Ligand-independent activation of an endogenous or non-endogenous GPCR of the present invention is determined by the ability of the receptor to bind to a G protein 6a e.g., Gq, Gs, Gi, Gz or Go and catalyse one or more reactions associated with said binding in the absence of an endogenous ligand of the receptor. In one example, ligand-independent activation of the GPCR is determined by binding to a labeled GTP analog e.g., a non-hydrolyzable analog such as [ 3 ss]GTPyS. 5 By way of non-limiting examples, data presented in Table H hereof indicate that the receptors designated hRUP13 and hRUP15 bind [ 35 s]GTPyS when they are expressed independently as fusion proteins with Gs. In another example for determining ligand-independent activation of a GPCR that is alternative to such GTP analog binding, or supplemental to such GTP analog 10 binding, a decrease in cAMP production is determined e.g., to thereby determine an ability of the GPCRs of the invention to couple to Gi. Preferably, a decrease in cAMP production is determined in the presence of a GPCR that causes accumulation of cAMP e.g., in the presence of a GPCR that is coupled to Gs such as TSHR or a variant thereof such as TSHR-A6231. By way of non-limiting examples, data presented in Table G 15 hereof indicate utility of this assay with respect to the endogenous form of the receptor designated hRUP 13 and the non-endogenous form of the receptor designated hRUP 15. In another example for determining ligand-independent activation of a GPCR that is alternative to such GTP analog binding, or supplemental to such GTP analog binding, an accumulation of cAMP is determined e.g., to determine an ability of a 20 GPCR of the invention to couple to Gs. For example, a second messenger reporter based assay may be performed to determine the ability of a GPCR of the invention to activate cAMP-mediated expression of a reporter gene such as luciferase or p galactosidase wherein said activation is indicative of cAMP accumulation in the presence of a GPCR that is coupled to Gs. By way of non-limiting examples, data 25 presented in Table G hereof indicate utility of this assay with respect to the non 6b endogenous forms of the receptors designated hRUP13, hRUP14, hRUP15 and hRUP23. In another example, a method of determining a ligand-independent activation of a GPCR that is alternative to such GTP analog binding, or supplemental to such GTP 5 analog binding, activation of phospholipase C and/or concomitant accumulation of inositol-1,4,5-triphosphate

(IP

3 ) is determined e.g., to determine an ability of a GPCR of the invention to couple to Gq and/or Go. For example, a second messenger reporter based assay may be performed to determine the ability of a GPCR of the invention to activate AP1-mediated expression of a reporter gene such as luciferase or p 10 galactosidase wherein said activation is indicative of phospholipase C activation and/or concomitant accumulation of IP 3 in the presence of a GPCR that is coupled to Gq. Alternatively, a second messenger reporter-based assay may be performed to determine the ability of a GPCR of the invention to activate serum response factor (SRF)-mediated expression of a reporter gene such as luciferase or p-galactosidase wherein said 15 activation is indicative of phospholipase C activation and/or concomitant accumulation of IP 3 in the presence of a GPCR that is coupled to Gq. Alternatively, the direct accumulation of IP 3 may be determined in the absence of endogenous ligand, wherein an accumulation of IP 3 is indicative of Gq coupling to the receptor i.e., ligand independent activation. By way of non-limiting examples, data presented in Table G 20 hereof indicate utility of measuring second messenger IP 3 production with respect to the non-endogenous forms of the receptors designated hRUP 11, hRUP17, and hRUP21. In another example, the present invention provides fusion proteins comprising the disclosed endogenous GPCRs, ligand-independent active versions thereof or ligand independent active variants thereof and a G protein (i.e., "GPCR fusion proteins"). In 25 the construction of such fusions, the G protein may be a cognate or non-cognate G 6c protein with respect to the GPCR, from which the ligand-independent active version or variant is derived, however it may be preferred to use a cognate G protein e.g., Gs. Preferably, the G protein is coupled to a C-terminal portion of the ligand-independent active version of a GPCR or variant thereof. The ligand-independent active version of a 5 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. Exemplary fusion proteins comprise a sequence selected from SEQ ID NO: 100 and SEQ ID NO: 104. In another example, the present invention provides an isolated nucleic acid 10 encoding an endogenous GPCR or non-endogenous GPCR according to any example hereof, and an isolated nucleic acid encoding a GPCR fusion protein comprising said endogenous GPCR or non-endogenous GPCR. This clearly extends to nucleic acids encoding a ligand-independent active version of a human GPCR polypeptide of the present invention. In one example, the isolated nucleic acid is amplifable using one or 15 more primers directed to an endogenous or non-endogenous GPCR or to nucleic acid encoding said endogenous or non-endogenous GPCR. Accordingly, a further embodiment of the present invention provides an isolated polynucleotide encoding an endogenous GPCR polypeptide (GPCR) disclosed herein or a non-endogenous constitutively-activated i.e., ligand-independent active version thereof or a variant 20 thereof. For the purposes of nomenclature, nucleotide sequences encoding the endogenous and non-endogenous versions of GPCRs of the present invention are disclosed in the Sequence Listing e.g., selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, 25 and 95. The sequences of oligonucleotide primers for amplifying a polynucleotide 6d encoding a GPCR of the present invention are disclosed in the Sequence Listing e.g., selected from the group consisting of SEQ ID NOs: 41-82. In a further example, the present invention provides a vector comprising a polynucleotide encoding an endogenous GPCR or non-endogenous GPCR according to 5 any example hereof. This includes any expression vectors for producing an endogenous or non-endogenous version of a GPCR, including any constitutively activated (i.e., a ligand-independent active) version of the GPCR i.e., wherein the polynucleotide encoding the GPCR is operably linked to a promoter. In a further example, the present invention provides a vector comprising a 10 polynucleotide encoding a GPCR fusion protein as described herein. Again, this includes an expression vector wherein the polynucleotide encoding the endogenous GPCR or non-endogenous GPCR e.g., a ligand-independent active version of a GPCR wherein the polynucleotide encoding the GPCR fusion protein is operably linked to a promoter. 15 In a further example, the present invention provides a recombinant host cell comprising a vector described in the preceding paragraphs. Preferably, the host cell is a mammalian cell e.g., a mammalian cell selected from the group consisting of a COS-7 cell, a 293 cell, and a 293T cell. In another embodiment the host cell is a melanophore or CHO cell. 20 In a further example, the present invention provides an isolated membrane of the recombinant host cell supra wherein said membrane comprises an endogenous or non-endogenous GPCR according to any example hereof. In a further example, the present invention provides an isolated membrane of a recombinant host cell supra wherein said membrane comprises a GPCR fusion protein 25 according to any example hereof. 6e In a further example, the present invention provides a method of producing an endogenous GPCR or a non-endogenous GPCR of the invention e.g., a ligand independent active version of a human GPCR or variant thereof or a GPCR fusion protein, wherein said method comprises: 5 (a) transfecting an expression vector comprising nucleic acid encoding an endogenous GPCR or a non-endogenous GPCR or a GPCR fusion protein according to any example hereof into a host cell thereby producing a transfected host cell; and (b) culturing the transfected host cell under conditions sufficient to thereby express the endogenous GPCR or non-endogenous GPCR or GPCR fusion protein from the 10 expression vector. In one preferred form, this method further comprises obtaining the transfected host cell. In another preferred form, the method further comprises obtaining or isolating a membrane fraction from the transfected host cell. In a further example, the present invention provides a method of producing a 15 non-endogenous ligand-independent version of a human GPCR or ligand-independent active variant thereof, wherein said method comprises comprising introducing a mutation into the coding region of nucleic acid comprising the nucleotide sequence of an endogenous human GPCR, isolating or recovering the modified nucleic acid, and expressing the modified nucleic acid to thereby produce a non-endogenous 20 constitutively-activated or ligand-independent active receptor. Preferably, the nucleic acid is DNA, e.g., cDNA. In an alternative embodiment, the nucleic acid is RNA from a tissue in which the endogenous receptor is expressed. As will be apparent from the disclosure herein, it is particularly preferred that the mutation produces an amino acid substitution referred to in Table D or E hereof. 25 6f As used herein and unless the context requires otherwise, the term "expression" shall be taken to mean the level or rate of transcription and/or translation of mRNA encoding the GPCR to produce an active protein. As used herein and unless the context requires otherwise, the term "activity" shall be taken to mean any activity of a GPCR 5 protein or a ligand-independent active version of a GPCR or variant thereof, including a function mediated by binding of a GPCR to a ligand such as, for example, its cognate ligand, and any signalling effected thereby. The present inventors have found that isolated or recombinant human GPCRs and any non-endogenous ligand-independent active versions thereof or ligand 10 independent active variants thereof have particular utility in screens to identify and/or isolate pharmaceutical agents e.g., for the treatment of a disease or disorder associated with aberrant endogenous GPCR activity and/or expression in a subject. The present inventors have also found that the tissue in which an endogenous GPCR or a polynucleotide encoding an endogenous GPCR disclosed herein is expressed in vivo 15 provides an indication of the endogenous pathway(s) in which the receptor is involved, thereby suggesting one or more diseases or disorders associated with aberrant endogenous GPCR activity and/or aberrant expression in a subject. Endogenous GPCR expression in specific tissues or organs is readily determined e.g., using a process that comprises performing RT-PCR. By way of non-limiting examples, Table J hereof 20 indicates tissue expression profiles of the exemplified GPCRs of the invention designated hRUP1O, hRUPll, hRUP12, hRUP13, hRUP16, hRUP18, hRUP21, hRUP22, hRUP23, hRUP26 and hRUP27. Accordingly, in another example, the present invention provides for the use of an endogenous and non-endogenous version of a GPCR described herein in drug 6g screening to identify and/or isolate compounds and to formulate those compounds as pharmaceutical compositions e.g., for the treatment of disorders of a tissue or organ in which the endogenous GPCR is expressed in vivo. In more specific embodiments, the present invention provides the following 5 examples. In one specific example, the present invention provides an isolated polynucleotide encoding a G protein-coupled receptor (GPCR) polypeptide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: 10 (a) a sequence encoding a polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96; (b) a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, and 95; 15 (c) the sequence of a polynucleotide that hybridizes under stringent conditions to (a) or (b) or a complementary sequence thereto: (d) the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using one or more oligonucleotide primer molecules each comprising a sequence selected from the group consisting of SEQ ID NOs: 41-82; and 20 (e) a sequence at any one of (a) or (b) or (c) or (d) wherein said sequence encodes a ligand-independent active version or constitutively-activated variant of a GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of the corresponding endogenous version of the 25 GPCR. 6h Preferred isolated polynucleotides of the invention comprise a nucleotide sequence encoding an exemplified GPCR described herein, or a nucleotide sequence exemplified herein, or a variant of an exemplified nucleotide sequence comprising a substitution of one or two or three nucleotides within a codon at positions 1192 to 1194 5 of SEQ ID NO: 15 for other nucleotide residue(s) thereby producing a codon encoding an amino acid residue that is positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of an endogenous version of the encoded GPCR polypeptide. Preferred isolated polynucleotides of the invention are obtained from a cell, 10 tissue or organ of human origin. In another specific example, the present invention provides an isolated polynucleotide encoding a GPCR fusion protein comprising a nucleotide sequence selected from the group consisting of: (a) a sequence encoding a polypeptide that comprises an amino acid 15 sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96; (b) a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, and 95; (c) the sequence of a polynucleotide that hybridizes under stringent 20 conditions to (a) or (b) or a complementary sequence thereto: (d) the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using one or more oligonucleotide primer molecules each comprising a sequence selected from the group consisting of SEQ ID NOs: 41-82; and (e) a sequence at any one of (a) or (b) or (c) or (d) wherein said sequence 25 encodes a ligand-independent active version or constitutively-activated variant of a 6i GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of the corresponding endogenous version of the GPCR. 5 In another specific example, the present invention provides a vector comprising a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a sequence encoding a polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 10 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96; (b) a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, and 95; (c) the sequence of a polynucleotide that hybridizes under stringent conditions to (a) or (b) or a complementary sequence thereto: 15 (d) the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using one or more oligonucleotide primer molecules each comprising a sequence selected from the group consisting of SEQ ID NOs: 41-82; (e) a sequence at any one of (a) or (b) or (c) or (d) wherein said sequence encodes a ligand-independent active version or constitutively-activated variant of a 20 GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of the corresponding endogenous version of the GPCR; and (f) a sequence encoding a GPCR fusion protein wherein said sequence 25 comprises any one of (a) or (b) or (c) or (d) or (e) and a sequence encoding a G protein. 6j Preferably, the vector is an expression vector, wherein the GPCR-encoding polynucleotide or GPCR fusion protein-encoding polynucleotide is operably linked to a promoter. In another specific example, the present invention provides a recombinant host 5 cell comprising a vector according to any example hereof, wherein the host cell is a mammalian cell e.g., selected from the group consisting of a COS-7 cell, a 293 cell, and a 293T cell, or a melanophore or CHO cell. In another specific example, the present invention provides a recombinant host cell or isolated membrane thereof comprising a recombinant GPCR polypeptide 10 according to any example hereof e.g., a non-endogenous GPCR or GPCR fusion protein according to any example hereof, wherein the host cell is a mammalian cell e.g., selected from the group consisting of a COS-7 cell, a 293 cell, and a 293T cell, or a melanophore or CHO cell. In another specific example, the present invention provides a method for 15 producing a GPCR or a GPCR fusion protein comprising the steps of: (a) obtaining a transfected host cell comprising an expression vector comprising nucleic acid encoding an endogenous GPCR or non-endogenous GPCR or GPCR fusion protein as described according to any example hereof, e.g., wherein the host cell is a mammalian cell or a melanophore or CHO cell e.g., wherein the 20 mammalian cell is selected from the group consisting of a COS-7 cell, a 293 cell, and a 293T cell; and (b) culturing the transfected host cell under conditions sufficient to express a GPCR or GPCR fusion protein from the expression vector. In another specific example, the present invention provides a method for 25 producing a GPCR or a GPCR fusion protein comprising the steps of: 6k (a) transfecting the expression vector comprising nucleic acid encoding an endogenous GPCR or non-endogenous GPCR or GPCR fusion protein as described according to any example hereof into a host cell thereby producing a transfected host cell, wherein the host cell is a mammalian cell e.g., selected from the group consisting 5 of a COS-7 cell, a 293 cell, and a 293T cell, or a melanophore or CHO cell; and (b) culturing the transfected host cell under conditions sufficient to express a GPCR or GPCR fusion protein from the expression vector. The present invention also provides an isolated or recombinant GPCR polypeptide comprising an amino acid sequence selected from the group consisting of: 10 (a) a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96; (b) a sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a sequence that is complementary to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 15 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, and 95: (c) a sequence encoded by a polynucleotide that is amplifiable e.g., by polymerase chain reaction (PCR) using one or more oligonucleotide primer molecules each comprising a sequence selected from the group consisting of SEQ ID NOs: 41-82; and 20 (d) a sequence at any one of (a) or (b) or (c) wherein said sequence is the structure of a ligand-independent active version or constitutively-activated variant of a GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of the corresponding endogenous version of the 25 GPCR. 61 Preferably, the isolated or recombinant GPCR is of human origin. Preferably the GPCR comprises an amino acid exemplified herein or the amino acid sequence of a ligand-independent active version or constitutively-activated variant of a GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises 5 a mutation positioned 16 amino residues N-terminal from a conserved proline residue within the TM6 domain of the corresponding endogenous version of the GPCR. In another specific example, the present invention provides an isolated or recombinant GPCR fusion protein comprising a GPCR polypeptide and a G protein, wherein the GPCR polypeptide comprises an amino acid sequence selected from the 10 group consisting of: (a) a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 84, 88, 92, and 96; (b) a sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a sequence that is complementary to a 15 sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 83, 87, 91, and 95: (c) a sequence encoded by a polynucleotide that is amplifiable e.g., by polymerase chain reaction (PCR) using one or more oligonucleotide primer molecules each comprising a sequence selected from the group consisting of SEQ ID NOs: 41-82; 20 and (d) a sequence at any one of (a) or (b) or (c) wherein said sequence is the structure of a ligand-independent active version or constitutively-activated variant of a GPCR wherein said ligand-independent active version or said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from a conserved 6m proline residue within the TM6 domain of the corresponding endogenous version of the GPCR. In another specific example, the present invention provides for the use of an endogenous or non-endogenous GPCR polypeptide or GPCR fusion protein according 5 to any example hereof to isolate or identify a pharmaceutical agent for the treatment of a disease or disorder related to a cell, tissue or organ in which the GPCR is expressed in humans. In one example, the endogenous or non-endogenous GPCR polypeptide or GPCR fusion protein is used to identify or isolate a pharmaceutical agent for the treatment of a disease or disorder state related to a function selective to a cell, tissue or 10 organ in which the endogenous version of the GPCR is expressed in humans e.g., as set forth in Table J hereof. In another specific example, the present invention provides a method of identifying a modulator of a G protein-coupled receptor (GPCR) comprising: (a) contacting a candidate compound with a recombinant host cell that 15 expresses the GPCR or a non-endogenous version thereof or a GPCR fusion protein comprising the GPCR or an isolated membrane comprising said GPCR or a non endogenous version thereof or a GPCR fusion protein; and (b) measuring the ability of the compound to inhibit or stimulate functionality of the GPCR or a non-endogenous version thereof or a GPCR fusion 20 protein wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide. In another specific example, the present invention provides a method of identifying a modulator of a G protein-coupled receptor (GPCR) comprising: 6n (a) providing a recombinant host cell that expresses the GPCR or a non endogenous version thereof or a GPCR fusion protein comprising the GPCR or an isolated membrane comprising said GPCR or a non-endogenous version thereof or a GPCR fusion protein; 5 (b) contacting a candidate compound with the recombinant host cell or isolated membrane; and (c) measuring the ability of the compound to inhibit or stimulate functionality of the GPCR or a non-endogenous version thereof or a GPCR fusion protein wherein inhibition or stimulation of said functionality indicates that the 10 candidate compound is a modulator of the G protein-coupled receptor polypeptide. In preferred forms of these methods, the non-endogenous version of the receptor is a ligand-independent active version or constitutively-activated version of the endogenous GPCR and/or the GPCR fusion protein comprises said ligand-independent active version or constitutively-activated version. In a preferred embodiment, the 15 method described supra for identifying a modulator of a GPCR further comprises providing the host cell or membrane. Preferably, the host cell comprises an expression vector capable of expressing the GPCR or GPCR fusion protein comprising endogenous RUP15 or a non-endogenous variant of RUP15 e.g., RUP15(A398K) or other variant according to any embodiment described herein. 20 It is preferred that the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide or the G protein-coupled receptor polypeptide portion of the GPCR fusion protein comprises determining inositol triphosphate (IP3), diacylglycerol (DAG), cyclic AMP (cAMP) or cyclic GMP (cGMP), and more preferably comprises determining intracellular cAMP level. 25 6o Preferred modulatory compounds are agonists, partial agonists or inverse agonists. Such screening methods are useful for identifying a modulatory compound e.g., an agonist, partial agonist or inverse agonist that modulates signal transduction mediated by the GPCR in a cell or tissue. Accordingly, the present invention further 5 provides processes for identifying and/or isolating compounds that modulate signal transduction. In another example, the present invention provides processes for identifying and/or isolating compounds for the treatment of a condition associated with expression or aberrant expression of the GPCR. Such processes comprise performing a screening method of the invention as described herein. 10 In yet another example, the present invention provides a process of formulating a compound for therapy of a condition ameliorated by modulating signal transduction mediated by a GPCR polypeptide, said process comprising performing a method according to any example hereof for identifying a modulator of a GPCR to thereby identify a modulator of a GPCR polypeptide and formulating the modulator with a 15 pharmaceutically acceptable carrier or excipient. In yet another example, the present invention provides a process for isolating a modulator of a GPCR polypeptide comprising performing a method according to any example hereof for identifying a modulator of a GPCR on a panel of candidate compounds and isolating a compound that modulates a GPCR polypeptide from the 20 panel. In yet another example, the present invention provides a process of formulating a compound for therapy of a condition ameliorated by modulating signal transduction mediated by a GPCR polypeptide, said process comprising performing a process according to any example hereof to thereby isolating a compound that modulates a 6p GPCR and formulating the compound with a pharmaceutically acceptable carrier or excipient. In yet further specific examples, the present invention provides compositions of matter and processes substantially as described herein with reference to the 5 accompanying drawings and/or any one or more of Examples 1-10. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides an illustration of second messenger IP3 production from endogenous version RUP12 ("RUP 12") as compared with the control ("CMV"). 10 Figure 2 is a graphic representation of the results of a second messenger cell based cyclic AMP assay providing comparative results of constitutive signalling of endogenous RUP13 ("RUP13") and a control vector ("CMV"). Figure 3 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP 13 ("RUP 13 wt") and non-endogenous, constitutively activated 15 RUP13 ("RUP13(A268K)"), utilizing 8XCRE-Luc reporter plasmid. Figure 4 is a graphic representation of the results of a [ 3 5 S]GTPyS assay providing comparative results for constitutive signaling by RUP13:Gs Fusion Protein ("RUP13-Gs") and a control vector ("CMV"). Figure 5 is a diagrammatic representation of the signal measured comparing 20 CMV, endogenous RUP 14 ("RUP 14 wt") and non-endogenous, constitutively activated RUP13 ("RUP14 (L246K)"), utilizing 8XCRE-Luc reporter plasmid. Figure 6 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP15 ("RUP15 wt") and non-endogenous, constitutively activated RUP15 ("RUP15 (A398K)"), utilizing 8XCRE-Luc reporter plasmid. 25 6q Figure 7 is a graphic representation of the results of a second messenger cell based cyclic AMP assay providing comparative results for constitutive signaling of endogenous RUP15 ("RUP15 wt"), non-endogenous, constitutively activated version of RUP15 ("RUP15 (A398K)") and a control vector ("CMV"). 5 Figure 8 is a graphic representation of the results of a [ 3 5 S]GTPyS assay providing comparative results for constitutive signaling by RUP15:Gs Fusion Protein ("RUP15-Gs") and a control vector ("CMV"). Figure 9 provides an illustration of second messenger IP 3 production from endogenous version RUP 17 ("RUP 17") as compared with the control ("CMV"). 10 Figure 10 provides an illustration of second messenger IP 3 production from endogenous version RUP21 ("RUP21") as compared with the control ("CMV"). Figure 11 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP23 ("RUP23 wt") and non-endogenous, constitutively activated RUP23 ("RUP23 (W275K)"), utilizing 8XCRE-Luc reporter plasmid. 15 Figure 12 is a graphic representation showing the level of IP 3 production in a cell expressing RUP 18 or RUP 1 8(L294K) in the presence or absence of Gq(del)/Gi. Figure 13 is a copy of a photographic representation showing RT-PCR analysis or expression of RUP 18 in various tissues. Tissues analysed were lane 1, brain; lane 2, colon; lane 3, heart; lane 4, kidney; lane 5, leukocytes; lane 6, liver; lane 7, lung; lane 8, 20 ovary; lane 9, pancreas, lane 10, placenta; lane 11, prostate; lane 12, skeletal muscle; lane 13, small intestine; lane 14, spleen; lane 15, thyroid and lane 16, testis. Figure 14 is a copy of a photographic representations showing RT-PCR analysis of RUP18 expression in insulin secreting cell lines HIT-T15, NIT-I and $TC-6 and pancreatic p islet cells isolated from mice (as indicated). The arrow indicates the 25 position of the RUP18 amplification product. 6r Figure 15 is a graphic representation of results from primary screen of several candidate compounds against RUP13; results for "Compound A" are provided in well A2 and "Compound "B" are provided in well G9. 5 DETAILED DESCRIPTION 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 10 following definitions shall control: AGONISTS shall mean material (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding membranes. PARTIAL AGONISTS shall mean materials (e. g., ligands, candidate 15 compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists. AMINO ACID ABBREVIATIONS used herein are set out in Table A. ANTAGONIST shall mean materials (e. g., ligands, candidate compounds) 20 that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists. ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist. 25 6s TABLE A 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 THREONTNE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V 5 CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique and, preferably, were not known previously to have inverse agonist, agonist or antagonist 10 activity with respect to a receptor and/or were not known to have therapeutic efficacy. 6t COMPOSITION means a material comprising at least one component; a "pharmaceutical composition" is an example of a composition. COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding 5 affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document. CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to a phosphate group and which, 10 when translated, encodes an amino acid. 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 15 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 together, whether in an in vitro system or an in vivo system. DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to 20 the phrase "candidate compound", shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound. This phrase is, under no circumstances, to be interpreted or understood to be encompassed by 25 or to encompass the phrase "indirectly identifying" or "indirectly identified." 7 ENDOGENOUS shall mean a material that a mammal naturally produces. ENDOGENOUS in reference to, for example and not limitation, the term "receptor," shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. By contrast, the term NON-ENDOGENOUS in this 5 context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. For example, and not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a "non-endogenous, constitutively activated receptor." Both terms can be utilized to describe both "in vivo" 10 and "in vitro" systems. For example, and not limitation, in a screening approach, the endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in vivo system is viable. 15 G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a non endogenous protein comprising an endogenous, constitutively activate GPCR or a non endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha (a) subunit of such G protein (this being the subunit that binds 20 GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. For example, and not limitation, in an endogenous state, if the G protein "Gsa' is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein based upon the specific GPCR would be a non endogenous protein comprising the GPCR fused to Gsa; in some circumstances, as will 25 be set forth below, a non-predominant G protein can be fused to the GPCR. The G 8 protein can be fused directly to the c-terminus of the constitutively active GPCR or there may be spacers between the two. HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein. In the case of a prokaryotic Host Cell, a Plasmid is typically 5 replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates. Preferably, for the purposes of the invention disclosed herein, the Host Cell is eukaryotic, more preferably, 10 mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells. INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate 15 compounds against the receptor for determination of those which interfere and/or compete with the ligand-receptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor. INHIBIT or INHIBITING, in relationship to the term "response" shall mean 20 that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound. INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response 25 initiated by the active form of the receptor below the normal base level of activity which 9 is observed in the absence of agonists or.partial agonists, or decrease GTP binding to membranes. Preferably, the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, 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 5 agonist. KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified. LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor. 10 MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor. In terms of equivalents to specific sequences, a subsequent mutated form of a human receptor is 15 considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor, and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 80%, 20 more preferably at least about 90% and most preferably at least 95%. Ideally, and owing to the fact that the most preferred cassettes disclosed herein for achieving constitutive activation includes a single amino acid and/or codon change between the endogenous and the non-endogenous forms of the GPCR, the percent sequence homology should be at least 98%. 10 NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway. ORPHAN RECEPTOR shall mean an endogenous receptor for which the 5 endogenous ligand specific for that receptor has not been identified or is not known. PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human). Those of ordinary skill in the art will understand and appreciate the 10 techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan. PLASMID shall mean the combination of a Vector and cDNA. Generally, a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein. 15 SECOND MESSENGER shall mean an intracellular response produced as. a result of receptor activation. A second messenger can include, for example, inositol triphosphate (IP 3 ), diacycglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP). Second messenger response can be measured for a determination of receptor activation. In addition, second messenger response can be measured for the direct 20 identification of candidate compounds, including for example, inverse agonists, agonists, partial agonists and antagonists. STIMULATE or STIMULATING, in relationship to the term "response" shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound. 11 VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell. The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to 5 follow. A. Introduction The traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before 10 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 15 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. 20 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. 25 B. Identification of Human GPCRs 12 The efforts of the Human Genome project has led to the identification of a plethora of information regarding nucleic acid sequences located within the human genome; it has been the case in this endeavor that genetic sequence information has been made available without an understanding or recognition as to whether or not any 5 particular genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleic acid sequences within the human genome are within the purview of those having ordinary skill in the art. For example, and not limitation, a variety of human GPCRs, disclosed herein, were discovered by reviewing the GenBankTM database. Table B, below, lists several 10 endogenous GPCRs that we have discovered, along with other GPCR's that are homologous to the disclosed GPCR. TABLE B Disclosed ' Accession Open Reading Reference To Per Cent Human Number Frame Homologous Homology Orphan GPCRs Identified (Base Pairs) GPCR To Designated GPCR hRUP8 AL121755 -1,152bp NPY2R 27% hRUP9 AC0113375 1,260bp GAL2R 22% hRUP10 AC008745 1,014bp C5aR 40% hRUP11 AC013396 1,272bp HM74 36% hRUP12 AP000808 966bp Masl 34% hRUP13 ACO1780 1,356bp Fish GPRX- 43% ORYLA hRUP14 AL137118 1,041bp CysLTIR 35% hRUP15 AL016468 l,527bp RE2 30% hRUP16 AL136106 1,068bp GLR101 37% hRUP17 AC023078 969bp MasI 37% hRUP18 AC008547 1,305bp Oxytocin 31% hRUP19 AC026331 1,041bp HM74 52% hRUP20 AL161458 1,011bp GPR34 25% hRUP21 AC026756 1,014bp P2YIR 37% hRUP22 AC027026 993bp RUP17 67% Masi 37% 13 hRUP23 AC007104 1,092bp Rat GPR26 31% hRUP24 AL355388 1,125bp SALPR 44%0 hRUP25 AC026331 1,092bp HM74 95% hRUP26 AC023040 1,044bp Rabbit 5HT1D 27% hRUP27 A027643 158,700 MCH 38% Receptor homology is useful in terms of gaining an appreciation of a role of the receptors within the human body. As the patent document progresses, we will disclose techniques for mutating these receptors to establish non-endogenous, constitutively 5 activated versions of these receptors. The techniques disclosed herein have also been applied to other human, orphan GPCRs known to the art, as will be apparent as the patent document progresses. C. Receptor Screening 10 Screening candidate compounds against a non-endogenous, constitutively activated version of the human GPCRs disclosed herein allows for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand. Using routine, and often commercially available techniques, one can determine areas within the body where the 15 endogenous version of human GPCRs disclosed herein is expressed and/or over expressed. It is also possible using these techniques to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor, such an approach is disclosed in this patent document. With respect to creation of a mutation that may evidence constitutive activation 20 of the human GPCR disclosed herein is based upon the distance from the proline residue at which is presumed to be located within TM6 of the GPCR; this algorithmic technique is disclosed in co-pending and commonly assigned patent document PCT Application 14 Number PCT/US99/23938, published as WO 00/22129 on April 20, 2000, which, along with the other patent documents listed herein, is incorporated herein by reference. The algorithmic technique is not predicated upon traditional sequence "alignment" but rather a specified distance from the aforementioned TM6 proline residue (or, of course, 5 endogenous constitutive substitutionf for such proline residue). 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. 10 D. Disease/Disorder Identification and/or Selection As will be set forth in greater detail below, most preferably inverse agonists and agonists to the non-endogenous, constitutively activated GPCR can be identified by the methodologies of this invention. Such inverse agonists and agonists are ideal candidates 15 as lead compounds in drug discovery programs for treating diseases related to this receptor. Because of the ability to directly identify inverse agonists to the GPCR, thereby allowing for the development of pharmaceutical compositions, a search for diseases and disorders associated with the GPCR is relevant. For example, scanning both diseased and normal tissue samples for the presence of the GPCR now becomes 20 more than an academic exercise or one which might be pursued along the path of identifying an endogenous ligand to the specific GPCR. Tissue scans can be conducted across a broad range of healthy and diseased tissues. Such tissue scans provide a preferred first step in associating a specific receptor with a disease and/or disorder. Preferably, the DNA sequence of the human GPCR is used to make a probe for 25 (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue 15 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 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 5 technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced. E. Screening of Candidate Compounds 1. Generic GPCR screening assay techniques 10 When a G protein receptor becomes constitutively active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP'to the G protein. The G protein then acts as a GTPase and 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 15 GTP, [SJGTPyS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [ 5 SJGTPyS 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 20 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 25 receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the 16 receptor site is preferred. For example, a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain. a. Gs, Gz and GI. 5 Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz 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 Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. 10 See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain ( 3 rd 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.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; 15 a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA based 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 20 factor (CREB) that 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.g., p-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of 17 the reporter protein. The reporter protein such as P-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995). b. Go and Gq. 5 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 inistol 1,4,5-triphoisphate

(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 (3r 10 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.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ). Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API 15 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 20 The use of an endogenous, constitutively activate orphan GPCR or a non endogenous, constitutively activated orphan GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to 25 differentiate between, e.g., the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an 18 aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial agonist or have no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation. A preferred approach is the use of a GPCR Fusion Protein. 5 Generally, once it is determined that a non-endogenous orphan GPCR has been constitutively activated using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian 10 expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of 15 screening, between the receptor when it is contacted with the inverse agonist. The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening 20 techniques. 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 25 expression vectors and systems offer a variety of approaches that can fit the particular 19 needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the endogenous GPCR sequence and the G protein sequence both be in frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence) and that the "stop" codon of the GPCR must be deleted or replaced such that 5 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 Wil, effectively, upon 10 expression, become a spacer. Most preferably, the G protein that couples to the non endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is prefened that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; 15 this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences. As noted above, constitutively activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation thus making the 20 direct identification of, e.g, inverse agonists (which would further decrease this signal), interesting. As will be disclosed herein, we have ascertained that for these types of receptors, it is possible to create a GPCR Fusion Protein that is not based upon the endogenous GPCR's endogenous G protein, in an effort to establish a viable cyclase based assay. Thus, for example, an endogenous Gi coupled receptor can be fused to a Gs 25 protein - we believe that such a fusion construct, upon expression, "drives" or "forces" 20 the endogenous GPCR to couple with, e.g., Gs rather than the "natural" Gi protein, such that a cyclase-based assay can be established. Thus, for Gi, Gz and Go coupled receptors, we prefer that that when a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with 5 Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase). Equally effective is a G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. A most preferred fusion construct can be accomplished with a Gq Protein wherein the first six (6) amino acids of the G-protein a-subunit 10 ("Gaq") is deleted and the last five (5) amino acids at the C-tenninal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest. For example, a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct". We believe that this fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G 15 protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylgycerol, can be measured in lieu of cAMP production. 4. Co-transfection of a Target Gi Coupled GPCR with a Signal Enhancer Gs Coupled GPCR (cAMP Based Assays) 20 A Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging. An effective technique in measuring the decrease in production of cAMP as an indication of constitutive activation of a receptor that predominantly couples Gi upon 25 activation can be accomplished by co-transfecting a signal enhancer, e.g., a non endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A6231, disclosed below), with the Gi linked GPCR. As is 21 apparent, constitutive activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP. Constitutive activation of a Gi coupled receptor leads to a decrease in production cAMP. Thus, the co-transfection approach is intended to advantageously exploit these "opposite" affects. For example, co-transfection of a 5 non-endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with the endogenous Gi coupled receptor (the "target receptor") provides a baseline cAMP signal (i.e., although the Gi coupled receptor. will decrease cAMP levels, this "decrease" will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co-transfecting the signal 10 enhancer with a constitutively activated version of the target receptor, cAMP would be expected to further decrease (relative to base line) due to the increased functional activity of the Gi target (i.e., which decreases cAMP). Screening of candidate compounds using a cAMP based assay can then be accomplished, with two provisos: first, relative to the Gi coupled target receptor, 15 "opposite" effects will result, i.e., an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal; second, as would be apparent, candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or 20 after screening against the co-transfected receptors). F. Medicinal Chemistry Generally, but not always, direct identification of candidate compounds is preferably conducted in conjunction with compounds generated via combinatorial 25 chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. Generally, the results of such screening will be compounds having 22 unique core structures; thereafter, these compounds are preferably subjected to additional chemical modification around a preferred core structure(s) to further enhance the medicinal properties thereof. Such techniques are known to those in the art and will not be addressed in detail in this patent document. 5 G. Pharmaceutical compositions Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see 10 Remington's Pharmaceutical Sciences, 16"' Edition, 1980, Mack Publishing Co., (Oslo et al., eds.). H. Other Utility Although a preferred use of the non-endogenous versions the human GPCRs 15 disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as phannaceutical agents), these versions of human 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 20 diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. The value in non-endogenous human GPCRs is that their utility as a research tool is enhanced in that, because of their unique features, non-endogenous human GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefore is identified. Other uses of the 25 disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document. 23 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 5 ability to make minor modifications to these sequences while achieving the same or substantially similar results reported below. The traditional approach to application or understanding of sequence cassettes from one sequence to another (e.g. from rat receptor to human receptor or from human receptor A to human receptor B) is generally predicated upon sequence alignment techniques whereby the sequences are aligned in an 10 effort to determine areas of commonality. The mutational approach disclosed herein does not rely upon this approach but is instead based upon an algorithmic approach and a positional distance from a conserved proline residue located within the TM6 region of human GPCRs. Once this approach is secured, those in the art are credited with the ability to make minor modifications thereto to achieve substantially the same results (i.e., 15 constitutive activation) disclosed herein. Such modified approaches are considered within the purview of this disclosure. // / // 20 // Example 1 ENDOGENOUS HUMAN GPCRS 1. Identification of Human GPCRs The disclosed endogenous human GPCRs were identified based upon a review 25 of the GenBankTM database information. While searching the database, the following cDNA clones were identified as evidenced below (Table C). 24 TABLE C Disclosed- Accession Com-plete DNA- Open Readin N.... ucleic A-Xmino Human Number Sequence Frame Acid Acid Orphan Identified (Base Pairs) (Base Pairs) SEQ.JD. SEQ.I1D. GPCRs NO. NO. JIU8 AL121755 147,566b 1, 15lbp 1 2 hRJ9 AC0113375 143,1811i 1,260bp 3 4 RU10 AC008745 94,194bp 1,Ol4bp 5 6 hRUP11 AC013396 155,086bp 1,272bp 7 8 hRUPl2 AP000808 177,764bp 966bp 9 10 IIRUP13 A00 11780 -1-67,819bp 1,356bp 11 12 IRJ14 AL137118 168,297bp- 1,O)4lbP 13- 14 bRUPiS AL016468 138,828bp l,527bp 1-5 1-6 hRU.P16 AL136106 0-8,042bp 1,068bp 17 18 hRUP17 AC023078 16 ,735bp 969bp 19 20 JIRUP18 Z08 -547 11 -7304bp 1,305bp 2-1 22 hRUP19 A026331 145,183bp 1,O41bp 23 24 IIRUP2O ALI61458 163,Sllbp l,Ollbp- 25 26 hRJ21 A067 6 l56,534bp 1,Ol4bp 27 2-8 JIR T22 AC027026 151,811bp 993bp 219 3-0 hRUP23 AC007104 200,-OObp 1-T,092bp 3-1 3-2 hRIJP4 AL355388 19 -,538bp 1, 125bp 33 34 IIRUP25 ACO 26331- 145,T836b 1,092b 3-5 3-6 IIRUI2 523640- 1-i78,508bp 1,044b 37 38 hRP7 AC27643 158 7001p1OOb 94 5 2. Full Length Cloning a. hRUP8 (Seq. Id. Nos. 1& 2) The disclosed human RUP8 was identified based upon the use of EST database (dbESl) information. While searching the dbEST, a cDNA clone with accession number 25 AL121755 was identified to encode a novel GPCR. The following PCR primers were used for RT-PCR with human testis Marathon-Ready cDNA (Clontech) as templates: 5'-CTTGCAGACATCACCATGGCAGCC-3'(SEQ.ID.NO.:41; sense) and 5'-GTGATGCTCTGAGTACTGGACTGG-3' (SEQ.D.NO.: 42; antisense). 5 PCR was performed using Advantage cDNA polymerase (Clontech; manufacturing instructions will be followed) in 50ul reaction by the following cycles: 94*C for 30 see; 94*C for 10 sec; 65*C for 20 see, 72*C for 1.5 min, and 72 0 C for 7 min. Cycles 2 through 4 were repeated 35 times. A 1.2kb PCR fragment was isolated and cloned into the pCRII-TOPO vector 10 (Invitrogen) and sequenced using the ABI Big Dye Terminator kit (PE. Biosystem). See, SEQ.ID.NO.:1. The putative amino acid sequence for RUP8 is set forth in SEQ.ID.NO.:2. b. hRUP9 (Seq. Id. Nos. 3 & 4) The disclosed human RUP9 was identified based upon the use of GeneBank 15 database information. While searching the database, a cDNA clone with Accession Number ACO 1375 was identified as a human genomic sequence from chromosome 5. The full length RUP9 was cloned by PCR using primers: 5'-GAAGCTGTGAAGAGTGATGC-3' (SEQ.ID.NO.:43; sense), 5'GTCAGCAATATTGATAAGCAGCAG-3' (SEQ.ID.NO.:44; antisense) 20 and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase (Stratagene) was used for the amplification in a 100pil reaction with 5% DMSO by the following cycle with step 2 to step 4 repeated 35 times: 94*C for 1 minute; 94*C for 30 seconds; 56 0 C for 30 seconds; 72*C for 2 minutes; 72*C for 5 minutes. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector 25 (Invitrogen) from 1% agarose gel and completely sequenced using the ABI Big Dye 26 Terinator kit (P.E. Biosystem). See, SEQ.ID.NO.:3. The putative amino acid sequence for RUP8 is set forth in SEQ.ID.NO.:4. The sequence of RUP9 clones isolated from human genonic DNA matched with the sequence obtained from data base. c. hRUP10 (Seq. Id. Nos. 5 & 6) 5 The disclosed human RUPlO was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with accession number AC008754 was identified as a human genomic sequence from chromosome 19. The full length RUPlO was cloned by RT-PCR using primers: 5'-CCATGGGGAACGATTCTGTCAG.CTACG-3' (SEQ.ID.NO.:45; sense) and 10 5'-GCTATGCCTGAAGCCAGTCT'GTG-3' (SEQ.ID.NO.:46; antisense) and human leukocyte Marathon-Ready cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50pl reaction by the following cycle with step 2 to step 4 repeated 35 times: 94C for 30 seconds; 94*C for 10 seconds; 62*C for 20 seconds; 72 0 C for 1.5 minutes; 72 0 C for 7 minutes. A 1.0 15 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). The nucleic acid sequence of the novel human receptor RUPlO is set forth in SEQ.ID.NO.:5 and the putative amino acid sequence thereof is set forth in SEQ.ID.NO.:6. 20 d. hRUPI 1 (Seq. Id. Nos. 7 & 8) The disclosed human RUPI I was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with accession 25 number AC013396 was identified as a human genomic sequence from chromosome 2. 27 The full length RUPI I was cloned by PCR using primers: 5'-CCAGGATGTTGTGTCACCGTGGTGGC-3' (SEQ.ID.NO.:47; sense), 5'-CACAGCGCTGCAGCCCTGCAGCTGGC-3' (SEQ.ID.NO.:48; antisense) and human genomic DNA (Clontech) as a template. TaqPlus Precision DNA 5 polymerase (Stratagene) was used for the amplification in a 50sl reaction by the following cycle with step 2 to step 4 repeated 35 times: 94*C for 3 minutes; 94*C for 20 seconds; 67*C for 20 seconds; 72*C for 1.5 minutes; 72*C for 7 minutes. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). The 10 nucleic acid sequence of the novel human receptor RUPI I is set forth in SEQ.ID.NO.:7 and the putative amino acid sequence thereof is set forth in SEQ.ID.NO.:8. e. hRUP12 (Seq. Id. Nos. 9 & 10) The disclosed human RUP12 was identified based upon the use of GenBank database. While searching the database, a cDNA clone with accession number 15 AP000808 was identified to encode a new GPCR, having significant homology with rat RTA and human mas1 oncogene GPCRs. The full length RUP12 was cloned by PCR using primers: 5'-CTICCTCTCGTAGGGATGAACCAGAC-3'(SEQ.ID.NO.:49; sense) S'-CTCGCACAGGTGGGAAGCACCTGTGG-3' (SEQ.ID.NO.:50; antisense) 20 and human genomic DNA (Clontech) as template. TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94*C for 3 min; 940C for 20 sec; 65"C for 20sec; 72*C for 2 min and 72*C for 7 min, A 1.0kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Tenninator kit 28 (P.E. Biosystem) (see, SEQ.ID.NO.:9 for nucleic acid sequence and SEQ.ID.NO.: 10 for deduced amino acid sequence). f. hRUP13 (Seq. Id. Nos. 11 & 12) The disclosed human RUP13 was identified based upon the use of 5 GenBank database. While searching the database, a cDNA clone with accession number ACO 11780 was identified to encode a new GPCR, having significant homology with GPCR fish GPRX-ORYLA. The full length RUP13 was cloned by PCR using primers: 5'-GCCTGTGACAGGAGGTACCCTGG-3' (SEQ.ID.NO.:51; sense) 5'-CATATCCCTCCGAGTTCCAGCGGC-3'(SEQ.ID.NO.:52; antisense) 10 and human genomic DNA (Clontech) as template. TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 940C for 3 min; 94*C for 20 sec; 65*C for 20sec; 72*C for 2 min and 72*C for 7 min. A 1.35kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit 15 (P.E. Biosystem) (see, SEQ.ID.NO.: II for nucleic acid sequence and SEQ.ID.NO.:12 for deduced amino acid sequence). g. hRUP14 (Seq. Id. Nos. 13 & 14) The disclosed human RUP14 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 20 Number AL137118 was identified as a human genomic sequence from chromosome 13. The full length RUP14 was cloned by PCR using primers: 5'-GCATGGAGAGAAAATTTATGTCCTGCAACC-3' (SEQ.ID.NO.:53; sense) 5'-CAAGAACAGGTCICATCTAAGAGCTCC- 3 ' (SEQ.lD).NO.:54; antisense) and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase 25 (Stratagene) and 5% DMSO were used for the amplification by the following cycle 29 with step 2 and step 3 repeated 35 times: 94*C for 3 minute; 94*C for 20 seconds; 58*C for 2 minutes; 72 0 C for 10 minutes. A 1.1 Kb PCR fragment was isolated and cloned into the pCR11-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. 5 Biosystem) (see, SEQ.ID.NO.:13 for nucleic acid sequence and SEQ.ID.NO.:14 for deduced amino acid sequence). The sequence of RUP14 clones isolated from human genomic DNA matched with the sequence obtained from database. h. hRUP15 (Seq. Id. Nos. 15 & 16) The disclosed human RUP15 was identified based upon the use of GeneBank 10 database information. While searching the database, a cDNA clone with Accession Number AC016468 was identified as a human genomic sequence. The full length RUP15 was cloned by PCR using primers: 5'-GCTGTrGCCATGACGTCCACCTGCAC-3' (SEQ.ID.NO.:55; sense) 5'-GGACAGTTCAAGGTTIGCCITAGAAC-3'(SEQ.ID.NO.:56; antisense) 15 and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase (Stratagene) was 'used for the amplification by the following cycle with step 2 to 4 repeated 35 times: 94"C for 3 minute; 940C for 20 seconds; 65*C for 20 seconds; 72*C for 2 minutes and 72*C for 7 minutes. A 1.5 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector 20 (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). See, SEQ.ID.NO.:15 for nucleic acid sequence and SEQ.ID.NO.:16 for deduced amino acid sequence. The sequence of RUP15 clones isolated from human genomic DNA matched with the sequence obtained from database. i. hRUP16 (Seq. Id. Nos. 17 & 18) 30 The disclosed human RUP16 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AL136106 was identified as a human genomic sequence from chromosome 13. The full length RUP16 was cloned by PCR using primers: 5 5'-CrICGATACTGCTCCTATGCTC.

3 ' (SEQJD.NO.:57; sense, 5' of initiation codon), 5'-GTAGTCCACTGAAAGTCCAGTGATCC-3'(SEQ.D.NO.:58; antisense, 3' of stop codon) and human skeletal muscle Marathon-Ready cDNA (Clontech) as template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50ul reaction by the following cycle with step 2 to 4 repeated 35 times: 94*C for 30 seconds; 94*C for 5 10 seconds; 69*C for 15 seconds; 72*C for 1 minute and 72*C for 5 minutes. A 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the T7 sequenase kit (Amsham). See, SEQ.ID.NO.: 17 for nucleic acid sequence and SEQ.ID.NO.: 18 for deduced amino acid sequence. The sequence of RUP16 clones matched with four unordered segments of 15 AL136106, indicating that the RUP16 cDNA is composed of 4 exons. j. hRUP17 (Seq. Id. Nos. 19 & 20) The disclosed human RUP17 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC023078 was identified as a human genomic sequence from chromosome 20 11. The full length RUP17 was cloned by PCR using primers: 5'-TTTCTGAGCATGGATCCAACCATCTC-3' (SEQ.ID.NO.:59; sense, containing initiation codon) 5'-CTGTCTGACAGGGCAGAGGCTCTI-3'(SEQ.ID.NO.:60; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix 25 (Clontech) was used for the amplification in a 100ul reaction with 5% DMSO by the 31 following cycle with step 2 to 4 repeated 30 times: 94 0 C for 1 min; 94 0 C for 15 see; 67*C for 20 sec; 72*C for 1 min and 30 sec; and 72 0 C for 5 min. A 970bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye 5 Ternilantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:19 for nucleic acid sequence and SEQ.ID.NO.:20 for deduced amino acid sequence. k. hRUP18 (Seq. Id. Nos. 21 & 22) The disclosed human RUP18 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 10 Number AC008547 was identified as a human genomic sequence from chromosome 5. The full length RUP18 was cloned by PCR using primers: 5'-GGAACTCGTATAGACCCAGCGTCGCTrCC-3'(SEQ.ID.NO.:61; sense, 5' of the initiation codon), 5'-GGAGGTGOCGCCTTAGCGACAGATGACC-3'(SEQ.ID.NO.:62; antisense, 3' of stop 15 codon) and human genomic DNA (Promega) as template. TaqPlus precision DNA polymerase (Stratagene) was used for the amplification in a 100ul reaction with 5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 95*C for 5 min; 95*C for 30 sec; 65*C for 30 sec; 72 0 C for 2 min; and 72 0 C for 5 min. 20 A 1.3kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:21 for nucleic acid sequence and SEQ.ID.NO.:22 for deduced amino acid sequence. I. hRUP19 (Seq. Id. Nos. 23 & 24) 32 The disclosed human RUP19 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC026331 was identified as a human genomic sequence from chromosome 12. The full length RUP19 was cloned by PCR using primers: 5 5'-CTGCACCCGGACACTTGCTCTG-3' (SEQ.ID.NO.:63; sense, 5' of initiation codon), 5'-GTCTGCTTGTTCAGTGCCACTCAAC-3' (SEQ.ID.NO.:64; antisense, containing the stop codon) and human genomic DNA (Promega) as template. TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification with 5% DMSO by the 10 following cycle with step 2 to 4 repeated 35 times: 94*C for 1 min; 94*C for 15 sec; 70*C for 20 sec; 72 0 C for 1 min and 30 sec; and 72 0 C for 5 min. A I.lkp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:23 for nucleic acid sequence and 15 SEQ.ID.NO.:24 for deduced amino acid sequence. m. hRUP20 (Seq. Id. Nos. 25 & 26) The disclosed human RUP20 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AL161458 was identified as a human genomic sequence from chromosome 20 1. The full length RUP20 was cloned by PCR using primers: 5'-TATCTGCA AT CTAGCTCCTG-3' (SEQ.ID.NO.:65; sense, 5' of initiation codon), 5'-TGTCCCTAATAAAGTCACA T GC-3' (SEQ.ID.NO.:66; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clonetech) was used for the amplification with 5% DMSO by the following cycle with 33 step 2 to 4 repeated 35 times: 940C for 1 min; 94 0 C for 15 sec; 600C for 20 sec; 720C for I min and 30 sec; and 72 0 C for 5 min. A 1.0 kp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye 5 Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:25 for nucleic acid sequence and SEQ.ID.NO.:26 for deduced amino acid sequence, n. hRUP21 (Seq. Id. Nos. 27 & 28) The disclosed human RUP21 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 10 Number AC026756 was identified as a human genomic sequence from chromosome 13. The full length RUP21 was cloned by PCR using primers: 5'-GGAGACAACCATGAATGAGCCAC-3'(SEQ.ID.NO.:67; sense) 5'-TAmCAAGGGTGTITGAGTAAC-3'(SEQ.ID.NO.:68; antisense) and human genomic DNA (Promega) as template. Taq Plus Precision polymerase 15 (Stratagene) was used for the amplification in a 100ul reaction with 5% DMSO by the following cycle with step 2 to 4 repeated 30 times: 94*C for I min; 94*C for 15 sec; 55*C for 20 sec; 72*C for 1 min and 30 sec; and 720C for 5 min. A 1,014 bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye 20 Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:27 for nucleic acid sequence and SEQ.ID.NO.:28 for deduced amino acid sequence. o. hRUP22 (Seq. Id. Nos. 29 & 30) The disclosed human RUP22 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 34 Number AC027026 was identified as a human genomic sequence from chromosome 11. The full length RUP22 was cloned by PCR using primers: 5'- GGCACCAGTGGAGGTMCTGAGCATG -3' (SEQ.ID.NO.:69; sense, containing initiation codon) 5 5'-CTGATGGAAGTAGAGGCTGTCCATCTC-3'(SEQ.ID.NO.:70; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification in a 100ul reaction with 5% DMSO by the following cycle with step 2 to 4 repeated 30 times: 94*C, 1 minutes 94*C, 15 seconds 10 55 0 C, 20 seconds 720C, 1.5 minute 720C, 5 minutes. A 970bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.B. Biosystem). See, SEQ.ID.NO.:29 for nucleic acid sequence and SEQ.ID.NO.:30 for deduced amino acid sequence. 15 p. hRUP23 (Seq. Id. Nos. 31 & 32) The disclosed human RUP23 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC007104 was identified as a human genomic sequence from chromosome 4. The full length RUP23 was cloned by PCR using primers: 20 5'-CCTGGCGAGCCGCTAGCGCCATG-3' (SEQID.NO.:71; sense, ATG as the initiation codon), 5'-ATGAGCCCTGCCAGG(J CAGT-3' (SEQ.ID.NO.:72; antisense, TCA as the stop codon) and human placenta Marathon-Ready cDNA (Clontech) as template. Advantage cDNA 25 polymerase (Clontech) was used for the amplification in a 50ul reaction by the following 35 cycle with step 2 to 4 repeated 35 times: 95*C for 30 see; 95*C for 15 see; 66*C for 20 see; 720C for 1 min and 20 see; and 72 0 C for 5 min. A 1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator Kit (P.E. 5 Biosystem). See, SEQ.ID.NO.:31 for nucleic acid sequence and SEQ.ID.NO.:32 for deduced amino acid sequence. q. hRUP24 (Seq. Id. Nos. 33 & 34) The disclosed human RUP25 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 10 Number AC026331 was identified as a human genomic sequence from chromosome 12. The full length RUP25 was cloned by PCR using primers: 5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ.ID.NO.:73; sense, 5'of initiation codon), 5'-AGATCC[GGTICITGGTGACAATG-3'(SEQ.ID.NO.:74; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix 15 (Clontech) was used for the amplification with 5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 94C for 1 minute; 94*C for 15 seconds; 56*C for 20 seconds 72*C for 1 minute 30 seconds and 720C for 5 minutes. A 1.2kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye 20 Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:33 for nucleic acid sequence and SEQ.ID.NO.:34 for deduced amino acid sequence. r. hRUP25 (Seq. Id. Nos. 35 & 36) The disclosed human RUP25 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession 36 Number AC026331 was identified as a human genomic sequence from chromosome 12. The full length RUP25 was cloned by PCR using primers: 5'-GCTGGAGCATCACTAGGCGAG-3' (SEQ.ID.NO.:75; sense, 5'of initiation codon), 5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.:76; antisense, 3' of stop codon) 5 and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clontech) was used for the amplification with 5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 94 0 C for 1 minute; 94 0 C for 15 seconds; 56*C for 20 seconds 72 0 C for I minute 30 seconds and 72 0 C for 5 minutes. A 1.2kb PCR fragment was isolated from 1% agarose gel and cloned into the 10 pCRII-TOPO vector (Invitrogen) and completely sequenced using the A131 Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:35 for nucleic acid sequence and SEQ.ID.NO.:36 for deduced amino acid sequence. s. hRUP26 (Seq. Id. Nos. 37 & 38) The disclosed human RUP26 was identified based upon the use of GeneBank 15 database information. While searching the database, a cDNA clone with Accession Number AC023040 was identified as a human genomic sequence from chromosome 2. The full length RUP26 was cloned by RT-PCR using RUP26 specific primers: 5'-AGCCATCCTGCCA GAACATGG-3' (SEQ.ID.NO.:77; sense, containing initiation codon) 20 5'-CCAGACTGTGGACTCAAGAACCTAGG-3' (SEQ.ID.NO.:78; antisense, containing stop codon) and human pancreas Marathon - Ready cDNA (Clontech) as template. Advantage cDNA polymerase mix (Clontech) was used for the amplification in a 100ptl reaction with 5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 94*C for 5 minute; 25 95 0 C for 30 seconds; 65*C for 30 seconds 72 0 C for 2 minute and 72 0 C for 5 minutes. 37 A 1.1kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:37 for nucleic acid sequence and SEQ.ID.NO.:38 for deduced amino acid sequence. 5 t. hRUP27 (Seq. Id. Nos. 39 & 40) The disclosed human RUP27 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC027643 was identified as a human genomic sequence from chromosome 12. The full length RUP27 was cloned by PCR using RUP27 specific primers: 10 S'-AGTCCACGAACAATGAATCCAITATG-3'(SEQ.ID.NO.;79; sense, containing initiation codon), 5'-ATCATG'CTAGACTCATGGTGATCC3' (SEQ.ID.NO.:30; antisense, 3' of stop codon) and the human adult brain Marathon-Ready cDNA (Clontech) as template. Advantage cDNA polymerase mix (Clontech) was used for the amplification in a 50pl reaction with 5% 15 DMSO by the following cycle with step 2 to 4 repeated 35 times: 94*C for 1 minute; 940C for 10 seconds; 58 0 C for 20 seconds 72*C for 1 minute 30 seconds and 72*C for 5 minutes. A 1.1kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye 20 Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.:35 for nucleic acid sequence and SEQ.ID.NO.:36 for deduced amino acid sequence. The sequence of RUP27 cDNA clone isolated from human brain was determined to match with five unordered segments ofAC027643, indicating that the RUP27 cDNA is composed of 5 exons. 38 Example 2 PREPARATION OF NON-ENDOGENOUS, CONSTITUTIVELY ACTIVATED GPCRS Those skilled in the art are credited with the ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches utilized to 5 create non-endogenous versions of several of the human GPCRs disclosed above. The mutations disclosed below are based upon an algorithmic approach whereby the 16th amino acid (located in the IC3 region of the GPCR) from a conserved proline (or an endogenous, conservative substitution therefore) residue (located in the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, preferably to an alanine, 10 histidine, arginine or lysine amino acid residue, most preferably to a lysine amino acid residue. 1. Transformer Site-Directed TM Mutagenesis Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using Transformer Site-DirectedTm Mutagenesis Kit (Clontech) according to the 15 manufacturer instructions. Two mutagenesis primers are utilized, most preferably a lysine mutagenesis oligonucleotide that creates the lysine mutation, and a selection marker oligonucleotide. For convenience, the codon mutation to be incorporated into the human GPCR is also noted, in standard form (Table D): 20 TABLE D Receptor Identifier Codon Mutation hRUPS V274K RUP9 T249K hRUP10 R232K hRUP11 M294K hRUP12 F220K hRUP16 A238K 39 hRUP17 Y215K hRUP18 L294K hRUP19 T219K RUPn20 K248A K248H K248R hRUP21 R240K hRUP22 Y222K ~ IiRUP24 A245K hRUP25 1230K hRUP26 V285K hRUP27 T248K 2. QuikChangeTm Site-DirectedT" Mutagenesis Preparation of non-endogenous human GPCRs can also be accomplished by 5 using QuikChangeTM Site-DirectedTM Mutagenesis Kit (Stratagene, according to manufacturer's instructions). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a selection marker oligonucleotide (included in kit). For convenience, the codon mutation incorporated into the novel human GPCR and the 10 respective oligonucleotides are noted, in standard form (Table E): TABLE E Receptor C odon 5'3 rlnai(ense), 5'-3' Orienta-tion Cycle Conditions Identifier aaon E .N) tato (antisense) (SEQ.ID.NO.) M (Co Sec C) underlined Cycles 2-4 repeated 16 times hRUP13 A268K GGGGAGGA CAA CCAGGAGAACCACCT 980 for2' AGGTGTU crGC ~ T 98* for 30" (81) (82) 560C for 30" 720 for 11'40" 720 for 5' hRUPi4 L246K CAGGAAGGCAGAC GATGATGATGGTGGT 980 for 2' CAOCATCATCATC (85) CITCCITCCTG (86) 98* for 30" 55*C for 30" 72* for 11'40" 72 for 5' 40 hRUP15 A398K CCAGTGCAAAGCIAAG GAAGATCACTITCITA 98* for 2' AAAGTGATCrrC (89) GCTITGCACrGG (90) 98* for 30" 55 0 C for 30" 720 for 11' 40" 720 for 5' hRUP23 W275K GCCGCCACCGCGCCAA GCCAATCTICCTCITG 98* for 2' GAGGAAGATIGC(93) GCGCOGTGGOGGC 980 for 30" (94) 56 0 C for 30" 720 for 11' 40" 72* for 5' The non-endogenous human GPCRs were then sequenced and the derived and verified nucleic acid and amino acid sequences are listed in the accompanying 5 "Sequence Listing" appendix to this patent document, as summarized in Table F below: TABLE F Non Endogenous Human Nucleic Acid Sequence Listing Amino Add Sequence GPCR Listing hRUP13 SEQ.IDNO.:83 SEQ.ID.NO.:84 hRUP14 SEQJD.NO.:87 SEQ.D.NO.:88 -RUP15 SEQ.D.NO.:91 SEQ.ID.NO.:92 hRUP23 SEQ.ID.NO.:95 SEQ.ID.NO.:96 Example 3 10 RECEPTOR EXPRESSION Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the 15 expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems - thus, results obtained in non-mammalian cells, while of 41 potential use, are not as preferred as that obtained from mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. 5 a. Transient Transfection On day one, 6x10 6 / 10 cm dish of 293 cells well were plated out. On day two, two reaction tubes were prepared (the proportions to follow for each tube are per plate): tube A was prepared by mixing 4pig DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B was prepared by 10 mixing 24pl lipofectamine (Gibco BRL) in 0.5ml serum free DMEM. Tubes A and B were admixed by inversions (several times), followed by incubation at room temperature for 3 0-45min. The admixture is referred to as the "transfection mixture". Plated 293 cells were washed with 1XPBS, followed by addition of 5 ml serum free DMEM. I ml of the transfection mixture were added to the cells, followed by incubation for 4hrs at 15 370C/5% CO2. The transfection mixture was removed by aspiration, followed by the addition of 10ml of DMEM/1 0% Fetal Bovine Serum. Cells were incubated at 37*C/5%

CO

2 . After 48hr incubation, cells were harvested and utilized for analysis. b. Stable Cell Lines: Gs Fusion Protein Approximately 12x106 293 cells are plated on a 15cm tissue culture plate. 20 Grown in DME High Glucose Medium containing ten percent fetal bovine serum and one percent sodium pyruvate, L-glutamine, and anti-biotics. Twenty-four hours following plating of 293 cells to -80% confluency, the cells are transfected using 12sg of DNA. The 12ptg of DNA is combined with 60ul of lipofectamine and 2mL of DME High Glucose Medium without serum. The medium is aspirated from the plates and the 25 cells are washed once with medium without serum. The DNA, lipofectamine, and 42 medium mixture is added to the plate along with 10mL of medium without serum. Following incubation at 37 degrees Celsius for four to five hours, the medium is aspirated and 25ml of medium containing serum is added. Twenty-four hours following transfection, the medium is aspirated again, and fresh medium with serum is added. 5 Forty-eight hours following transfection, the medium is aspirated and medium with serum is added containing geneticin (G418 drug) at a final concentration of 500pg/mL. The transfected cells now undergo selection for positively transfected cells containing the G418 resistant gene. The medium is replaced every four to five days as selection occurs. During selection, cells are grown to create stable pools, or split for stable clonal 10 selection. Example 4 ASSAYS FOR DETERMINATION OF CoNsTiTUTIVE ACTIVITY OF NON-ENDOGENOUS GPCRs 15 A variety of approaches are available for assessment of constitutive activity of the non-endogenous human GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan. 1. Membrane Binding Assays: [ 35 SJGTPTS Assay 20 When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes 25 the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, [ 35 S]GTPyS, can be utilized to demonstrate enhanced binding of [ 5 S]GTPyS to membranes expressing constitutively activated receptors. The advantage of using 43

[

3 5 SJGTPyS binding to measure constitutive activation is that: (a) it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade. The assay utilizes the ability of G protein coupled receptors to stimulate 5 [ 3 S]GTPyS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to known, orphan and constitutively activated G protein-coupled receptors. The assay is generic and has application to drug discovery at all G protein-coupled receptors. The [ 5 S]GTPyS assay was incubated in 20 mM HEPES and between 1 and 10 about 20mM MgCl 2 (this amount can be adjusted for optimization of results, although 20mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [S]GTPyS (this amount can be adjusted for optimization of results, although 1.2 is preferred ) and 12.5 to 75 jig membrane protein (e.g, 293 cells expressing the Gs Fusion Protein; this amount can be adjusted for optimization) and 10 pM GDP (this amount can 15 be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 pl; Amersham) were then added and the mixture incubated for another 30 minutes at room temperature. The tubes were then centrifuged at 1500 x g for 5 minutes at room temperature and then counted in a scintillation counter. 2. Adenylyl Cyclase 20 A Flash PlateTm Adenylyl Cyclase kit (New England Nuclear, Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP 44 antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors. Transfected cells were harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10ml of PBS is gently 5 added to each dish of cells followed by careful aspiration. Il of Sigma cell dissociation buffer and 3ml of PBS are added to each plate. Cells were pipeted off the plate and the cell suspension was collected into a 50m] conical centrifuge tube. Cells were then centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet was carefully re-suspended into an appropriate volume of PBS (about 3ml/plate). The cells 10 were then counted using a hemocytometer and additional PBS was added to give the appropriate number of cells (with a final volume of about 50 pl/well). cAMP standards and Detection Buffer (comprising 1 pCi of tracer [2cAMP (50 p1] to 11 ml Detection Buffer) was prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer was prepared fresh for screening and 15 contained 50pl of Stimulation Buffer, 3ul of test compound (12uM final assay concentration) and 50pl cells, Assay Buffer was stored on ice until utilized. The assay was initiated by addition of 50pl of cAMP standards to appropriate wells followed by addition of 50ul of PBSA to wells H-I1 and H12. 50p of Stimulation Buffer was added to all wells. DMSO (or selected candidate compounds) was added to appropriate wells 20 using a pin tool capable of dispensing 3p] of compound solution, with a final assay concentration of 12pM test compound and 100pi total assay volume. The cells were then added to the wells and incubated for 60 min at room temperature, 100pl of Detection Mix containing tracer cAMP was then added to the wells. Plates were then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation 45 counter. Values of cAMP/well were then extrapolated from a standard cAMP curve which was contained within each assay plate. 3. Cell-Based cAMP for Gi Coupled Target GPCRs 5 TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation. TSHR will be constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue). A Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging. An effective 10 technique for measuring the decrease in production of cAMP as an indication of constitutive activation of a Gi coupled receptor can be accomplished by co-transfecting, most preferably, non-endogenous, constitutively activated TSHR (TSHR-A6231) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP. Upon creating a non 15 endogenous version of the Gi coupled receptor, this non-endogenous version of the target GPCR is then co-transfected with the signal enhancer, and it is this material that can be used for screening. We will utilize such approach to effectively generate a signal when a cAMP assay is used; this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled 20 GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal. On day one, 2X10 4 293 and 293 cells/well will be plated out. On day two, two reaction tubes will be prepared (the proportions to follow for each tube are per plate): tube A will be prepared by mixing 2sg DNA of each receptor transfected into the 25 mammalian cells, for a total of 4pg DNA (e.g., pCMV vector, pCMV vector with mutated THSR (TSHR-A6231); TSHR-A6231 and GPCR, etc.) in 1.2ml serum free 46 DMEM (Irvine Scientific, Irvine, CA); tube B will be prepared by mixing 120p1 lipofectanine (Gibco BRL) in 1,2ml serum free DMEM. Tubes A and B will then be admixed by inversions (several times), followed by incubation at room temperature for 3 0-45min. The admixture is referred to as the "transfection mixture". Plated 293 cells 5 will be washed with IXPBS, followed by addition of loml serum free DMEM. 2.4ml of the transfection mixture will then be added to the cells, followed by incubation for 4hrs at 37 0 C/5%.CO 2 . The transfection mixture will then be removed by aspiration, followed by the addition of 25ml of DMEMI/0% Fetal Bovine Serum. Cells will then be incubated at 37*C/5% CO 2 . After 24hr incubation, cells will then be harvested and 10 utilized for analysis. A Flash PlateTm Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMPO04A) is designed for cell-based assays, however, can be modified for use with crude plasma membranes depending on the need of the skilled artisan. The Flash Plate wells will contain a scintillant coating which also contains a specific antibody 15 recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors. Transfected cells will be harvested approximately twenty four hours after 20 transient transfection. Media will be carefully aspirated off and discarded. 10ml of PBS will be gently added to each dish of cells followed by careful aspiration. Imil of Sigma cell dissociation buffer and 3ml of PBS will be added to each plate. Cells will be pipeted off the plate and the cell suspension will be collected into a 50ml conical centrifuge tube. Cells will then be centrifuged at room temperature at 1,100 rpm for 5 min. The cell 25 pellet will be carefully re-suspended into an apropriate volume of PBS (about 47 3ml/plate). The cells will then be counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50g1/well). cAMP standards and Detection Buffer (comprising 1 pCi of tracer [125 cAMP 5 (50 p1] to II ml Detection Buffer) will be prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer should be prepared fresh for screening and contained 50pl of Stimulation Buffer, 3ul of test compound (12uM final assay concentration) and 50p4 cells, Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50pl of cAMP standards to appropriate wells followed by 10 addition of 50pl of PBSA to wells H-11 and H12. 50ul of Stimulation Buffer will be added to all wells. Selected compounds (e.g., TSH) will be added to appropriate wells using a pin tool capable of dispensing 3pl of compound solution, with a final assay concentration of 12iM test compound and 100p1 total assay volume. The cells will then be added to the wells and incubated for 60 min at room temperature. 100pl of Detection 15 Mix containing tracer cAMP will then be added to the wells. Plates were then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well will then be extrapolated from a standard cAMP curve which is contained within each assay plate. 4. Reporter-Based Assays 20 a. CRE-LUc Reporter Assay (Gs-associated receptors) 293 and 293T cells are plated-out on 96 well plates at a density of 2 x 104 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA/lipid mixture is prepared for each 6 well transfection as follows: 260ng of plasmid DNA in 100pl of DMEM were gently 25 mixed with 2pl of lipid in 100pl of DMEM (the 260ng of plasmid DNA consisted of 48 2 00ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and 1Ong of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc reporter plasmid was prepared as follows: vector SRIF-P-gal was obtained by cloning the rat somatostatin 5 promoter (-71/+51) at BgIV-HindII site in the ppgal-Basic Vector (Clontech). Eight (8) copies of cAMP response element were obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-p-gal vector at the Kpn-BglV site, resulting in the 8xCRE-p-gal reporter vector. The 8xCRE-Luc reporter plasmid was generated by replacing the 10 beta-galactosidase gene in the 8xCRE-p-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindIII-BamHI site. Following 30 min. incubation at room temperature, the DNA/lipid mixture was diluted with 400 pl of DMEM and 100pl of the diluted mixture was added to each well. 100 pl of DMEM with 10% FCS were added to each well after a 4hr incubation 15 in a cell culture incubator. The following day the transfected cells were changed with 200 pl/well of DMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100 pl /well of DMEM without phenol red, after one wash with PBS. Luciferase activity were measured the next day using the LucLiteTm reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBetaTM 20 scintillation and luminescence counter (Wallac). b. AP1 reporter assay (Gq-associated receptors) A method to detect Gq stimulation depends on the known property of Gq dependent phospholipase C to cause the activation of genes containing APi elements 25 in their promoter. A PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue # 219073) can be utilized following the protocol set forth above with respect to the 49 CREB reporter assay, except that-the components of the calcium phosphate precipitate were 410 ng pAP I-Luc, 80 ng pCMV.-receptor expression plasmid, and 20 ng CMV SEAP. c. SRF-Luc Reporter Assay (Gq- associated receptors) 5 One method to detect Gq stimulation depends on the known property of Gq dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A PathdetectTm SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, e.g., COS7 cells. Cells are transfected with the plasmid components of the system and the indicated 10 expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to 15 control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells in a serum free media for 24 hours. The last 5 hours the cells are incubated with I pM Angiotensin, where indicated. Cells are then lysed and assayed for luciferase activity 20 using a LucliteTM Kit (Packard, Cat, # 6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. The data can be analyzed using GraphPad PrismTm 2.0a (GraphPad Software Inc.). 25 50 d. Intracellular

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3 Accumulation Assay (Gq-associated receptors) On day 1, cells comprising the receptors (endogen6us and/or non-endogenous) 5 can be plated onto 24 well plates, usually 1x105 cells/well (although his umber can be optimized. On day 2 cells can be transfected by firstly mixing 0.25pg DNA in 50 i serum free DMEM/weII and 2 pl lipofectamine in 50 pl serumfree DMBM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS and 400 pl of serum free media is mixed with the transfection 10 media and added to the cells. The cells are then incubated for 3-4 hrs at 370C/5%CO2 and then the transfection media is removed and replaced with Iml/well of regular growth media. On day 3 the cells are labeled with 3 H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 pCi of 3 H-myo-inositol/ well and the cells 15 are incubated for 16-18 hrs o/n at 370C/5%C02. On Day 4 the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositol-free/serum free media 10 pM pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50Il of l0x ketanserin (ket) to final concentration of 10pM. The cells are then incubated for 30 min at 370C. The cells are then washed with 0.5 ml PBSand 200pI of fresh/icecold stop 20 solution (IM KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The solution is kept on ice for 5-10 min or until cells were lysed and then neutralized by 200 pL of fresh/ice cold neutralization sol. (7.5 % HCL). The lysate is then transferred into 1.5 ml eppendorf tubes and I ml of chloroform/methanol (1:2) is added/tube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8Th anion 25 exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded onto the column. The column is washed with 10 mIs of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60mM Na-formate. The inositol 51 tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/ I M ammonium formate. The columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsed twice with dd H 2 0 and stored at 4 0 C in water. 5 Exemplary results are presented below in Table G: TABLE G Receptor Mutation Assay Signal Signal Signal Difference Utilized Generated: Generated: Generated: (<( Figure No.) CMV Endogenous Non- Between Version Endogenous . CMV v. (Relative Light Version Wild-type Units) (Relative . Wild-type Light Units) v. Mutant hRUP12 N/A 1P 3 317.03 3463.29 (Figure 1) cpm/mg protein cpm/ng protein 1. 11 Fold hRUP13 N/A cAMP 8.06 19.10 (Figure 2) pmol/cAMP/mg pmo/cAMP/mg 1. 2.4 Fold c protein protein A268K 8XCRE- 3665.43 83280.17 61713.6 1. 23 Fold LUC LCPS LPCS LCPS (Figure 3) 2. 26%( hRUP14 L246K 8XCRE- 86.07 1962.87 789.73 1. 23 Fold < LUC LCPS LCPS LCPS ---- 5- _.2. 60%( hRUP15 A398K 8XCRE 86.07 18286.77 17034.83 1. 212 Fold LUC LCPS LCPS LCPS <= (Figure 6) 2. 1% A398K cAMP 15.00 164.4 117.5 1. 11 Fold (Figure 7) pmolfcAMP/mg pmol/cAMP/mg pmol/cAMP/ 4= protein protein mg protein 2. 29%( hRUP17 N/A IIp, 317.03 741.07 .. 2.3 Fold (Figure 9) cpmnmg protein cpm/mg protein hRUP21 N/A

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3 730.5 1421.9 1. 2 Fold .10 cpmng protein cpm/mg protein hRUP23 W275K 8XCRE- 311.73 13756.00 9756.87 1. 44 Fold LUC pmol/cAMP/mg pmol/cAMP/nig pmol/cAMP/ ge 11 otein protein mng protein 2. 30% ( NA not applied 52 Exemplary results of GTPyS assay for detecting constitutive activation, as disclosed in Example 4(1) above, was accomplished utilizing Gs:Fusion Protein Constructs on human RUP13 and RUP15. Table H below lists the signals generated from this assay and the difference in signals as indicated: 5 TABLE H Receptor: Assay | Signal Signal Signal Signal Difference Gs Fusion Utilized Generated: Generated: Generated: Generated: Between: Protein . CMV Fusion CMV+ Fusion 1. CMV v. Fusion (cpm bound Protein 10pMGDP Protein + Protein GTP) (cpm bound (cpm bound 10pM GDP 2.CMV+GDP GTP) GTP) (cpm bound vs. GTP) Fuslon+GDP 3. Fusion vs. Fusion+GDP (cpm bound GTP) hRUP13-Gs GTPyS 32494.0 49351.30 1114830 28834.67 1. 1.5 Fold < (Figure 4) 2. 2.6 Fold < 3. 42%( hRUP15-Gs GTPyS 30131.67 32493.67 7697.00 14157.33 1. 1.1 Fold< (Figure 8) 2. 1.8 Fold < 3. 56%( Example 5 FUSION PROTEIN PREPARATION 10 a. GPCR:Gs Fusion Constuct The design of the constitutively activated GPCR-G protein fusion construct was accomplished as follows: both the 5' and 3' ends of the rat G protein Gsa (long form; Itoh, H. et al, 83 PNAS 3776 (1986)) were engineered to include a HindlIH (5' 15 AAGCTT-3') sequence thereon. Following confirmation of the correct sequence (including the flanking HindlII sequences), the entire sequence was shuttled into pcDNA3.1(-) (Invitrogen, cat. no. V795-20) by subcloning using the HindlH restriction site of that vector. The correct orientation for the Gsa sequence was determined after 53 subeloning into pcDNA3.1(-). The modified pcDNA3.1(-) containing the rat Gsa gene at HindIII sequence was then verified; this vector was now available as a "universal" Gsa protein vector. The pcDNA3.1(-) vector contains a variety of well-known restriction sites upstream of the HindlI site, thus beneficially providing the ability to 5 insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach can be utilized to create other "universal" G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized - the important criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein. 10 RUP13 couples via Gs. For the following exemplary GPCR Fusion Proteins, fusion to Gsa was accomplished. A RUP13-Gsx Fusion Protein construct was made as follows: primers were designed as follows: 5'-gtc[TCTAGAAT]GGAGTCCTCACCCATCCCCCAG -3' (SEQ.ID.NO.:97; sense) 15 5'-gat[GATATC]CGTGACTCCAGCCGGGGTGAG GCGGC-3'(SEQ.ID.NO.:98; antisense). Nucleotides in lower caps are included as spacers in the restriction sites (designated in brackets) between the G protein and RUP13. The sense and anti-sense primers included the restriction sites for XbaI and EcoRV, respectively, such that spacers (attributed to the restriction sites) exists between the G protein and RUP 15. 20 PCR was then utilized to secure the respective receptor sequences for fusion within the Gsa universal vector disclosed above, using the following protocol for each: 1OOng cDNA for RUP15 was added to separate tubes containing 21l of each primer (sense and anti-sense), 3p.L of 10mM dNTPs, 10pL of 1OXTaqPlusTM Precision buffer, 1pL of TaqPlusTM Precision polymerase (Stratagene: #600211), and 8OpL of water. 25 Reaction temperatures and cycle times for RUP15 were as follows with cycle steps 2 54 through 4 were repeated 35 times: 94 0 C for I min; 94 0 C for 30 seconds; 62*C for 20 sec; 72 0 C 1 min 40sec; and 720 C 5 min. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was digested with XbaI and EcoRV and the desired inserts purified and ligated into the Gs universal vector at the 5 respective restriction site. The positive clones was isolated following transformation and determined by restriction enzyme digest; expression using 293 cells was accomplished following the protocol set forth infra. Each positive clone for RUPI 5-Gs Fusion Protein was sequenced to verify correctness. (See, SEQ.ID.NO.:99 for nucleic acid sequence and SEQ.ID.No.: 100 for amino acid sequence). 10 RUP15 couples via Gs. For the following exemplary GPCR Fusion Proteins, fusion to Gsa was accomplished. A RUP15-Gsa Fusion Protein construct was made as follows: primers were designed as follows: 5'-TCTAGAATGACGTCCACCTGCACCAACAGC-3' (SEQ.ID.NO.:101; sense) 15 S'-gtatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3'(SEQID.NO.:102; antisense). Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and RUPI5. The sense and anti-sense primes included the restriction sites for EcoRV and Xbal, respectively, such that spacers (attributed to the restriction sites) exists between the G protein and RUP15. 20 PCR was then utilized to secure the respective receptor sequences for fusion within the Gsx universal vector disclosed above, using the following protocol for each: 100ng cDNA for RUP15 was added to separate tubes containing 2pl of each primer (sense and anti-sense), 3iL of lOmM dNTPs, 10iL of 1OXTaqPlusTM Precision buffer, luL of TaqPlusTM Precision polymerase (Stratagene: #600211), and 80pL of water. 25 Reaction temperatures and cycle times for RUP15 were as follows with cycle steps 2 55 through 4 were repeated 35 times: 94*C for I min; 9400 for 30 seconds; 62*C for 20 sec; 720C 1 min 40sec; and 72* C 5 min. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was digested). The purified product was digested with EcoRV and Xbal and the desired inserts purified and 5 ligated into the Gs universal vector at the respective restriction site. The positive clones was isolated following transformation and determined by restriction enzyme digest; expression using 293 cells was accomplished following the protocol set forth infra. Each positive clone for RUP15-Gs Fusion Protein was sequenced to verify correctness. (See, SEQ.ID.NO.: 103 for nucleic acid sequence and SEQ.ID.NO.: 104 for amino acid 10 sequence). b. Gq(6 amino acid deletion)/Gi Fusion Construct The design of a Gq (del)/Gi fusion construct can be accomplished as follows: the N-terminal six (6) amino acids (amino acids 2 through 7, having the sequence of TLESIM (SEQ.ID.NO.: 129) Gaq-subunit will be deleted and the C-terminal five (5) 15 amino acids, having the sequence EYNLV (SEQ.ID.NO.:130) will be replace with the corresponding amino acids of the Goi Protein, having the sequence DCGLF (SEQ.ID.NO.:131). This fusion construct will be obtained by PCR using the following primers: 2 '-gtcaagettcCATGGCGTGCTGCCTGAGCGAGGAG-3' (SEQ.ID.NO.: 132) and 20 5'-gateggatecTTAGAACAGGCCGCAGTCCITCAGGTTCAGCTGCAGGATGGIU-3' (SEQ.ID.NO.: 133) and Plasmid 63313 which contains the mouse Gaq-wild type version with a 25 hemagglutinin tag as template. Nucleotides in lower caps are included as spacers. TaqPlus Precision DNA polymerase (Stratagene) will be utilized for the amplification by the following cycles, with steps 2 through 4 repeated 35 times: 95 0 C 56 for 2 min; 95*C for 20 sec; 56*C for 20 sec; 72*C for 2 min; and 72*C for 7 min. The PCR product is cloned into a pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). Inserts from a TOPO clone containing the sequence of the fusion construct are shuttled into the 5 expression vector pcDNA3.1(+) at the HindIII/BamHI site by a 2 step cloning process. Example 6 Preferential coupling of RUP18 to Gi: 10 IP 3 assay in 293 cells co-expressing hRUP18 and Gq(DEL)/Gi To determine coupling preference for RUP18 and Gq(del)/Gi fusion construct ("GqdelGi") were used in a method of transient transfection in conjunction with an IP 3 assay in 293 cells. IP 3 assay was carried out approximately 24h post-transfection, essentially as described. In a first experimental group, 293 15 cells were transiently transfected with empty expression vector pCMV, with or without co-transfection of GqdelGi in pCMV. In a second experimental group, 293 cells were transiently transfected with endogenous ("WT") RUP 18 in pCMV, with or without co-transfection of GqdelGi in pCMV. In a third experimental group, 293 cells were transiently transfected with non-endogenous ("L294K") RUP18 in 20 pCMV, with our without co-transfection of GqdelGi in pCMV. Transiently transfected cells were split into 96-well plates (50,000 cells/well) and allowed to attach for a period of 6 h. The growth medium was then replaced with medium supplemented with 4 pCi/ml [ 3 H]myo-inositol (100pl; Perkin Elmer Life Sciences) and the cells were allowed to incubate for approximately 20 h. The plates were 25 then frozen overnight at -80*C to achieve complete cell lysis. The following day, the assay plates were thawed at room temperature. The thawed contents were then 57 transferred to 96-well filter plates (Millipor, Multiscreen) pre-loaded with resin (Biorad, AGl-X8 100-200 mesh, formate form). The plate was filtered using a vacuum manifold and the resin was washed multiple times with water. An elution buffer was then applied (200 pl, 0.2 M ammonium formate/0.1 M formic acid) and 5 the resulting eluent was collected, under vacuum, in a 96-well collection plate. Aliquots of the eluent (80 pl) were transferred to filter plates (Whatman, Unifilter GF/C and dried in a 45'C oven overnight. Dried plates were counted on a scintillation counter following the addition of an appropriate scintillant (Perkin Elmer Life Sciences, Optiphase Supermix or Hi-Safe 3). 10 Results are presented in Figure 12. Both endogenous RUP18 and non endogenous RUP18 (L294K) stimulated IP3 production to a much greater extent when co-expressed with GqdelGi. GqdelGi alone did not enhance IP3 production, indicating that the observed stimulation required the presence of RUP18. The results indicate that RUP18 evidences a detectable level of constitutive activity in 15 its endogenous form and that RUP 18 has a coupling preference for Gi. 58 Example 7 Tissue distribution of the disclosed human GPCRs: RT-PCR RT-PCR was applied to confirm the expression and to determine the tissue distribution of several novel human GPCRs. Oligonucleotides utilized were GPCR 5 specific and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA ploymerase (Stratagene) were utilized for the amplification in a 40ptl reaction according to the manufacturer's instructions. 20pl of the reaction will be loaded on a 1.5% agarose gel to analyze the RT-PCR products. Table J below lists the receptors, the cycle conditions and the primers 10 utilized. TABLE J Receptor Cycle 5' Primer 3' Primer DNA Tissue Identifier Conditions (SEQ.ID.NO.) (SEQ.ID.NO.) Fragment Expression Min ('), Sec (") Cycles 2-4 repeated 30 times hRUPIO 940 for 30" CATGTATGCCA GCTATGCCTGA 730bp Kidney, 940 for 10" GCGTCCTGCTC AGCCAGTCTTG leukocyte, 62"C for 20" C(105) TG(106) liver, placenta 720 for ' and spleen 720 for 7' *cycles 2-4 repeated 35 times hRUP 11 940 for 2' GCACCTGCTCC CACAGCGCTG 630bp Liver, kidney, 940 for 15" TGAGCACCTTC CAGCCCTGCA pancreas, 67"C for 15" TCC (107) GCTGGC colon, small 720 for 45" (108) intestinal, 720 for 5' spleen and prostate hRUP12 940 for 2' CCAGTGATGA CAGACACTTG 490bp Brain, colon, 940 for 15" CTCTGTCCAGC GCAGGGACGA heart, kidney, 66"C for 15" CTG(109) GGTG(1 10) leukocyte, 72' for 45" pancreas, 720 for 5' prostate, small intestine, spleen, testis, and thymus 58a hRUP13 94* for ' CTGTGGTCT CATATCCCrC 700bp Placenta and 940 for 15" ACTGCAGCA CGAGTGTCC lung 68 0 C for 20" TGTTCCG AGCGGC (112) 72* for 1'45" (111) 72* for 5' hRUP14 940for I' ATGGATCCT CAAGAACAG 700bp Not yet 94* for 15" TATCATGGC GTCTCATCTA detennined 68 0 C for 20" TTCCTC (113) AGAGCTCC 720 for 1'45" (114) 720 for 5' hRUP16 940 for 30" CTUTGATGC GTAGTCCACT 370bp Fetal brain, fetal 940 for 5" CATCTCCG GAAAGTCCA kidney and fetal 69*C for 15" GATTCCTG GTGATCC skeletal muscle 720 for 30" (115) (116) 720 for 5' hRUP18 940for2' TGGTGGCGA GTTGCGCCTT 330bp Pancreas 940 for 15" TGGCCAACA AGCGACAGA 60*C for 20" GCGCTC (117) TGACC (118) 72* for I' 72* for 5' hRUP21 94* for 1' TCAACCTGT AAGGAGTAG Kidney, lung 94* for 15" ATAGCAGCA CAGAATGGT and testis 56*C for 20" TCCTC (119) TAGCC (120) 720 for 40" *cycles 2-3 repeated 30 times hRUP22 94* for 30" GACACCTGT CITGATGGAA Testis, thymus 940 for 15" CAGCGGTCG GTAGAGGCT and spleen 69 0 C for 20" TGTGTG (121) GTCCATCTC 72* for 40" (122) *cycles 2-3 repeated 30 times hRUP23 940 for 2' GCGCTGAGC CACGGTGAC 520bp Placenta 940 for 15" GCAGACCAG GAAGGGCAC 60*C for 20" TGGCTG (123) GAGCTC (124) 72* for I' 72* for 5' hRUP26 940 for 2' AGCCATCCC CCAGGTAGG 470bp Pancreas 94* for 15" TGCCAGGAA TGTGCAGCA 65*C for 20" GCATGG (125) CAATGGC 720 for 1' (126) 720 for 5' hRUP27 94* for 30" CTGTICAAC ATCATGTCTA 890bp Brain 940 for 10" AGGGCTGGT GACICATGGT 55C for 20" TGGCAAC GATCC (128) 72* for l' (127) 720 for 3' *cycles 2-4 repeated 35 times 59 Example 8 Tissue distribution of human RUP18: RT-PCR RT-PCR was used to confirm the expression and to determine the tissue distribution of RUP 18. Oligonucleotides used has the following sequences: 5 5'-TGGTGGCGATGGCCAACAGCGCTC-3' (SEQ ID NO.: 117; sense), 5'-GTTGCGCCTTAGCGACAGATGACC-3' (SEQ ID NO.: 118; antisense), and the human multiple tissue cDNA panels (MTC, Clontech) were used as templates. PCR was performed using Taq DNA polymerase (Stratagene; manufacturer's instructions followed) in a 40 jfl reaction by the following cycles: 94"C for 2 min; 94"C 10 for 15 sec; 60'C for 20 sec, 72"C for 1 min, and 72"C for 5 min. Cycles 2 through 4 were repeated 30 times. Then, 20 pl of the reaction were loaded on a 1% agarose gel to analyze the RT-PCR products. A specific 3 3Obp DNA fragment representing RUP 18 was strongly detected in the pancreas (lane 9), while weakly detected in the leukocytes (lane 5), prostate (lane 15 11), spleen (lane 14) and testis (lane 16) (as shown in Figure 13). Example 9 Expression of RUP18 in mouse pancreatic beta cells RT-PCR was used to determine whether RUP18 is expressed in mouse 20 pancreatic islets and in rodent insulin-producing cell lines. The cell lines used were HIT-T15 9ATCC CRL-1777), NIT-1 (ATCC CRL-2055) and pTC-6 9ATCC CRL 11506). All of these cell lines are known to produce insulin. Pancreatic islets were isolated from C57B16/N mouse pancreata by standard methods. Total RNA was isolated from all cell sources TRIZOL@ Reagent, and cDNA 60 was prepared from these RNA samples by standard methods. The primers used to detect mouse RUP 18 were: 5'-TCCTTGCCCAGCGCGTTGCCCCAAGCCAAG-3' (SEQ ID NO: 117; sense) and 5'-CTAGAAGGCACTTTCACAGGAGCAGGGCGG-3' (SEQ ID NO: 118;antisense). 5 Taq DNA polymerase (Stratagene) was used in a 40 pl reaction according to the manufacturer's instructions. The amplification conditions were as follows: 1 cycle: 940 for 30 sec; 30 cycles: 940 for 30 sec, 530 for 30 sec, 720 for 2 min; and 1 cycle: 720 for 7 min. 201.l of each reaction were loaded on a 1.5% agarose gel to analyze the PCR products. 10 Results are presented in Figure 14. The arrow indicates the position of the 465bp amplification product corresponding to mouse RUP18. RUP18 was clearly detected in the mouse pancreatic islet sample as well as in the mouse insulin-producing pancreatic beta cell lines NIT-1 and PTC-6. 15 Example 10 Protocol: Direct Identification of Inverse Agonists and Agonists A. [ 35 S]GTPyS Assay Although we have utilized endogenous, constitutively active GPCRs for the direct identification of candidate compounds as, e.g., inverse agonists, for reasons that 20 are not altogether understood, intra-assay variation can become exacerbated. Preferably, then, a GPCR fusion protein, as disclosed above, is also utilized with a non endogenous, constitutively activated GPCR. We have determined that when such a protein is used, intra-assay variation appears to be substantially stabilized, whereby an effective signal-to-noise ration is obtained. This has the beneficial result of allowing for 25 a more robust identification of candidate compounds. Thus, it is preferred that for direct 61 identification, GPCR Fusion Protein be used and that when utilized, the following assay protocols be utilized. 1. Membrane Preparation Membranes comprising the constitutively active orphan GPCR Fusion Protein 5 of interest and for use in the direct identification of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows: a. Materials "Membrane Scrape Buffer" is comprised of 20mM HEPES and 10mM EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES and 0.1 mM EDTA, 10 pH 7.4; "Binding Buffer" is comprised of 20mM HEPES, 100 mM NaCl, and 10 mM MgCl2, pH 7.4. b. Procedure All materials will be kept on ice throughout the procedure. Firstly, the media are aspirated from a confluent monolayer of cells, followed by rinse with 10ml cold 15 PBS, followed by aspiration. Thereafter, 5ml of Membrane Scrape Buffer is added to scrape cells, followed by transfer of cellular extract into 50ml centrifuge tubes (centrifuged at 20, 000 rpm for 17 minutes at 4'C). Thereafter, the supernatants are aspirated and each pellet is resuspended in 30ml Membrane Wash Buffer followed by centrifugation at 20,000 rpm for 17 minutes at 4'C. The supernatants are then aspirated 20 and the pellets resuspended in Binding Buffer and homogenized using a Brinkman polytron homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as"Membrane Protein". 2. Bradford Protein Assay Following the homogenization, protein concentration of the membranes is 25 determined using the Bradford Protein Assay (protein can be diluted to about 1. 62 5mg/ml, aliquoted and frozen (-80'C) for later use. When frozen, the protocol for use is as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a polytron at about 12 x 1,000 rpm for about 5-10 seconds (for multiple preparations, the homogenizor should be 5 thoroughly cleaned between homogenization of different preparations). a. Materials Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard is utilized, following manufacturer instructions (Biorad, cat. no. 500- 0006). b. Procedure 10 Duplicate tubes are prepared, one including the membrane, and one as a control"blank". Each tube contains 800ul Binding Buffer. Thereafter, lOIl of Bradford Protein Standard (1 mg/ml) is added to each tube, and lOgl of membrane protein is added to just one tube (not the blank). Thereafter, 200pl of Bradford Dye Reagent is added to each tube, followed by vortex of each. After five (5) minutes, the tubes are re 15 vortexed and the material therein is transferred to cuvettes. The cuvettes are read using a CECIL 3041 spectrophotometer, at wavelength 595nm. 3. Direct Identification Assay a. Materials GDP Buffer consisted of 37. 5 ml Binding Buffer and 2mg GDP (Sigma, cat. 20 no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 pM GDP (final concentration of GDP in each well was 0.1 gM GDP); each well comprising a candidate compound has a final volume of 200 pl consisting of 100 pl GDP Buffer (final concentration, 0.1 pM GDP), 50pl membrane protein in Binding Buffer, and 50 pl [1 5 S]GTPyS (0.6 nM) in Binding Buffer (2.5 p.l [ 3 5 S]GTPyS per 10ml Binding Buffer). 63 b. Procedure Candidate compounds are preferably screened using a 96-well plate format (these can be frozen at -80'C). Membrane protein (or membranes with expression vector excluding the GPCR Fusion Protein, as control), are homogenized briefly until in 5 suspension. Protein concentration is determined using the Bradford Protein Assay set forth above. Membrane protein (and control) is diluted to 0.25 mg/ml in Binding Buffer (final assay concentration, 12.5 gg/well). Thereafter, 100 pl GDP Buffer is added to each well of a Wallac Scintistrip T M (Wallac). A 5 d pin-tool is used to transfer 5 pl of a candidate compound into such well (i.e., 5 1il in total assay volume of 200 R1 is a 1:40 10 ratio such that the final screening concentration of the candidate compound is 10 IM). Again, to avoid contamination, after each transfer step the pin-tool should be rinsed in three reservoirs comprising water (lX), ethanol (lX) and water (2X)-excess liquid should be shaken from the pin-tool after each rinse and dried with paper and kimwipes. Thereafter, 50 jl of membrane protein is added to each well (a control well comprising 15 membranes without the GPCR fusion protein is also utilized), and pre-incubated for 5 10 minutes at room temperature. Thereafter, 50 I of [ 3 5 S]GTPyS (0. 6 nM) in Binding Buffer is added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates are covered with foil). The assay is stopped by spinning of the plates at 4,000 r.p.m. for 15 minutes at 22'C. The plates are 20 aspirated with an 8-channel manifold and sealed with plate covers. The plates are read on a Wallace 1450 using setting "Prot. #37" (as per manufacturer instructions). B. Cyclic AMP Assay Another assay approach to directly identified candidate compound was accomplished by utilizing a cyclase-based assay. In addition to direct identification, this 64 assay approach can be utilized as an independent approach to provide confirmation of the results from the [35 ] GTPyS approach as set forth above. A modified Flash Plate Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) was preferably utilized for direct identification of candidate compounds as 5 inverse agonists and agonists to constitutively activated orphan GPCRs in accordance with the following protocol. Transfected cells were harvested approximately three days after transfection. Membranes were prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10mM MgCl 2 . Homogenization was performed on ice 10 using a Brinkman Polytron TM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 X g for 15 minutes at 4'C. The resulting pellet was then resuspended in buffer containing 20 mM HEPES, pH 7. 4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 X g for 15 minutes at 4'C. The resulting pellet was then stored at -80 0 C until utilized. On the day of direct 15 identification screening, the membrane pellet was slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 , to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes are placed on ice until use). cAMP standards and Detection Buffer (comprising 2 pCi of tracer [121I cAMP 20 (100 gl)] to 11 ml Detection Buffer) were prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer was prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 20 mM phosphocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 tM GTP (Sigma), and 0.2 mM ATP (Sigma) ; Assay Buffer was then stored on ice until utilized. 65 Candidate compounds identified as per above (if frozen, thawed at room temperature) were added, preferably, to 96-well plate wells (31/well; 12 gM final assay concentration), together with 40 gl membrane protein (30 pg/well) and 50 pl of Assay Buffer. This admixture was then incubated for 30 minutes at room temperature, with 5 gentle shaking. Following the incubation, 100 gl of Detection Buffer was added to each well, followed by incubation for 2-24 hours. Plates were then counted in a Wallac MicroBetaTM plate reader using "Prot. #31" (as per manufacturer instructions). A representative screening assay plate (96 well format) result is presented in 10 Figure 12. Each bar represents the results for a different compound in each well, plus RUP13-Gsa fusion protein construct, as prepared in Example 5(a) above. The representative results presented in Figure 12 also provide standard deviations based upon the mean results of each plate ("m") and the mean plus two arbitrary preference for selection of inverse agonists as "leads" from the primary screen involves selection of 15 candidate compounds that that reduce the per cent response by at least the mean plate response, minus two standard deviations. Conversely, an arbitrary preference for selection of an agonists as "leads" from the primary screen involves selection of candidate compounds that increase the per cent response by at least the mean plate response, plus the two standard deviations. Based upon these selection processes, the 20 candidate compounds in the following wells were directly identified as putative inverse agonist (Compound A) and agonist (Compound B) to RUP13 in wells A2 and G9, respectively. See, Figure 12. It is noted for clarity: these compounds have been directly identified without any knowledge of the endogenous ligand for this GPCR. By focusing on assay techniques that are based upon receptor function, and not compound binding 25 affinity, we are able to ascertain compounds that are able to reduce the functional 66 activity of this receptor (Compound A) as well as increase the functional activity of the receptor (Compound B). Based upon the location of these receptor in lung tissue (see, for example, hRUP13 and hRUP21 in Example 6), pharmaceutical agents can be developed for potential therapeutic treatment of lung cancer. 5 References cited throughout this patent document, including co-pending and related patent applications, unless otherwise indicated, are fully incorporated herein by reference. Modifications and extension of the disclosed inventions that are within the purview of the skilled artisan are encompassed within the above disclosure and the claims that follow. 10 Although a variety of expression vectors are available to those in the art, for purposes of utilization for both the endogenous and non-endogenous human GPCRs, it is most preferred that the vector utilized be 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 15 the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #20335 1. 67

Claims (29)

1. An isolated polynucleotide comprising a nucleic acid encoding a G protein-coupled receptor (GPCR) comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28.

2. An isolated polynucleotide comprising a nucleic acid encoding a G protein-coupled receptor (GPCR) comprising an amino acid sequence set forth in SEQ ID NO NO: 28.

3. The isolated polynucleotide according to claim I or 2, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 27.

4. The isolated polynucleotide according to claim I or 2, wherein the GPCR induces inositol triphosphate (IP 3 ) accumulation or G-protein coupling to a membrane.

5. A vector comprising a polynucleotide according to any one of claims I to 4.

6. The vector according to claim 5, wherein said vector is an expression vector and wherein the polynucleotide is operably linked to a promoter.

7. A recombinant host cell comprising a vector according to claim 5.

8. A recombinant host cell comprising an expression vector according to claim 6.

9. A process for making a recombinant host cell comprising: (a) introducing an expression vector according to claim 6 into a suitable host cell; and (b) culturing the host cell under conditions that allow for the expression of the G protein coupled receptor by said cell.

10. An isolated membrane of a recombinant host cell according to claim 9, wherein the isolated membrane comprises the G protein-coupled receptor.

I1. A process for producing a G protein-coupled receptor comprising: (a) obtaining a transfected host cell comprising an expression vector according to claim 6; and (b) culturing the transfected host cell under conditions that allow for the expression of said G protein-coupled receptor by said cell.

12. An isolated or recombinant G protein-coupled receptor (GPCR) comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28.

13. An isolated or recombinant G protein-coupled receptor comprising an amino acid sequence set forth in SEQ ID NO: 28.

14. The isolated or recombinant G protein-coupled receptor according to claim 12 or 13, wherein the GPCR induces inositol triphosphate (IP 3 ) accumulation or G-protein coupling to a membrane.

15. Use of an isolated or recombinant G protein-coupled receptor according to any one of claims 12-14 in drug screening.

16. The use according to claim 15, wherein the drug screening is to identify a pharmacological agent for the treatment of a disease or disorder related to a cell, tissue or organ in which a G protein-coupled receptor is expressed, and wherein the expressed G protein-coupled receptor comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28.

17. The use according to claim 15, wherein the drug screening is to identify a pharmaceutical agent for the treatment of a disease or disorder related to a function selective to a cell, tissue or organ in which a G protein-coupled receptor is expressed, and wherein the expressed G protein coupled receptor comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28.

18. A method for identifying a G protein-coupled receptor agonist, inverse agonist or partial agonist comprising: (a) contacting a candidate compound with a recombinant host cell or membrane thereof comprising a G protein-coupled receptor according to any one of claims 12 to 14; (b) measuring the ability of the compound to inhibit or stimulate the intracellular reaction of the G protein-coupled receptor; and (c) determining if the candidate compound is a G protein-coupled receptor agonist, inverse agonist, or partial agonist based on the ability of the candidate compound to inhibit or stimulate the intracellular reaction of the G protein-coupled receptor in (b).

19. A method for identifying a modulator of a G protein-coupled receptor comprising: (a) contacting a candidate compound with a recombinant host cell or membrane thereof comprising a G protein-coupled receptor according to any one of claims 12 to 14; and (b) 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 the G protein-coupled receptor.

20. The method according to claim 18 or 19, wherein the host cell is a mammalian host cell or a yeast host cell.

21. The method according to claim 18 or 19, wherein the method comprises using [ 35 S] GTPyS to monitor G-protein coupling to a membrane comprising the G protein-coupled receptor.

22. The method according to claim 18 or 19, wherein the method comprises detecting IP 3 .

23. The method according to claim 18 or 19, wherein the method comprises formulating the agonist, inverse agonist or partial agonist as a pharmaceutical composition.

24. The method according to claim 18 or 19, wherein the receptor is an endogenous receptor comprising an amino acid sequence set forth in SEQ ID NO: 28.

25. The method according to claim 18 or 19, wherein the receptor is a non-endogenous receptor having the arginine residue at amino acid position 240 of amino acid sequence set forth in SEQ ID NO: 28 mutated to a lysine residue.

26. A process for isolating a modulator of a G protein-coupled receptor comprising performing a method according to any one of claims 19 to 25 on a panel of candidate compounds and isolating a compound that modulates a G protein-coupled receptor from the panel.

27. A process of formulating a compound for therapy of a condition ameliorated by modulating signal transduction mediation by a G protein-coupled receptor, said process comprising performing a method according to any one of claims 19 to 25 to identify a modulator of a G protein-coupled receptor and formulating the modulator with a pharmaceutically acceptable carrier or excipient.

28. A process of formulating a compound for therapy of a condition ameliorated by modulating signal transduction mediated by a G protein-coupled receptor, said process comprising performing the process according to claim 26 to thereby isolate a compound that modulates a G protein coupled receptor and formulating the compound with a pharmaceutically acceptable carrier or excipient.

29. The isolated polynucleotide according to any one of claims I to 4 or the vector according to claim 5 or 6 or the recombinant host cell according to claim 7 or 8 or the process according to any one of claims 9 or I I or 26 to 28 or the isolated membrane according to claim 10 or the isolated or recombinant GPCR according to any one of claims 12 to 14 or the use according to any one of claims 15 to 17 or the method according to any one of claims 18 to 25 substantially as hereinbefore described with reference to the figures and/or examples. DATED this TENTH day of OCTOBER, 2011 Arena Pharmaceuticals, Inc. by patent attorneys for the applicant: FB Rice