AU782959B2 - Endogenous and non-endogenous versions of human G protein-coupled receptors - Google Patents

Description

WO 01/36471 PCT/US00/31509 ENDOGENOUS AND NON-ENDOGENOUS VERSIONS OF HUMAN G PROTEIN-COUPLED RECEPTORS 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 nonendogenous versions of the GPCRs that have been altered to establish or enhance constitutive activity of the receptor. Preferably, the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists having potential applicability as therapeutic agents.

BACKGROUND OF THE INVENTION Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately or 2,000 genes, are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as "known" receptors, while receptors for which the endogenous ligand has not been identified are referred to as "orphan" receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription 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 transmebrane-2 etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, SUBSTITUTE SHEET (RULE 26) WO 01/36471 PCT/US0O/31509 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 transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and 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 ICrespectively). The "carboxy" terminus of the receptor lies in the intracellular space within the cell, and the "amino" terminus of the receptor lies in the extracellular space outside of the cell.

Generally, when an endogenous ligand binds with the receptor (often referred to as "activation" of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular "Gprotein." It has been reported that GPCRs are "promiscuous" with respect to G proteins, that a GPCR can interact with more than one G protein. See, Kenakin, 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 ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.

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

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

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.

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.

I0 25 The presently-claimed invention particularly relates to endogenous and nonendogenous versions of a novel GPCR designated RUP18 and variants thereof. It will be apparent from the description herein that the isolated polynucleotide encoding a RUP18 polypeptide is preferably of human origin. Preferably, the polynucleotide is obtained from pancreatic tissue, more preferably human pancreata. As shown in Figure 13 and 14 and Table J, the isolated polynucleotide encoding the RUP18 polypeptide is selectively expressed endogenously in the pancreas of a human, and in the beta-islet cells of rat or mouse pancreas, as determined using a process that comprises performing RT-PCR. The present invention also provides isolated polynucleotides encoding endogenous and non-endogenous versions of RUP18 and variants thereof RUP18(L294K). The present invention extends to GPCR fusion proteins comprising endogenous and non-endogenous forms of the RUP18 polypeptide or variant thereof preferably further comprising a G protein Gs, and to polynucleotides encoding such fusion proteins. The present invention extends further to a vector comprising a polynucleotide encoding an endogenous or non-endogenous form of the RUP 18 polypeptide or variant thereof or a GPCR fusion protein comprising said RUP18 polypeptide or variant. The present invention extends further to a host cell comprising a vector encoding an endogenous or non-endogenous form of the RUP18 polypeptide or variant thereof or a GPCR fusion protein comprising same, and to membrane fractions of such host cells. The present invention further provides methods of producing an endogenous or non-endogenous form of a RUP18 polypeptide in transfected host cells. The present invention further provides for the use of the endogenous and non-endogenous forms of the RUP18 polypeptide and variants thereof in drug screening to identify compounds for the treatment of disorders of the 20 pancreas.

For the purposes of nomenclature, the term "RUP18" relates to endogenous and non-endogenous forms of the exemplary polypeptide of humans comprising the sequence set forth in SEQ ID NO: 22 hRUP18) and variants thereof. The sequence set forth in SEQ ID NO: 21 is an example of a sequence encoding a RUP18 25 polynucleotide of humans.

Accordingly, one embodiment the present invention provides an isolated polynucleotide encoding a G protein-coupled receptor polypeptide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 22; the sequence set forth in SEQ ID NO: 21; the sequence of a polynucleotide that hybridizes under stringent conditions to or or a complementary sequence thereto: the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 61 and/or SEQ ID NO: 62; a sequence encoding a constitutively-activated variant of SEQ ID NO: 22 wherein said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22 and a variant of SEQ ID NO: 21 comprising a mutation positioned at a codon encoding an amino acid positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22.

Preferably, the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; the nucleotide sequence set forth in SEQ ID NO: 21; and a variant of comprising a substitution of one or two or three nucleotides within the codon at positions 880 to 882 of SEQ ID NO: 21 for other 20 nucleotide residue(s) thereby producing a codon encoding a lysine residue.

Preferably, the isolated polynucleotide is obtained from a cell, tissue or organ of human origin.

•A further embodiment of the invention provides an isolated polynucleotide encoding a GPCR fusion protein comprising a RUP18-encoding polynucleotide or 25 variant thereof according to any embodiment described herein. For example, a polynucleotide encoding such a fusion protein may comprise nucleic acid encoding a G a protein, Gs.

A further embodiment of the invention provides a vector comprising a RUP18encoding polynucleotide or variant thereof, or encoding a RUP18 fusion protein, according to any embodiment described herein. Preferably, the vector is an expression vector, wherein the RUP18-encoding polynucleotide or variant or RUP18 fusion protein-encoding polynucleotide is operably linked to a promoter.

A further embodiment of the present invention provides a recombinant host cell comprising a vector comprising a RUP18-encoding polynucleotide or variant thereof, or encoding a RUP18 fusion protein, according to any embodiment described herein.

The present invention additionally provides a membrane of a recombinant host cell according to any embodiment described herein, wherein said membrane comprises a recombinant RUP 18 polypeptide or variant thereof or RUP 18 fusion protein.

In a further embodiment, the present invention provides a method for producing a GPCR or a GPCR fusion protein comprising the steps of: transfecting the expression vector comprising nucleic acid encoding a RUP18 polypeptide or a variant thereof or RUP18 fusion protein as described according to any embodiment described herein into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a G protein-coupled receptor or GPCR fusion protein from the expression vector.

Preferably, the method supra for producing a GPCR or GPCR fusion protein further comprises obtaining the transfected host cell. In one embodiment, the host cell is a mammalian cell, such as, for example, a pancreatic cell or a mammalian cell selected from the group consisting of a COS-7 cell, a 293 cell and a 293T cell.

The present invention also provides an isolated or recombinant GPCR polypeptide comprising an amino acid sequence selected from the group consisting of: a sequence comprising the amino acid sequence set forth in SEQ ID NO: 25 22; :oo* a sequence encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 21 or a complementary sequence thereto; i a sequence encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 61 and/or SEQ ID NO: 62; and the sequence of a constitutively-activated variant of SEQ ID NO: 22 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22.

Preferably, the isolated or recombinant GPCR is of human origin. Preferably the GPCR comprises an amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 wherein the leucine residue at position 294 of SEQ ID NO: 22 is substituted for a lysine residue.

A further embodiment of the present invention provides an isolated or recombinant GPCR fusion protein a RUP18 fusion protein). For example, the isolated or recombinant GPCR fusion protein comprises an endogenous or nonendogenous RUP18 sequence covalently linked to the sequence of a G protein or variant thereof, Gs.

Another embodiment of the invention provides for the use of a RUP18 polypeptide or variant thereof or RUP18 fusion protein according to any embodiment herein to identify a pharmaceutical agent for the treatment of a disease or disorder state related to the pancreas. In one embodiment, the RUP18 or variant thereof or RUP18 fusion protein is used to identify a pharmaceutical agent for the treatment of a disease or disorder state related to a function selective to the pancreas.

20 In yet another embodiment, the present invention provides a method of identifying a modulator of a G protein-coupled receptor (GPCR) comprising: contacting a candidate compound with a recombinant host cell that expresses a RUP 18 polypeptide or variant thereof or RUP18 fusion protein or an isolated membrane comprising said RUP18 polypeptide or variant 25 thereof or RUP18 fusion protein; and measuring the ability of the compound to inhibit or stimulate functionality of the RUP18 polypeptide or variant thereof or RUP18 fusion protein *0 :1 wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

In a preferred embodiment, the 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 RUP18 or a non-endogenous variant of RUP18 RUP18(L294K) or other variant according to any embodiment described herein.

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 (IP 3 diacylglycerol (DAG), cyclic AMP (cAMP) or cyclic GMP (cGMP), and more preferably comprises determining intracellular cAMP level.

Alternatively, when assayed in the presence of a Gq/Gi fusion protein e.g., Gq(del)/Gi, such as by co-transfecting nucleic acid encoding a Gq/Gi fusion with nucleic acid encoding the G protein-coupled receptor polypeptide or GPCR fusion protein, it is possible to assay functionality of the G protein-coupled receptor polypeptide or the G protein-coupled receptor polypeptide portion of the GPCR fusion protein by a process comprising determining intracellular accumulation of inositol triphosphate (IP 3 This is made possible by virtue of the ability of hRUP18 to couple Gi and the ability of the Gq/Gi fusion protein to activate intracellular inositol triphosphate (IP 3 accumulation.

20 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 hRUP18-mediated signal transduction in a cell or tissue, such as, for example, the pancreas, for example, the 25 signal transduction modulates the concentration of blood glucose, glucagon or insulin.

Accordingly, the present invention further provides processes for identifying compounds that modulate hRUP18-mediated signal transduction in the pancreas, such as, for example, the modulation of blood glucose, glucagon or insulin. In another embodiment, the present invention provides processes for identifying compounds for the treatment of diabetes or a condition associated with diabetes, such as, for example, a defect in glucagon regulation or insulin regulation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides an illustration of second messenger IP 3 production from endogenous version RUP12 ("RUP12") as compared with the control Figure 2 is a graphic representation of the results of a second messenger cellbased cyclic AMP assay providing comparative results of constitutive signalling of endogenous RUP13 ("RUP13") and a control vector Figure 3 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP13 ("RUP13 wt") and non-endogenous, constitutively activated RUP13 ("RUP13(A268K)"), utilizing 8XCRE-Luc reporter plasmid.

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*o ooo WO 01/36471 PCT/US00/31509 Figure 4 is a graphic representation of the results of a 3 S]GTPyS assay providing comparative results for constitutive signaling by RUP13:Gs Fusion Protein ("RUP I3-Gs") and a control vector Figure 5 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP14 ("RUP14 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(A398K)"), utilizing 8XCRE-Luc reporter plasmid.

Figure 7 is a graphic representation of the results of a second messenger cellbased cyclic AMP assay providing comparative results for constitutive signaling of endogenous RUP15 ("RUP15 non-endogenous, constitutively activated version of ("RUP15(A398K)") and a control vector Figure 8 is a graphic representation of the results of a 3 S]GTPyS assay providing comparative results for constitutive signaling by RUP15:Gs Fusion Protein ("RUP 15-Gs") and a control vector Figure 9 provides an illustration of second messenger IP3 production from endogenous version RUP 17 ("RUP 17") as compared with the control Figure 10 provides an illustration of second messenger IP 3 production from endogenous version RUP21 ("RUP21") as compared with the control 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.

Figure 12 is a graphic representation showing the level of IP 3 production in a cell expressing RUP18 or RUP 18(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 RUP18 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, 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-1 and BTC-6 and pancreatic 3 islet cells isolated from mice (as indicated). The arrow indicates the position of the RUP18 amplification product.

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 are provided in well G9.

*e DETAILED DESCRIPTION 20 The scientific literature that has evolved around receptors has adopted a number of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document.

To the extent that these definitions conflict with other definitions for these terms, the following definitions shall control: AGONISTS shall mean material ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding membranes.

AMINO ACID ABBREVIATIONS used herein are set out in Table A: 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 a a a a.

a a a a a..

a a a a. a a WO 01/36471 PCT/US00/31509 PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V PARTIAL AGONISTS shall mean materials ligands, candidate 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.

ANTAGONIST shall mean materials ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists.

ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.

CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique. Preferably, the phrase "candidate compound" does not include compounds which were publicly known to be compounds selected from the group consisting of inverse agonist, agonist or antagonist to a receptor, as previously determined by an indirect identification process ("indirectly identified compound"); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at least one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans.

WO 01/36471 PCT/US00/31509 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 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 guanosine cytidine uridine and thymidine coupled to a phosphate group and which, 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 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 the phrase "candidate compound", shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound. This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase "indirectly identifying" or "indirectly identified." WO 01/36471 PCT/US00/31509 ENDOGENOUS shall mean a material that a mammal naturally produces.

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

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a nonendogenous protein comprising an endogenous, constitutively activate GPCR or a nonendogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR. For example, and not limitation, in an endogenous state, if the G protein "Gsa" is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein based upon the specific GPCR would be a nonendogenous protein comprising the GPCR fused to Gsa; in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR. The G WO 01/36471 PCT/US00/31509 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 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, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells.

INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the 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 that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.

INVERSE AGONISTS shall mean materials ligand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which WO 01/36471 PCT/US00/31509 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 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.

MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor. In terms of equivalents to specific sequences, a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 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%.

WO 01/36471 PCT/US00/31509 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 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 techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

PLASMID shall mean the combination of a Vector and cDNA. Generally, a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein.

SECOND MESSENGER shall mean an intracellular response produced as a result of receptor activation. A second messenger can include, for example, inositol triphosphate (IP 3 diacycglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP). Second messenger response can be measured for a determination of receptor activation. In addition, second messenger response can be measured for the direct 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.

WO 01/36471 PCT/US00/31509 VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell.

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

A. Introduction The traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the receptor. Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been heretofore recognized is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists, and inverse agonists of the receptor. For those diseases which result from an overly active receptor or an under-active receptor, what is desired in a therapeutic drug is a compound which acts to diminish the active state of a receptor or enhance the activity of the receptor, respectively, not necessarily a drug which is an antagonist to the endogenous ligand.

This is because a compound that reduces or enhances the activity of the active receptor state need not bind at the same site as the endogenous ligand. Thus, as taught by a method of this invention, any search for therapeutic compounds should start by screening compounds against the ligand-independent active state.

B. Identification of Human GPCRs WO 01/36471 WO 0136471PCTIUSOOI31509 The efforts of the Human Genome project has led to the identification of a plethora of information regarding nucleic acid sequences located within the human genome; it has been the case in this endeavor that genetic sequence information has been made available without an understanding or recognition as to whether or not any particular genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleic acid sequences within the human genome are within the purview of those having ordinary skill in the art. For example, and not limitation, a variety of human GPCRs, disclosed herein, were discovered by reviewing the GenBankm database. Table B, below, lists several 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

hRIJP8 AL121755 1, 152bp NPY2R 27% hRTJP9 ACO 113 375 1,260bp GAL2R 22% bRUPIO AC008745 l,Ol4bp C5aR bRUPI 1 AC013396 1,272bp HM474 36% hRUP12 APOD0808 966bp Masi 34% hRUP13 AC01 1780 1,356bp Fish GPRX- 43% ORYLA hRUP14 AL137118 1,04l1bp CysLT1R AL0 16468 l,52Thp RE2 htRUP16 AL136106 l,068bp GLR101 37% hRUP17 AC023078 969bp Mast 37% hLRUPI8 AC008547 1,305bp Oxytocin 31% hRUP19 AC026331 1,O.41bp HM74 52% bRUP2O AL161458 1,01 lbp GPR34 hRIJP21 AC026756 I,0l4bp P2YIR 37% hRUP22 AC027026 993bp RUP 17 67% Mas] 37% WO 01/36471 PCT/US00/31509 hRUP23 AC007104 1,092bp Rat GPR26 31% hRUP24 AL355388 1,125bp SALPR 44% AC026331 1,092bp HM74 hRUP26 AC023040 1,044bp Rabbit 5HTID 27% hRUP27 AC027643 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 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 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 endogenous version of human GPCRs disclosed herein is expressed and/or overexpressed. 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 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 WO 01/36471 PCT/US00/31509 Number PCT/US99/23938, published as WO 00/22129 on April 20, 2000, which, along with the other patent documents listed herein, is incorporated herein by reference. The algorithmic technique is not predicated upon traditional sequence "alignment" but rather a specified distance from the aforementioned TM6 proline residue (or, of course, endogenous constitutive 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.

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 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 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 dot-blot analysis against tissue-mRNA, and/or RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue WO 01/36471 PCT/US00/31509 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 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 When a G protein receptor becomes constitutively active, it binds to a G protein 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 GTP, ["S]GTPyS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that 3 SS]GTPyS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. The preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.

2. Specific GPCR screening assay techniques Once candidate compounds are identified using the "generic" G protein-coupled receptor assay an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the WO 01/36471 PCT/US00/31509 receptor site is preferred. For example, a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, 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.

See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 r 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, an inverse agonist to the receptor such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISAbased format. Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) 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, P-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of WO 01/36471 PCT/US0O/31509 the reporter protein. The reporter protein such as p-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP3). 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 (3" 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, an inverse agonist to a Gq- or Go-associated receptor such a compound would decrease the levels of IP3). Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.

Commercially available assays for such detection are available.

3. GPCR Fusion Protein The use of an endogenous, constitutively activate orphan GPCR or a nonendogenous, 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 differentiate between, the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an WO 01/36471 PCT/US00/31509 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.

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 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, an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening 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 expression vectors and systems offer a variety of approaches that can fit the particular WO 01/36471 PCT/US00/31509 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 inframe (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 upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). We have a preference (based upon convenience) of use of a spacer in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the nonendogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.

As noted above, constitutively activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging the cAMP signal decreases upon activation thus making the direct identification of, 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 cyclasebased assay. Thus, for example, an endogenous Gi coupled receptor can be fused to a Gs protein we believe that such a fusion construct, upon expression, "drives" or "forces" WO 01/36471 PCT/US00/31509 the endogenous GPCR to couple with, 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 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 amino acids of the G-protein a-subunit is deleted and the last five amino acids at the C-terminal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest For example, a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct". We believe that this fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylgycerol, 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) 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 activation can be accomplished by co-transfecting a signal enhancer, a nonendogenous, constitutively activated receptor that predominantly couples with Gs upon activation TSHR-A623I, disclosed below), with the Gi linked GPCR. As is WO 01/36471 PCT/US00/31509 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 non-endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with the endogenous Gi coupled receptor (the "target receptor") provides a baseline cAMP signal although the Gi coupled receptor will decrease cAMP levels, this "decrease" will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co-transfecting the signal enhancer with a constitutively activated version of the target receptor, cAMP would be expected to further decrease (relative to base line) due to the increased functional activity of the Gi target which decreases cAMP).

Screening of candidate compounds using a cAMP based assay can then be accomplished, with two provisos: first, relative to the Gi coupled target receptor, "opposite" effects will result, an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal; second, as would be apparent, candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or 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 chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. Generally, the results of such screening will be compounds having WO 01/36471 PCT/US00/31509 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.

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 Remington's Pharmaceutical Sciences, 16 th Edition, 1980, Mack Publishing Co., (Oslo et al., eds.).

H. Other Utility Although a preferred use of the non-endogenous versions the human GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical 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 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 disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.

WO 01/36471 PCT/US00/31509

EXAMPLES

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

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

WO 01/36471 WO 0136471PCTUS00131509 TABLE C Disclosed Accession Complete DNA Open Reading Nucleic Amino Human Number Sequence Frame Acid Acid Orphan Identifted (Base Pairs) (Base Pairs) SEQ.ID. SEQ.ID.

GPCRs NO. NO.

hRUP8 AL 121755 147,566bp l,152bp 1 2 hRUP9 AC01 13375 143,I8lbp 1,260bp 3 4 bRUPlO AC008745 94,194bp 1,0Ol4bp 5 6 bRUP1 I AC013396 155,086bp l,272bp 7 8 hRUP12 AP000808 177,764bp 966bp 9 hRUP13 ACOl 11780 167,819bp 1,356bp 11 12 hRUP14 AL137118 l68,297bp 1,O4lbp 13 14 AL016468 138,828bp l,527bp 15 16 hRUP16 AL 136106 208,042bp l,068bp 17 18 hRUP17 AC023078 161,735bp 969bp 19 bRUP18 AC008547 I 17,304bp 1,305bp 21 22 hRU'P19 AC026331 145,183bp 1,04l1bp 23 24 hRUP2O AL161458 163,511lbp 1,01llbp 25 26 hRUP21 AC026756 156,534bp 1,Ol4bp 27 28 hRUP22 AC027026 151,811bp 993bp 29 hRUP23 AC007104 200,OO0bp 1,092bp 31 32 hiRUP24 AL355388 190,538bp 1,125bp 33 34 AC026331 145,183bp 1,092bp 35 36 hRUP26 AC023040 178,508bp 1,044bp 37 38 hRUP27 AC027643 158,700bp 1,02Obp 39 2. Full Length Cloning a. hRUP8 (Seq. Id. Nos. I 2) The disclosed human RUP8 was identified based upon the use of EST database (dbEST) information. While searching the dbEST, a cDNA clone with accession numnber WO 01/36471 PCT/US00/31509 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.ID.NO.: 42; antisense).

PCR was performed using Advantage cDNA polymerase (Clontech; manufacturing instructions will be followed) in 50ul reaction by the following cycles: 94 0 C for 30 sec; 94°C for 10 sec; 65 0 C for 20 sec, 72 0 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 (Invitrogen) and sequenced using the ABI Big Dye Terminator kit 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 database information. While searching the database, a cDNA clone with Accession Number AC011375 was identified as a human genomic sequence from chromosome 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) and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase (Stratagene) was used for the amplification in a 100p.l reaction with 5% DMSO by the following cycle with step 2 to step 4 repeated 35 times: 94C for 1 minute; 94 0 C for seconds; 56°C for 30 seconds; 72 0 C for 2 minutes; 72 0 C for 5 minutes.

A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) from 1% agarose gel and completely sequenced using the ABI Big Dye WO 01/36471 PCT/US00/31509 Terminator kit 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 genomic DNA matched with the sequence obtained from data base.

c. hRUP10 (Seq. Id. Nos. 5 6) The disclosed human RUP10 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 RUPIO was cloned by RT-PCR using primers: 5'-CCATGGGGAACGATICTGTCAGCTACG-3' (SEQ.ID.NO.:45; sense) and 5'-GCTATGCCTGAAGCCAGTCTTGTG-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: 94 0 C 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 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator kit Biosystem).

The nucleic acid sequence of the novel human receptor RUP10 is set forth in and the putative amino acid sequence thereof is set forth in SEQ.ID.NO.:6.

d. hRUP 11 (Seq. Id. Nos. 7 8) The disclosed human RUP11 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with accession number AC013396 was identified as a human genomic sequence from chromosome 2.

WO 01/36471 PCT/US00/31509 The full length RUP11 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 polymerase (Stratagenc) was used for the amplification in a 50pl reaction by the following cycle with step 2 to step 4 repeated 35 times: 94 0 C for 3 minutes; 94°C for seconds; 67 0 C for 20 seconds; 72 0 C for 1.5 minutes; 72 0 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 Biosystem). The nucleic acid sequence of the novel human receptor RUP11 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 The disclosed human RUP12 was identified based upon the use of GenBank database. While searching the database, a cDNA clone with accession number AP000808 was identified to encode a new GPCR, having significant homology with rat RTA and human masi oncogene GPCRs. The full length RUP12 was cloned by PCR using primers: 5'-CITCCTCTCGTAGGGATGAACCAGAC-3' (SEQ.ID.NO.:49; sense) 5'-CTCGCACAGGTGGGAAGCACCTGTGG-3' (SEQ.ID.NO.:50; antisense) 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 0 C for 3 min; 94°C for 20 sec; 65°C for 20sec; 72 0 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 Terminator kit WO 01/36471 PCT/US00/31509 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 GenBank database. While searching the database, a cDNA clone with accession number AC011780 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'-CATATCCCTCCGAGTGTCCAGCGGC-3' (SEQ.ID.NO.:52; antisense) 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; 94 0 C for 20 sec; 65 0 C for 20sec; 72 0 C for 2 min and 72 0 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 Biosystem) (see. SEQ.ID.NO.:11 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 Number AL137118 was identified as a human genomic sequence from chromosome 13. The full length RUP 14 was cloned by PCR using primers: 5'-GCATGGAGAGAAAATITATGTCCTTGCAACC-3' (SEQ.ID.NO.:53; sense) 5'-CAAGAACAGGTCTCATCTAAGAGCTCC-3' (SEQ.ID.NO.:54; antisense) and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase (Stratagene) and 5% DMSO were used for the amplification by the following cycle WO 01/36471 PCT/US00/31509 with step 2 and step 3 repeated 35 times: 94 0 C for 3 minute; 94 0 C for 20 seconds; 58 0 C for 2 minutes; 72 0 C for 10 minutes.

A 1.1 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) (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. hRUPIS (Seq. Id. Nos. 15 16) The disclosed human RUP15 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC016468 was identified as a human genomic sequence. The full length was cloned by PCR using primers: S'-GCTGTTGCCATGACGTCCACCTGCAC-3' (SEQ.ID.NO.:55; sense) 5'-GGACAGTTCAAGGTTGCCTTAGAAC-3' (SEQ.ID.NO.:56; antisense) 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; 94°C for 20 seconds; 65 0 C for 20 seconds; 72 0 C for 2 minutes and 72 0 C for 7 minutes.

A 1.5 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). 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) WO 01/36471 PCT/US00/31509 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'-CTITCGATACTGCTCCTATGCTC-3' (SEQ.ID.NO.:57; sense, 5' of initiation codon), 5'-GTAGTCCACTGAAAGTCCAGTGATCC-3' (SEQ.ID.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 seconds; 69 0 C for 15 seconds; 72 0 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 ALl36106, indicating that the RUP16 cDNA is composed of 4 exons.

j. hRUP17 (Seq. Id. Nos. 19 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 11. The full length RUP 17 was cloned by PCR using primers: 5'-TTTCTGAGCATGGATCCAACCATCTC-3' (SEQ.ID.NO.:59; sense, containing initiation codon) 5'-CTGTCTGACAGGGCAGAGGCTCTC-3' (SEQ.ID.NO.:60; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clontech) was used for the amplification in a 100ul reaction with 5% DMSO by the WO 01/36471 PCT/US00/31509 following cycle with step 2 to 4 repeated 30 times: 94 0 C for 1 min; 94°C for 15 sec; 67C for 20 sec; 72 0 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 Termiantor Kit Biosystem). See, SEQ.ID.NO.:19 for nucleic acid sequence and 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 Number AC008547 was identified as a human genomic sequence from chromosome The full length RUP18 was cloned by PCR using primers: 5'-GGAACTCGTATAGACCCAGCGTCGCTCC-3' (SEQ.ID.NO.:61; sense, 5' of the initiation codon), 5'-GGAGGTTGCGCCTTAGCGACAGATGACC-3' (SEQ.ID.NO.:62; 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 DMSO by the following cycle with step 2 to 4 repeated 35 times: 95 0 C for 5 min; for 30 sec; 65 0 C for 30 sec; 72 0 C for 2 min; and 72 0 C for 5 min.

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 Biosystem). See, SEQ.ID.NO.:21 for nucleic acid sequence and SEQ.ID.NO.:22 for deduced amino acid sequence.

1. hRUP19 (Seq. Id. Nos. 23 24) WO 01/36471 PCT/US00/31509 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 RUP 19 was cloned by PCR using primers: 5'-CTGCACCCGGACACTrGCTCTG-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 following cycle with step 2 to 4 repeated 35 times: 94°C for 1 min; 94°C for 15 sec; 0 C for 20 sec; 72 0 C for 1 min and 30 sec; and 72 0 C for 5 min.

A 1.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 Tcrmiantor Kit Biosystem). See, SEQ.ID.NO.:23 for nucleic acid sequence and 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 1. The full length RUP20 was cloned by PCR using primers: 5'-TATCTGCAATTCTATTCTAGCTCCTG-3' (SEQ.ID.NO.:65; sense, 5' of initiation codon), 5'-TGTCCCTAATAAAGTCACATGAATGC-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 WO 01/36471 PCT/US00/31509 step 2 to 4 repeated 35 times: 94 0 C for 1 min; 94*C for 15 sec; 60 0 C for 20 sec; 72°C for 1 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 Termiantor Kit 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 Number AC026756 was identified as a human genomic sequence from chromosome 13. The full length RUP21 was cloned by PCR using primers: GGAGACAACCATGAATGAGCCAC (SEQ.ID.NO.:67; sense) TAITTCAAGGGTTGTITIGAGTAAC (SEQ.ID.NO.:68; antisense) and human genomic DNA (Promega) as template. Taq Plus Precision 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 0 C for 1 min; 94 0 C for 15 sec; 0 C for 20 sec; 72°C for 1 min and 30 sec; and 72°C 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 Termiantor Kit 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 The disclosed human RUP22 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession WO 01/36471 PCT/US00/31509 Number AC027026 was identified as a human genomic sequence from chromosome 11. The full length RUP22 was cloned by PCR using primers: GGCACCAGTGGAGGTTTICTGAGCATG (SEQ.ID.NO.:69; sense, containing initiation codon) 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 55 0 C, 20 seconds 72 0 C, 1.5 minute 72 0 C, 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 Biosystem). See, SEQ.ID.NO.:29 for nucleic acid sequence and for deduced amino acid sequence.

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: 5'-CCTGGCGAGCCGCTAGCGCCATG-3' (SEQ.ID.NO.:71; sense, ATG as the initiation codon), 5'-ATGAGCCCTGCCAGGCCCTCAGT-3' (SEQ.ID.NO.:72; antisense, TCA as the stop codon) and human placenta Marathon-Ready cDNA (Clontech) as template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50ul reaction by the following WO 01/36471 PCT/US00/31509 cycle with step 2 to 4 repeated 35 times: 95*C for 30 sec; 95°C for 15 sec; 66°C for sec; 72°C for 1 min and 20 sec; 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.

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 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'-AGATCCTGGTrCTTGGTGACAATG-3' (SEQ.ID.NO.:74; antisense, 3' of stop codon) 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°C for 1 minute; 94C for 15 seconds; 56°C for seconds 72°C for 1 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 pCRII-TOPO vector (Invitrogen) and completely sequenced using the ABI Big Dye Termiantor Kit Biosystem). See, SEQ.ED.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 WO 01/36471 PCT/US00/31509 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.:75; sense, 5'of initiation codon), 5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.:76; antisense, 3' of stop codon) and human genomic DNA (Prmega) 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°C for 1 minute; 94°C for 15 seconds; 56°C for 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 Termiantor Kit 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 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'-AGCCATCCCTGCCAGGAAGCATGG-3' (SEQ.ID.NO.:77; sense, containing initiation codon) 5'-CCAGACTGTGGACTCAAGAACTCTAGG-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 100± reaction with DMSO by the following cycle with step 2 to 4 repeated 35 times: 94°C for 5 minute; 950C for 30 seconds; 650C for 30 seconds 720C for 2 minute and 72°C for 5 minutes.

WO 01/36471 PCT/US00/31509 A l.lkb 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 Biosystem). See, SEQ.ID.NO.:37 for nucleic acid sequence and SEQ.ID.NO.:38 for deduced amino acid sequence.

t. hRUP27 (Seq. Id. Nos. 39 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: 5'-AGTCCACGAACAATGAATCCATITCATG-3' (SEQ.ID.NO.:79; sense, containing initiation codon), 5'-ATCATGTCTAGACTCATGGTGATCC-3' (SEQ.ID.NO.:80; 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 50tl reaction with DMSO by the following cycle with step 2 to 4 repeated 35 times: 94°C for 1 minute; 94C for 10 seconds; 58 0 C for 20 seconds 72 0 C for 1 minute 30 seconds and 72 0 C for 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 Termiantor Kit 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 of AC027643, indicating that the RUP27 cDNA is composed of 5 exons.

WO 01/36471 PCT/US00/31509 Example 2 PREPARATION OF NON-ENDOGENOUS, CONSTITUTIVELY ACTIVATED GPCRS Those skilled in the art are credited with the ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches utilized to create non-endogenous versions of several of the human GPCRs disclosed above.

The mutations disclosed below are based upon an algorithmic approach whereby the 16'" amino acid (located in the IC3 region of the GPCR) from a conserved proline (or an endogenous, conservative substitution therefore) residue (located in the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, preferably to an alanine, histidine, arginine or lysine amino acid residue, most preferably to a lysine amino acid residue.

1. Transformer Site-Directed T Mutagenesis Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using Transformer Site-Directed T Mutagenesis Kit (Clontech) according to the 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): TABLE D Receptor Identifier Codon Mutation hRUP8 V274K hRUP9 T249K R232K hRUP11 M294K hRUP1 2 F220K hRUP16 A238K WO 01/36471 WO 0136471PCTUSOO/31509 hRUPl7 Y215K hRUP18 L294K hRUP19 T219K hRUP2O K248A K248H ___K248R hRUP2I 11240K hRUP22 Y222K hRUP24 A245K 1230K hRUIP26 V285K hRUP27 T248K 2. QuikChange t m Site-Directedrm Mutagenesis Preparation of non-endogenous human GPCRs can also bc accomplished by using QuikChangeTrm 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 respective oligonucleotides are noted, in standard form (Table E): TABLE E Receptor Codon 51-3' orientation (sense), orientation Cycle Conditions Identifier Mutation (SEQ.ID.NO.) mutation (antisense) (SEQ.I]D.NO.) Min Sec underlined Cycles 2-4 repeated 16 times hRJPI 13 A268K GGGGAGGGAAAGCAA CCAGGAGAACCACCr' 980 for 2' AGGTGGTCCrFCCTGG ITGCITrCCCTCCCC 980 for (81) (82) 56 0 C for 720 for I11,4V" for hRIJPI4 L246K CAGGAAGGCAAAGAC GAT)GATGATGGTGGT 980 for 2' CACCATCATCATC (85) CTIGCCITCCTG (86) 980 for for 720 for 11' 1 1 1 720 for WO 01/36471 WO 01/6471PUS0O/3 1509 bRUPiS A398K CCAGTGCAAAGCrAAG GAAGATCACITIrC[TA 980 for 2' AAAGTGATCTTC (89) GCnTGCACrGG (90) 980 for for 720 for 11' for bIUP23 W275Kf GCCGCCACCGCGCCAA GCCAATCTITCCTC'ITG 98' for 2' GAGGAAGATGGC GCGCGGTGGCGGC 980 for (94) 56*C for 720 for 11' 1720 for The non-endogenous human GPCRs were then sequenced and the derived and verified nucleic acid and amino acid sequences are listed in the accompanying "Sequence Listing" appendix to this patent document, as summarized in Table F below: TABLE F Non Endogenous Human Nucleic Acid Sequence Listing Amino Acid Sequence GPCR Listing hRUP13 SEQ.TD.NO.:83 SEQ.ID.NO.:94 hRUP14 SEQ.ID.NO.:87 SEQ.ID.NO.:88 SEQ.ID.NO.:91 SEQ.ID.NO.:92 hRUP23 SEQ.[D.NO.:95 SEQ.ID.NO.:96 Example 3 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, utilization of, yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptorcoupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems thus, results obtained in non-mammalian cells, while of WO 01/36471 PCT/US00/31509 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.

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 4pg DNA pCMV vector, pCMV vector with receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B was prepared by mixing 24pl lipofcctamine (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 30-45min. 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. 1 ml of the transfection mixture were added to the cells, followed by incubation for 4hrs at 37°C/5% CO2. The transfection mixture was removed by aspiration, followed by the addition of 10ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 370C/5% CO2. After 48hr incubation, cells were harvested and utilized for analysis.

b. Stable Cell Lines: Gs Fusion Protein Approximately 12x10 6 293 cells are plated on a 15cm tissue culture plate.

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 12tg of DNA. The 12gig 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 cells are washed once with medium without serum. The DNA, lipofectamine, and WO 01/36471 PCT/US00/31509 medium mixture is added to the plate along with lOmL 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.

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 selection.

Example 4 ASSAYS FOR DETERMINATION OF CONSTITUTIVE ACTIVITY OF NON-ENDOGENOUS GPCRs A variety of approaches are available for assessment of constitutive activity of the 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: 3 SIGTPyS Assay When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, 3 5 S]GTPyS, can be utilized to demonstrate enhanced binding of 3 5 S]GTPyS to membranes expressing constitutively activated receptors. The advantage of using WO 01/36471 PCT/US00/31509 35 S]GTPyS binding to measure constitutive activation is that: it is generically applicable to all G protein-coupled receptors; it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.

The assay utilizes the ability of G protein coupled receptors to stimulate 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 35 S]GTPyS assay was incubated in 20 mM HEPES and between 1 and about 20mM MgCI 2 (this amount can be adjusted for optimization of results, although is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM 3 S]GTPyS (this amount can be adjusted for optimization of results, although 1.2 is preferred and 12.5 to 75 gpg membrane protein 293 cells expressing the Gs Fusion Protein; this amount can be adjusted for optimization) and 10 pM GDP (this amount can 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 A Flash Plate T M 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 WO 01/36471 PCT/US00/31509 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 added to each dish of cells followed by careful aspiration. Iml 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 50ml 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 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 Il/well).

cAMP standards and Detection Buffer (comprising 1 pCi of tracer [I(I cAMP pl] to 11 ml Detection Buffer) was prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer was prepared fresh for screening and 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-11 and H12. 501p of Stimulation Buffer was added to all wells. DMSO (or selected candidate compounds) was added to appropriate wells using a pin tool capable of dispensing 3ld of compound solution, with a final assay concentration of 12pM test compound and 1001l total assay volume. The cells were then added to the wells and incubated for 60 min at room temperature. 1001 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 WO 01/36471 PCT/US00/31509 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 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 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 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-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP. Upon creating a nonendogenous 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 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 21ig DNA of each receptor transfected into the mammalian cells, for a total of 4gg DNA pCMV vector, pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) in 1.2ml serum free WO 01/36471 PCT/US00/31509 DMEM (Irvine Scientific, Irvine, CA); tube B will be prepared by mixing 120pl lipofectamine (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 30-45min. The admixture is referred to as the "transfection mixture". Plated 293 cells will be washed with IXPBS, followed by addition of 10ml 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 DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37 0 C/5% CO 2 After 24hr incubation, cells will then be harvested and utilized for analysis.

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

SMP004A) 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 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 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. Iml 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 pellet will be carefully re-suspended into an appropriate volume of PBS (about WO 01/36471 PCT/US0O/31509 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 0Sl/well).

cAMP standards and Detection Buffer (comprising 1 Ci of tracer [1251 cAMP (50 gl] to 11 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 50pl cells, Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50gl of cAMP standards to appropriate wells followed by addition of 50l of PBSA to wells H-ll and H12. 50ul of Stimulation Buffer will be added to all wells. Selected compounds TSH) will be added to appropriate wells using a pin tool capable of dispensing 31l of compound solution, with a final assay concentration of 12 1 M test compound and 100pl total assay volume. The cells will then be added to the wells and incubated for 60 min at room temperature. 100pl of Detection 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 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 6well transfection as follows: 260ng of plasmid DNA in 100ll of DMEM were gently mixed with 2pl of lipid in 1Opl of DMEM (the 260ng of plasmid DNA consisted of WO 01/36471 PCT/US00/31509 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and 10ng 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 promoter at BglV-HindIII site in the ppgal-Basic Vector (Clontech). Eight 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-0-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 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 100l 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 in a cell culture incubator. The following day the transfected cells were changed with 200 pl/well of DMEM with 10% FCS. Eight 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 LucLite T M reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBeta

T

scintillation and luminescence counter (Wallac).

b. API reporter assay (Gq-associated receptors) A method to detect Gq stimulation depends on the known property of Gqdependent phospholipase C to cause the activation of genes containing API elements 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 WO 01/36471 PCT/US00/31509 CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-

SEAP.

c. SRF-LUC Reporter Assay (Gq- associated receptors) One method to detect Gq stimulation depends on the known property of Gqdependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A PathdetectT M SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, COS7 cells.

Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionT M Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed 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 1gM Angiotensin, where indicated. Cells are then lysed and assayed for luciferase activity using a Luclite T M Kit (Packard, Cat. 6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. The data can be analyzed using GraphPad Prism T 2.0a (GraphPad Software Inc.).

WO 01/36471 PCT/US00/31509 d. Intracellular IP 3 Accumulation Assay (Gq-associated receptors) On day 1, cells comprising the receptors (endogenous and/or non-endogenous) can be plated onto 24 well plates, usually Ixl0 s cells/well (although his umber can be optimized. On day 2 cells can be transfected by firstly mixing 0.25ug DNA in 50 pl serum free DMEM/well and 2 ul lipofectamine in 50 pll serumfree DMEM/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 media and added to the cells. The cells are then incubated for 3-4 hrs at 37 0 C/5%CO 2 and then the transfection media is removed and replaced with 1ml/well of regular growth media. On day 3 the cells are labeled with '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 are incubated for 16-18 hrs o/n at 37C/5%CO 2 On Day 4 the cells are washed with ml PBS and 0.45 ml of assay medium is added containing inositol-free/serum free media M pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50ul of ketanserin (ket) to final concentration of 10M. The cells are then incubated for 30 min at 37 0 C. The cells are then washed with 0.5 ml PBSand 200tl of fresh/icecold stop solution (1M 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 1 ml of chloroform/methanol is added/tube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AGI-X8 T anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1:1.25 W/ and 0.9 ml of upper phase is loaded onto the column. The column is washed with 10 mls of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60mM Na-formate. The inositol WO 01/36471 WO 01/647 1PCTUSOO/3 1509 ti-is phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0. 1 M formic acid/ 1 M ammonium formate. The columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammnonium formate and rinsed twice with dd H 2 0 and stored at 49C in water.

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 11P, 317.03 3463.29 (Figure 1) cpm/mg protein cplu/mg protein 1 I11 Fold hRUP13 N/A cAMP 8.06 19.10 (Figure 2) pmaol/cAMP/mg prnol/cAMP/mng 1 2.4 Fold protein protein A268K 8XCRE- 3665.43 83280.17 61713.6 1 23 Fold LUG LCPS LPCS LCPS<- (Figure 3) 26%( hRUPI4 L246K 8XCRE- 86.07 1962.87 789.73 1 23 Fold LUG LCPS LCPS LCPS (Figure 5) A398K 8XGRE- 86.07 18286.77 17034.83 1. 212 Fold LUC LCPS LGPS LCPS (Figure 6) 1%( A398K cAMP 15.00 164.4 117.5 1 I1I Fold (Figure 7) pmnol/cAMP/mg prnol/cAMPT/ig pmol/cAMP/ protein protein mg protein 29P/o( hRUF17 N/A 1P 3 317.03 741.07 1. 2.3 Fold (Figure 9) cpm/mg protein cpm/mg protein<bRUP21 N/A 1P 3 730.5 1421.9 I1. 2 Fold 10) cpg! Lin protein cpni/mg protein hRP3 W275K 8XGRE- 311.73 13756.00 9756.87 1. 44 Fold LUG pmnol/cAMP/nig pniol/cAMP/rng pmol/cAMP/ 11) protein protein mg protein 2. 30% N/A not applied WO 01/36471 WO 0136471PCTIUSOO/31509 Exemplary results of GTPyS assay for detecting constitutive activation, as disclosed in Example 4(l) above, was accomplished utilizing Gs:Fusion Protein Constructs on human RUJP13 and RUPl5. Table H below lists the signals generated from this assay and thc difference in signals as indicated: TABLE H Receptor: Assay Signal Signal Signal Signal Difference Gs Fusion Utilized Generated: Generated: Generated: Generated: Between: Protein CMIV Fusion CMV+ Fusion 1. CMV v. Fusion (cpm bound Protein IOpMGDP Protein Protein GTP) (cpmn bound (cpmn bound 10jOiM GDP 2. CMV+GDP GTP) GTP) (cpm bound vs.

GTP) Fusion+GDP 3. Fusion vs.

Fusion+GDP (cpm bound GTP) hRUPI3-Gs GTPyS 32494.0 4935130 11148.30 28834.67 1. 1.5 Foldc (Figure 4) 2. 2.6 Fold 3. 42%( GTPyS 30131.67 32493.67 7697.00 14157.33 1. 1. 1 Fold (Figure 8) 2. 1.8 Fold 56%( Example FUSION PROTEIN PREPARATION a. GPCR:Gs Fusion Constact 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 Hindll AAGCfT-3') sequence thereon. Following confirmation of the correct sequence (including the flanking HfindD[l sequences), the entire sequence was shuttled into pcDNA3.1I(-) (Invitrogen, cat. no. V795-20) by subcloning using the Hindfl restriction site of that vector. The correct orientation for the Gsax sequence was determined after WO 01/36471 PCT/US00/31509 subcloning into pcDNA3.1(-). The modified pcDNA3.1(-) containing the rat Gsa gene at HindIl 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 HindII site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach 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.

RUP13 couples via Gs. For the following exemplary GPCR Fusion Proteins, fusion to Gsa was accomplished.

A RUP13-Gsa Fusion Protein construct was made as follows: primers were designed as follows: (SEQ.1D.NO.:97; sense) 5'-gatc[GATATC]CGTGACTCCAGCCGGGGTGAGGCGGC-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 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: 100ng cDNA for RUP15 was added to separate tubes containing 21 of each primer (sense and anti-sense), 3pL of 10mM dNTPs, 10L of 10XTaqPlus T M Precision buffer, luL of TaqPlus T M Precision polymerase (Stratagene: #600211), and 80jL of water.

Reaction temperatures and cycle times for RUP15 were as follows with cycle steps 2 WO 01/36471 PCT/US00/31509 through 4 were repeated 35 times: 94°C for 1 min; 94C for 30 seconds; 62 0 C for sec; 72 0 C 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 with XbaI and EcoRV and the desired inserts purified and 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.:99 for nucleic acid sequence and SEQ.ID.NO.:100 for amino acid sequence).

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.1D.NO.:101; sense) 5'-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3' (SEQ.ID.NO.:102; antisense).

Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and RUP15. The sense and anti-sense primers included the restriction sites for EcoRV and Xbal, respectively, such that spacers (attributed to the restriction sites) exists between the G protein and 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: 100ng cDNA for RUP5I was added to separate tubes containing 2pl of each primer (sense and anti-sense), 3tL of 10mM dNTPs, 10pL of 10XTaqPlusT M Precision buffer, luL of TaqPlus T M Precision polymerase (Stratagene: #600211), and 80L of water.

Reaction temperatures and cycle times for RUP15 were as follows with cycle steps 2 WO 01/36471 PCT/US00/31509 through 4 were repeated 35 times: 94°C for 1 min; 94 0 C for 30 seconds; 62 0 C for sec; 72 0 C 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 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 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 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 amino acids, having the sequence EYNLV (SEQ.ID.NO.:130) will be replace with the corresponding amino acids of the Goti Protein, having the sequence DCGLF (SEQ.ID.NO.:131). This fusion construct will be obtained by PCR using the following primers: 5'-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3' (SEQ.ID.NO.: 132) and 5'-gatcggatccTTAGAACAGGCCGCAGTCCT'CAGGTTCAGCTGCAGGATGGTG-3' (SEQ.ID.NO.: 133) and Plasmid 63313 which contains the mouse Gccq-wild type version with a 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: 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 will be cloned into a pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit Biosystem). Inserts from a TOPO clone containing the sequence of the fusion construct will be shuttled into the expression vector pcDNA3.1(+) at the HindIII/BamHI site by a 2 step cloning process.

Example 6 PREFERENTIAL COUPLING OF RUP18 TO Gi: IP3 ASSAY IN 293 CELLS CO- EXPRESSING HUMAN RUP18 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 IP3 assay in 293 cells. IP3 assay was carried out approximately 24h post-transfection, essentially as described.

In a first experimental group, 293 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 RUP18 in pCMV, with or without co-transfection of GqdelGi in pCMV.

"0 C In a third experimental group, 293 cells were transiently transfected with non- 20 endogenous ("L294K") RUP18 in 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 .5 medium supplemented with 4 pCi/ml 3 H]myo-inositol (100tl; Perkin Elmer Life 25 Sciences) and the cells were allowed to incubate for approximately 20 h. The plates a..

were then frozen overnight at -80 0 C to achieve complete cell lysis.

The following day, the assay plates were thawed at room temperature. The thawed contents were then transferred to 96-well filter plates (Millipor, Multiscreen) pre-loaded with resin (Biorad, AG1-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 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 0 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).

Results are presented in Figure 12. Both endogenous RUP18 and nonendogenous 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 its endogenous form and that RUP18 has a coupling preference for Gi.

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 GPCRspecific and the human multiple tissue cDNA panels (MTC, Clontech) as templates.

Taq DNA ploymerase (Stratagene) were utilized for the amplification in a 40p1 reaction according to the manufacturer's instructions. 20gl of the reaction will be loaded on a 20 1.5% agarose gel to analyze the RT-PCR products. Table J below lists the receptors, the cycle conditions and the primers utilized.

o•* o• TABLE J Receptor Cycle 5' Primer 31 Primer DNA Tissue Identifier Conditions (SEQ.ID.NO.) (SEQ.ID.NO.) Fragment Expression Min Sec Cycles 244 repeated times 94* for 30" CATGTATGC GCTATGCCTG 730bp Kidney, 94' for 10" CAGCGTCCT AAGCCAGTC leukocyte, liver, 62'C for 20" GCTCC (10) TTGTG (106) placenta and 72' for P'II spleen 72' for 7' *cycles 2-4 repeated 35 times hRUP1 1 94' for 2' GCACCTGCT CACAGCGCT 630bp Liver, kidney, 94' for 15" CCTGAGCAC GCAGCCCTG pancreas, colon, 67'C for 15" CTTCTCC CAGCTGGC small intestinal, 72' for 45" (107) (108) spleen and 72* for 5' prostate .0 0 0 *0:00 :0, 000 .0.00 0 0 .00.

:0.

0.0 0000 00.0 0000 6000 00 0 0, WO 01/36471 PCTIUSOO/31509 hRUP12 940 for 2' CCAGTGATG CAGACACIT 490bp Brain, colon, 940 for 15" ACTCTGTCC GGCAGGGAC heart, kidney, 66'C for 15" AGCCIG (109) GAGGTG (1 10) leukocytc, 72' for 45" pancreas, 720 for 5' prostate, small intestinal, spleen, testis, thymus WO 01/36471 PCT/USOO/31509 hRUP13 940 for 1' CrGTGGTCT CATATCGCTC 700bp Placenta and 940 for 15" ACTGCAGCA CGAGTGTCC lung 68 0 C for 20" TGTTCCG AGCGGC (112) 72 0 for1'45" (111) 720 for RUPI4 940 for 1' ATGGATCCI CAAGAACAG 700bp Not yet 940 for 15" TATCATGGC GTCrCATCTA determined 68 0 C for 20" T'C~TC (113) AGAGCTCC 720 for 1'45" (114) 720 for hRUPI6 940 for 30" CTCrGATGC GTAGTCCACT 370bp Fetal brain, fetal 940 for 5" CATCTGCTG GAAAGTCCA kidney and fetal 69C for 15" GATTCC-X3 GTGATCC skeletal muscle 72* for 30" (115) (116) 720 for hRUP18 940 for 2' TGGTGGCGA GTrGCGCC'IT 330bp Pancreas 940 for 15" TGGCCAACA AGCGACAGA 600C for 20" GCGCTC(117) TGACC(118) 720 for 1' 720 for hRUP2I 940 for TCAACCTGT AAGGAGTAG Kidney, lung 940 for 15" ATAGCAGCA CAGAATGGT and testis 56C for 20" TCCTC (119) TAGCC (120) 720 for *cycles 2-3 repeated 30 times hRUP22 940 for 30" GACACCGT CTGATGGAA Testis, thymus 940 for 15" CAGCGGTCG GTAGAGGCT and spleen 69 0 C for 20" TGTGTG (121) GTCGATCTC 720 for 40" (122) *cycles 2-3 repeated 30 times hRUP23 940 for 2' GCGCFGAGC CACGGTGAC 520bp Placenta 940 for 15" GCAGACCAG GAAGGGCAC 0 C for 20" TGGC7G (123) GAGCTC (124) 720 for 720 fbr hRUP26 940 for 2' AGCCATCXC CCAGGTAGG 470bp Pancreas 940 for 15" TGCCAGGAA TGTGCAGCA 0 C for 20" GCATGG (125) CAATGGC 720 for 1' (126) 720 for hRUP27 940 for 30" CTGTTCAAC ATCATGICTA 890bp Brain 940 for 10" AGGGC'GGT GACTCATGGT for 20" TGGCAAC GATCC (128) 72 0 for 1' (127) 720 for 3' *cycles 2-4 repeated 35 times Example 8 TISSUE DISTRIBUTION OF HUMAN RUP18: RT-PCR RT-PCR was used to confirm the expression and to determine the tissue distribution of RUP18. Oligonucleotides used has the following sequences: 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 ul reaction by the following cycles: 94°C for 2 min; 94°C 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.

pl of the reaction were loaded on a 1% agarose gel to analyze the RT-PCR products, and a specific 330bp DNA fragment representing RUP18 was strongly detected in the pancreas (lane while weakly detected in the leukocytes (lane prostate (lane 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 i 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 25 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 o* 5'-CTAGAAGGCACTTTCACAGGAGCAGGGCGG-3' (SEQ ID NO: 118; antisense).

Taq DNA polymerase (Stratagene) was used in a 40 ul reaction according to the manufacturer's instructions. The amplification conditions were as follows: 1 cycle: 940 for 30 sec; 30 cycles: 94° for 30 sec, 53° for 30 sec, 720 for 2 min; and 1 cycle: 72° for 7 min. 20pl of each reaction was loaded on a 1.5% agarose gel to analyze the PCR products. 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.

Example Protocol: Direct Identification of Inverse Agonists and Agonists A. 3 5 SIGTPyS Assay Although we have utilized endogenous, constitutively active GPCRs for the direct identification of candidate compounds as, inverse agonists, for reasons that 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 a more robust identification of candidate compounds. Thus, it is preferred that for direct identification, GPCR Fusion Protein be used and that when utilized, the

O*

following assay protocols be utilized.

0 20 1. Membrane Preparation 000 o Membranes comprising the constitutively active orphan GPCR Fusion Protein 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 25 "Membrane Scrape Buffer" is comprised of 20mM HEPES and 10mM EDTA, 0 pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20mM HEPES, 100 mM NaCI, and 10 mM MgCl 2 pH 7.4.

b. Procedure All materials will be kept on ice throughout the procedure. Firstly, the media will be aspirated from a confluent monolayer of cells, followed by rinse with 10ml cold WO 01/36471 PCT/US00/31509 PBS, followed by aspiration. Thereafter, 5ml of Membrane Scrape Buffer will be added to scrape cells; this will be followed by transfer of cellular extract into 50ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4 0 Thereafter, the supematant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17 minutes at 4 0 C. The supernatant will then be aspirated and the pellet resuspended in Binding Buffer. This will then be homogenized using a Brinkman polytronTM 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 will be determined using the Bradford Protein Assay (protein can be diluted to about aliquoted and frozen for later use; when frozen, protocol for use will be 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; it was noted that for multiple preparations, the homogenizor should be thoroughly cleaned between homoginezation of different preparations).

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

b. Procedure Duplicate tubes will be prepared, one including the membrane, and one as a control "blank". Each contained 800ul Binding Buffer. Thereafter, 10pl of Bradford Protein Standard (Img/ml) will be added to each tube, and 10pl of membrane Protein WO 01/36471 PCT/US00/31509 will then be added to just one tube (not the blank). Thereafter, 200ul of Bradford Dye Reagent will be added to each tube, followed by vortex of each. After five minutes, the tubes will be re-vortexed and the material therein will be transferred to cuvettes. The cuvettes will then be read using a CECIL 3041 spectrophotometer, at wavelength 595.

3. Direct Identification Assay a. Materials GDP Buffer consisted of 37.5 ml Binding Buffer and 2mg GDP (Sigma, cat. no.

G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 pM GDP (final concentration of GDP in each well was 0.1 M GDP); each well comprising a candidate compound, has a final volume of 200ul consisting of 100ul GDP Buffer (final concentration, 0.1gM GDP), 50ul Membrane Protein in Binding Buffer, and 35 S]GTPyS (0.6 nM) in Binding Buffer (2.5 ul 35 S]GTPyS per 10ml Binding Buffer).

b. Procedure Candidate compounds will be preferably screened using a 96-well plate format (these can be frozen at Membrane Protein (or membranes with expression vector excluding the GPCR Fusion Protein, as control), will be homogenized briefly until in suspension. Protein concentration will then be determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) will then be diluted to 0.25mg/ml in Binding Buffer (final assay concentration, 12.5pg/well). Thereafter, 100 pl GDP Buffer was added to each well of a Wallac Scintistrip T M (Wallac). A 5ul pintool will then be used to transfer 5 pl of a candidate compound into such well 5 l in total assay volume of 200 pjl is a 1:40 ratio such that the final screening concentration of the candidate compound is 10pM). Again, to avoid contamination, after each transfer step the pin tool should be rinsed in three reservoirs comprising water ethanol (IX) WO 01/36471 PCT/US00/31509 and water (2X) excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes. Thereafter, 50 ul of Membrane Protein will be added to each well (a control well comprising membranes without the GPCR Fusion Protein was also utilized), and pre-incubated for 5-10 minutes at room temperature. Thereafter, 50.l of 3 S]GTPyS (0.6 nM) in Binding Buffer will be added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates were covered with foil). The assay will then be stopped by spinning of the plates at 4000 RPM for 15 minutes at 22 0 C. The plates will then be aspirated with an 8 channel manifold and sealed with plate covers. The plates will then be read on a Wallacc 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 assay approach can be utilized as an independent approach to provide confirmation of the results from the 5 S]GTPyS approach as set forth above.

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

SMP004A) was preferably utilized for direct identification of candidate compounds as 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 HEPES, pH 7.4 and 10mM MgC 2 Homogenization was performed on ice using a Brinkman PolytronTM 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 20mM HEPES, pH 7.4 and 0.1 mM EDTA, WO 01/36471 PCT/US00/31509 homogenized for 10 seconds, followed by centrifugation at 49,000 X g for 15 minutes at 4 0 C. The resulting pellet was then stored at -80 0 C until utilized. On the day of direct identification screening, the membrane pellet as slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and 10mM MgCL2, to yield a final protein concentration of 0.60mg/ml (the resuspended membranes are placed on ice until use).

cAMP standards and Detection Buffer (comprising 2 gCi of tracer [125I cAMP (100 pl] 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 20mM HEPES, pH 7.4, 10mM MgCI 2 20mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 pM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer was then stored on ice until utilized.

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

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

A representative screening assay plate (96 well format) result is presented in 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 and the mean plus two arbitrary preference for WO 01/36471 PCT/US00/31509 selection of inverse agonists as "leads" from the primary screen involves selection of 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 candidate compounds in the following wells were directly identified as putative inverse agonist (Compound A) and agonist (Compound B) to RUPI3 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 affinity, we are able to ascertain compounds that are able to reduce the functional activity of this receptor (Compound A) as well as increase the functional activity of the receptor (Compound Based upon the location of these receptor in lung tissue (see, for example, hRUP13 and hRUP21 in Example pharmaceutical agents can be developed for potential therapeutic treatment of lung cancer.

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.

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 WO 01/36471 PCT/US00/31509 Blvd., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

ff- EDITORIAL NOTE APPLICATION NUMBER 17696/01 The following Sequence Listing pages 1 to 74 are part of the description. The claims pages follow on pages 67 to WO 01/36471 WO 0136471PCTUSOO/3 1509 SEQUENCE LISTING <110> Arena Pharmaceuticals, Inc.

Chen, Rupong Dang, Huong T.

Lowitz, Kevin P.

<120> Non-Endogenous, Constitutively Activated Human G Protein-Coupled Receptors <130> AREN0087 <150> 60/166,088 <151> 1999-11-17 <150> 60/166,369 <151> 1999-11-17 <150> 60/166,099 <151> 1999-11-17 <150> 61/171,902 <151> 1999-12-23 <150> 60/171,901 <151> 1999-12-23 <150> 60/171,900 <151> 1999-12-23 <150> 60/181,749 <151> 2000-02-11 <150> 60/189,258 <151> 2000-03-14 <150> 60/189,259 <151> 2000-03-14 <150> 60/195,899 <151> 2000-04-10 <150> 60/196,078 <151> 2000-04-10 <150> 60/195,898 <151> 2000-04-10 <150> 60/200,419 <151> 2000-04-28 <150> 60/203,630 <151> 2000-05-12 <150> 60/210,741 <151> 2000-06-12 <150> 60/210,982 <151> 2000-06-12 <150> 60/226,760 <151> 2000-08-21 <150> 60/235,779 <151> 2000-09-26 Page 1 WO 01/36471 WO 0136471PCT/USOO/31509 <150> <151> <150> <151> <150> <151> <150> <151> <160> <170> 60/235, 418 2000-09-26 60/242, 332 2000-10 -2 0 60/242,343 2000-10-20 60/243, 019 2000-10-24 133 Patentln version <210> 1 <211> 1155 <212> DNA <213> Homc <400> 1 atggcagccc gcctcctccc gaggacatga gcaggcatca tataagaagt ctggtggcca tgggagcatg gtctccacca ttgaaaccac tccattctca aagagccagg aagtcctact ctgtgctatg gagcagattc ctcacggcct ttccccactg atcgccatga atgaagtact tccagtgctg atcaggctga <210> 2 <211> 384 sapiens agaatggaaa tctcctttaa ccaagacccg tgctggtctg tgcgcaacct tcatctgctg gccacgtgct atgccttgct ggatgaatta ttgccatccc agaagatctt tcctcttcat ccaggatctc gcaagcggct atgtgctgtg tgttcgtgaa gcaacagcat tcaagaagat accttgacct agtga caccagtttc cttcagttat gaccttcttc cggcatcggt caccaatctg ccccttcgag ctgtgcctcc ggccattgc tcaaacggcc atcggcttac ctgtggccag ctttggtgtc ccgggagctc gcgctgccgc ctgggcacc ggaaaagcac gatcaacacc gatgctgctg cagaaccaac acacccaact qgtgattatg gcagccaaga aactttgtct ctcat tgcca atggactact gtcaactacc attgacagat tccttcctga tttgcaacag atctggcctg gagttcgtgg tggttcaagg aggaagacgg ttctacggtt tacctcactg gtgtgcttcg cactggcgtc ggggtgccca ttaatccac acctccctat tcgtcattgg ttatcgctgc acctggccat acgtggtacg tgcgcaccgt atctcgccat tcgccttggt aaacggtcct tggatcagca gccctqtggt cagtccctgg tcctggtgct tcaccatcgt Ccttctacgt tgacggtcaa cctcccagcg ccacagaaga ccaagaccat ggatgaggat cattgcactg cctcacccgc ctccgacttc gcagctctcc ctccctctac cgttcacccc ctggatggtg ctttattgtc gctctactac caccatgacc gttccagacg catgtgcatt tcgtgacttc ggtcgagtgc gaacaacacc ggggagcaag ggtggactgt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1155 Page 2 WO 01/36471 WO 01/647 1PCTIUSOO/31509 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ala Gin Asn 1 5 Pro Gin Asp His Ala Tyr Asp Leu Pro Met Phe Phe Ala Ala Lys Leu Val Cys Gly Ile Tyr Lys Lys Leu Arg Ile Ser Asp Phe Leu 100 Tyr Tyr Val Val Arg 115 Ala Ser Vai Asn Tyr 130 Ala Leu Leu Ala Ile 145 Leu Lys Pro Arg Met 165 Val Trp Met Val Ser 180 Thr Giu Thr Val Leu 195 Gly Gin Ile Trp Pro 210 Leu Phe Ile Phe Gly 225 Leu Cys Tyr Ala Arg 245 Gly Phe Gin Thr Giu 260 Thr Val Leu Vai Leu 275 Ala Pro Phe Tyr Gly 290 Gly Asn Ser Ser Asp Glu Ile Val Giy Asn 70 Asn Leu Val Ala Gin Leu Legi Arg 135 Ala Ile 150 Asn Tyr Ile Leo Phe le Val Asp 215 Vai Giu 230 Ile Ser Gin Ile Met Cys Phe Thr 295 Thr Ser Phe Thr Leu Ser Phe Asn 25 Asp Glu Asp Met 40 Ile Gly Ile Ala Phe Val Phe Ile Thr Asn Leu Leu Ile Ile Cys Cys 105 Ser Trp, Giu His 120 Thr Val Ser Leu Asp Arg Tyr Leu 155 Gin Thr Ala Ser 170 Ile Aia Ile Pro 185 Val Lys Ser Gin 200 Gin Gin Leu Tyr Phe Val Gly Pro 235 Arg Glu Leu Trp 250 Arg Lys Arg Leu 265 Ile Leu Thr Ala 280 Ile Val Arg Asp Asn Phe Ser Tyr Lys Thr Ala Gly Ala Leo Ala Asn Phe Giu 110 His Val 125 Val Ser Ile Val Leu Ile Ala Tyr 190 Lys Ile 205 Lys Ser Val Thr Lys Ala Cys Arg 270 Val Leo 285 Phe Pro Val Val Val Lys Giu Lys His Tyr Leu Thr Ala Phe Paqe 3 WO 01/36471 WO 0136471PCTIUSOO/3 1509 Ile Ala Met Ser Asn 325 Lys Asn Asn Thr Met 340 Arg Pro Ser Gin Arg 355 Thr Asn Gly Val Pro 370 Ser Met Ile Asn Thr Vai Cys Phe Val Thr Val 330 335 Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp 345 350 Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg 360 365 Thr Thr Giu Giu Val Asp Cys Ile Arg Leu Lys <210> 3 <211> 1260 <212> DNA <213> Hcmo sapiens <400> 3 atgctggcag ctgccttti ctccactttg ccggagggi gCtctcttgg tggctgtcl ctccttcaca atgcttgg agcctggctg atctctcc aaaagtgttt gggatcta tgcatggCag ccaagagci agtgacccag ccaagcaa atctggactg tggctagc catgaaggtg tggaaatgl atgtttggta agctctacc tatttctgga gagcttat aaccagatac gctcaaagc ctcttgtggc tccccgaal ccggccccac cacaaggtl gcaaatcctc tcatttttc tggaaatgga tgataacc ggcaactcag agggtctt( gaaaaagaga aacccagcl cccatccttc ctgacgta gacaatgacc ctatccccl <210> 4 <211> 419 <212> PRT <213> Homo sapiens gc agactctaac tccagcagca tgaatgtgtc ctttgctcac ta tg aa ct 99 ct gt ct tg cc ga ca tg Lt ct aa cc tc ga tg cctgccctct gattcccagg cctggtgggc ttcgtgggaa aggaaagcca tccatgatcc cctgctgttt tctgcaccta ctggtttgtc tgcaagtcct gacaatcgtt gtggtggcca gaqtatccac aactacacca gttacccctg ccggaatggt cctcqtggat gtaccagctg actcctggca tttggccttc ccaatgtaaa aaacgaggaa agtcacagtg atgctgctga ggtagcttgg ctgtgggtat catagccctg tctcaagtct tgtgatgtcg gaagagttca aaaacctcca actgtctcag tgacaaggtt ccatctccag tccctcctct ggcaaaggga qcagttttgq catgagaggg ggaacatgaa gatcaagaga actggagaac acctgtgtgt actccctgat tccgagctac ctgactggtt aagtatgctt tctggtcagt tctttagcac tggctgaaga cattattttt ctaagactca gcattgccat ggcatctgaa tgatgttttc gggaaggct t agtctcagga aatccccaqc aaactgagaa acacagtccc caggggaagg catcatcccg gattggcatc tctgaatctc ggcgtactcc tatccacaca catgtatgca gctggtggcc catcaggcat gtttatgtcg tgccagcttt aaatcttaga catctctgct ggctgcaggc catctcttca gaaaggtgta aacaccagct atccatacca ggcagagatt ttctgtacag tgttaaatag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 Page 4 WO 01/36471 WO 0136471PCTUSOO/31509 <400> 4 Met Leu Ala Ala Ala Phe Ala Asp Ser Asn Ser Ser Set met Asn Val 1 5 10 Set Phe Ala His Leu His Phe Ala Gly Gly Tyr Leu Pro Ser Asp Ser 25 Gin Asp Trp Arg Thr Ile Ile Pro Ala Leu Leu Val Ala Val Cys Leu 40 Val Gly Phe Val Giy Asn Leu Cys Val Ile Gly Ile Leu Leu His Asn 55 Ala Trp Lys Gly Lys Pro Ser Met Ile His Set Leu Ile Leu Asn Leu 70 75 Ser Leu Ala Asp Leu Set Leu Leu Leu Phe Ser Ala Pro Ile Arg Ala 90 Thr Ala Tyr Ser Lys Ser Val Trp Asp Leu Giy Trp, Phe Val Cys Lys 100 105 110 Ser Ser Asp Trp Phe Ile His Tbr Cys Met Ala Ala Lys Set Leu Thr 115 120 125 Ile Val Val Val Ala Lys Val Cys Phe Met Tyr Ala Ser Asp Pro Ala 130 135 140 Lys Gin Val Ser Ile His Asn Tyr Thr Ile Trp Ser Val Leu Val Ala 145 150 155 160 le Trp Thr Val Ala Ser Leu Leu Pro Leu Pro Giu Trp Phe Phe Ser 165 170 175 Thr Ile Arg His His Giu Giy Val Glu Met Cys Leu Val Asp Val Pro 180 185 190 Ala Val Ala Giu Giu Phe Met Ser Met Phe Gly Lys Leu Tyr Pro Leu 195 200 205 Leu Ala Phe Giy Leu Pro Leu Phe Phe Ala Set Phe Tyr Phe Trp Arg 210 215 220 Ala Tyr Asp Gin Cys Lys Lys Arg Gly Tbr Lys Thr Gin Asn Leu Arg 225 230 235 240 Asn Gin Ile Arg Set Lys Gin Val Thr Val Met Leu Leu Set Ile Ala 245 250 255 Ile Ile Set Ala Leu Leu Trp Leu Pro Glu Trp Val Ala Trp Leu Trp 260 265 270 Val Trp His Leu Lys Ala Ala Gly Pro Ala Pro Pro Gin Gly Phe le 275 280 285 Ala Leu Set Gin Val Leu Met Phe Set Ile Set Set Ala Asn Pro Leu 290 295 300 Ile Phe Leu Val Met Set Giu Giu Phe Arg Giu Gly Leu Lys Gly Val 305 310 315 320 Trp Lys Trp Met Ile Thr Lys Lys Pro Pro Thr Val Set Glu Set Gin 325 330 335 Page WO 01/36471 WO 0136471PCTUSOO/3 1509 Glu Thr Pro Pro Glu Ser 355 Ser Ser Gly 370 Asp Val Giu Gly Asn Ser Glu Leu Pro Asp Lys Ala Ser Ile Lys Glu Lys Val Pro Ser 350 Ser Ser Pro Ile Leu Pro Lys Gly Lys Gin Phe Trp 390 Pro Ile Pro Thr Giu Lys Ala Glu 375 His Glu Arg Asp Thr 395 Trp Glu His Glu Asp 410 Pro Ser Val.

Asn Asp Gin Glu Thr Gly Val Lys <210> <211> 1014 <212> DNA <213> Homo sapiens <400> atggggaacg attctgtcag gtggactgcc tggatggcgc ctgtatgccg ccatcttcct gggaaggtgg cccgccgqag ttgctgtgct gtttgtctct ccgtatggtg cagtgggctg agcgtcctgc tcctggcagc tggtggtcta cggttcagcg ctggccttgc tgctcaccgt ccagcccggc tgcagtgtgt actgccatcc ggtttCtttt agtgccctcc tgtgctgggc gggttttttg tctgctgggc ccgaactccg cactcctggc ctcgcicaca gctgcctcaa cggtcactgc cagctgcctg gtggacaqca agaaatccac <210> 6 <211> 337 <212> PRT <213> Homo sapiens ctacgagtat ctgcctggcc ggtgggggtg qgtgggtgcc gcccatcctg tcgggcgctg tctcagtgcc ggcgtgcggg gCcctccgcc ggtggactac tggcttcctg agcccqacgc accctaccac cagggccctg tcccatgctc tcactgggcc cagccatgac ggggattaca atcgacccgc ccgggcaatg acctggttgc gcagtgccca ccctccatca gacctctgct gtgcaggtgg atctaccgcc ggcggCtcct gggcccctgg tgccggccgc ctgctggggc cgggctgaac ttcctgtatt ctgagggagt ctggtCtcgg gcgacctctc tgcgcgtggc ccatggtggc tccacctggc ttgcccgtgg tcctgctgac tcctggctct cctgtggggc ggctgcacca ccagcaccga tggccgtggc tgggcacagc tggtgctcac ccctcatcgt ttgggagggc cccagggcca agatggaggt ggaccgccct cccgctccca ctgggtggct cqtggcggat aggccactgg catgtatgcc cgggcctgcc agcctggaca ggagcacttc gaatgcggtg cagctgccac cattgtggtg tgtggcggcc gggccttgcc tcaactccgc ggacgaaagt gtag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1014 Page 6 WO 01/36471 PCTIUSOO/31509 <400> 6 Met Gly Asn Asp Ser Val Ser Tyr Glu Tyr Gly Asp Tyr Ser Asp Leu 1 5 10 Ser Asp Arg Pro Val Asp Cys Leu Asp Gly Ala Cys Leu Ala Ile Asp 25 Pro Leu Arg Val Ala Pro Leu Pro Leo Tyr Ala Ala Ile Phe Leu Val 40 Gly Val Pro Gly Asn Ala Met Val Ala Trp Val Ala Gly Lys Val Ala 55 Arg Arg Arg Val Gly Ala Thr Trp Leu Leu his Leo Ala Val Ala Asp 70 75 Leu Leu Cys Cys Leu Ser Leu Pro Ile Leu Ala Val Pro Ile Ala Arg 90 Gly Gly His Trp Pro Tyr Gly Ala Val Gly Cys Arg Ala Leu Pro Ser 100 105 110 Ile Ile Leu Leu Thr Met Tyr Ala Ser Val Leu Leu Leu Ala Ala Leu 115 120 125 Ser Ala Asp Leu Cys Phe Leu Ala Leo Gly Pro Ala Trp Trp Ser Thr 130 135 140 Val Gin Arg Ala Cys Gly Val Gin Val Ala Cys Gly Ala Ala Trp Thr 145 150 155 160 Leo Ala Leu Leu Leu Thr Val Pro Ser Ala Ile Tyr Arg Arg Leu His 165 170 175 Gin Glu His Phe Pro Ala Arg Leo Gin Cys Val Val Asp Tyr Gly Gly 180 185 190 Ser Ser Ser Thi- Glu Asn Ala Val Thr Ala Ile Arg Phe Leu Phe Gly 195 200 205 Phe Leu Gly Pro Leo Val Ala Val Ala Ser Cys His Ser Ala Leu Leo 210 215 220 Cys Ti-p Ala Ala Arg Arg Cys Arq Pro Leu Gly Thr Ala Ile Val Val 225 230 235 240 Gly Phe Phe Val Cys Trp Ala Pro Tyr His Leu Leu Gly Leo Val Leu 245 250 255 Thr Val Ala Ala Pro Asn Ser Ala Leu Leu Ala Arg Ala Leo Arg Ala 260 265 270 Glu Pro Leu Ile Val Gly Leu Ala Leu Ala His Ser Cys Leo Asn Pro 275 280 285 Met Leo Phe Leu Tyr Phe Gly Arg Ala Gin Leu Arg Arg Ser Leu Pro 290 295 300 Ala Ala Cys His T-p, Ala Leu Arq Glu Ser Gin Gly Gin Asp Giu Ser 305 310 315 320 Val Asp Ser Lys Lys Ser Thr Ser His Asp Leu Vai Ser Glu Met Glu 325 330 335 Page 7 WO 01/36471 WO 0136471PCTUSOO/31509 <210> 7 <211> 1272 <212> DNA <213> Homo sapiens <400> 7 atgttgtgtc accgtggtgg tcctgcaggg gtagaagact gaacttcata acctgagctc ttctctccct caccctcctc gggccctgcc accccacctc ctggagtttg tcctgggcct acgcggccct ggacctccaa ctgatCagca acctgcccct ggggctgctg cctgcaaagt gtcttcctca cagccatcgc ctgagccgtg cttccgtggg ctgctcctca. acgggcacct agggtgggca cgaagccctc ttcttcctgc cactggcgct aaccgtggtc tgggcgggca gtggccgtct acaccatcig gctttctggc tgtccgcctg ctggccttca cctacctcaa aacttcctcc accagagccg agcgacgaga gctcctacca ccagctgata ccagaacctt tccatctccc tgctccctct ttcctcgctg ggtggggaac cacggtgttc ccgcgtggac caacctcttc actcaaccgc ggcagctgcc gctcctgagc ggcctcqctc catcctcttt ggcaggcccg cttcttgcc ccgatccctg cagtgtcctg ggccttgctg accctccagg gtgccaatca ctctcaggcc tctctctcct gcctttacca gtgtctgcct agtttggccc ctggtcagcc tactacctcc atgctgtcca tacctgaagg cgggtggccg accttctccg cgctggcacc gctattgtga cagagggcca agcatcatct gacctctgca gaccccgtgc ggcctcacgc cagtggcgct tcccactttg catggcccaa cctctgttct ctgtgggggg tcctggcacc tcttcatctt tggtggccgc tccatgagac ccaaccgcac tggtgcagcc ggggactctg gcccctcctg aggcactgta gcattgggct tgcgtgtgct ttggcatggc cacagctctt tctactgctt ggggccggca accgggaggc ccctgagcac gcagcccatg ccctccctcc gtcctctgga aatcctggcc ctgcatccac tgacttcctc ctggcgcttt ggccagcgtt ccaccacgtg ggtgggcatc cctcagctac ectgctggag caccatccgg ggccatggtg ttccatggtg ccatggctcc ctctagcccc gggcccagtg ctctaggaag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1272 gcggaggcca tagggaagct gaaagtgcag ggcgaggtct ctctggaaaa ggaaggctcc tcccagggct ga <210> 8 <211> 423 <212> PRT <213> Homo sapiens <400> 8 Met Leu Cys His Arg Gly Gly Gin Leu Ile Val Pro Ile Ile Pro Leu 1 5 10 Cys Pro Glu His Ser Cys Arg Gly Arg Arg Lou Gin Asn Leu Lou Ser Page 8 WO 01/36471 WO 0136471PCTIUSOO/31509 Glu Leu His Leu Pro Pro Thr Thr Val Ser Leu Val Leu Gly Leu 105 Thr Arg Pro Ala Asp Phe Leu Leu His 155 Leu Phe Met 170 Ala Ile Ala 185 Leu Ser Arg Trp Val Gly Ser Gly Pro 235 Ser Leu Arq 250 Leu Ala Leu 265 Asn Arg Gly Leu Ala Met Ile Phe Gly 315 Ser Leu Asp 330 Tyr Leu Asn 345 Asn Leu Ser Phe Gly Gly Ser Ala Val Gly Trp Thr 125 Leu Leu 140 Glu Thr Leu Ser Leu Asn Ala Ser 205 Ile Leu 220 Ser Cys Trp His Ile Leu Leu Gly 285 Val Val 300 Met Ala Leu Cys Ser Val Ser Ser Pro Ser Pro Ser Ser Ser Gly Phe Leu Ala Asn Ser Leu 110 Ser Asn Thr Ile Ser Asn Trp Arg Phe 160 Thr Asn Arg 175 Arg Tyr Leu 190 Val Gly Ala Leu Leu Asn Leu Ser Tyr 240 Gin Ala Leu 255 Phe Ala Ile 270 Gly Gin Ala Ala Val Tyr Ser Met Val 320 Thr Gin Leu 335 Leu Asp Pro 350 Ser Arg Ala Val Leu Tyr 355 Cys Phe Ser Ser Pro Asn Phe Leu His 360 Page 9 WO 01/36471 WO 0136471PCTIUSOO/31509 Leu Leu Gly Leu Thr Arg Gly Arg Gin Gly Pro Val Ser Asp Giu Ser 370 375 380 Ser Tyr Gin Pro Ser Arg Gin Trp Arg Tyr Arg Glu Ala Ser Arg Lys 385 390 395 400 Ala Giu Ala Ile Gly Lys Leu Lys Val Gin Gly Glu Val Ser Leu Giu 405 410 415 Lys Giu Gly Ser Ser Gin Gly 420 <210> 9 <211> 966 <212> DNA <213> Homo sapiens <400> 9 atgaaccaga ctttgaatag agcacagtgc acacggccta gggatggcag gcaacagcai ttctgcatct atatcctcaa tccacgctca gcctggaaac atgaagagac tgatgtactt acccagcgct gtctctctgt ctgtcagcct gggtgtgtgg tcttccttct gcagcaagtt gtccaggccg ccctcatcat ctctttgtct gggtgcggag gtggtggtcc tggcctctgt tggtttgtgc tctactggtt tcacgcctct cctcgtccgt agccggagga gccacaggct cgcgaggagc ccgagctgga gcttga cagtgggacc cctggtgctg ggtgatctgg cctggcggca ccagccccig tgcctacaca cctcttccct cctgctgtgg cttgaaattc gggggtctta gagctcccag cctggtgttc gagcctgccg aagcagcagc gcccaccagg aggtggggag gtggagtcag agctccctgg ctgctgggct gccgacctcc gtcaatacca gtgggcctga atctggttca acactctgtc aatgaagatc accccagtga cagtggcggc ctcatctgtt cccqagatgc gccaaccccg tccctgggga acgcccaccg ccctaaacta ccatgttcac ttcgaatgca tcttcCtctt ctgacaaggt gcctgctgac agtgtcaccg tcctgatgaa ggtgcttcag tgactctgtc ggcagcccac ccctgcctct aggtcctgtg tcatctactt ctgtgctcca tgggcaccaa ttccagaggg ctgcctgtgc caggaacccc cagcatggct ccacgagctg ggccatcagc gcccaggcac cgggttgacc ggtggacatg cagcctgacc acggctgttc gagcatctac cttcagcttg cctggtgggc acaggcgctt tgagatgggg <210> <211> 321 <212> PRT <213> Homo sapiens <400> Met Asn Gin Thr Leu Asn Ser Ser Gly Thr Val Giu Ser Ala Leu Asn 1 5 10 Tyr Ser Arg Gly Ser Thr Val His Thr Ala Tyr Leu Val Leu Ser Ser 25 Page WO 01/36471 WO 0136471PCTIUSOO/31509 Len Ala Met Phe Thr Cys Leu Cys Gly Met Ala Gly Asn Set Met Val 40 Ile Trp Len Leu Gly Phe Arg Met His Arg Asn Pro Phe Cys Ile Tyr 55 Ile Leu Asn Leu Ala Ala Ala Asp Leu Len Phe Leu Phe Ser Met Ala 70 75 Ser Thr Leu Ser Leu Glu Thr Gin Pro Len Val Asn Thr Thr Asp Lys 90 Val His Gin Leu Met Lys Arg Len Met Tyr Phe Ala Tyr Thr Val Gly 100 105 110 Len Ser Leu Leu Thr Ala Ile Ser Thr Gin Arg Cys Leu Ser Val Leu 115 120 125 Phe Pro Ile Trp Phe Lys Cys His Arg Pro Arg His Leu Ser Ala Trp 130 135 140 Val Cys Gly Leu Leu Trp Thr Leu Cys Leu Len Met Asn Gly Leu Thr 145 150 155 160 Ser Set Phe Cys Ser Lys Phe Leu Lys Phe Asn Glu Asp Arg Cys Phe 165 170 175 Arg Val Asp Met Val Gin Ala Ala Len Ile Met Gly Val Leu Thr Pro 180 185 190 Val Met Thr Leu Ser Ser Leu Thr Leu Phe Val Trp Val Arg Arg Set 195 200 205 Ser Gin Gln Trp Arg Arg Gin Pro Thr Arg Leu Phe Val Val Val Leu 210 215 220 Ala Ser Val Leu Val Phe Leu Ile Cys Ser Len Pro Leu Set Ile Tyr 225 230 235 240 Trp Phe Val Leu Tyr Trp Leu Set Leu Pro Pro Gin Met Gin Val Leu 24.5 250 255 Cys Phe Set Leu Set Arg Leu Set Set Set Val Ser Set Set Ala Asn 260 265 270 Pro Val Ile Tyr Phe Len Val Gly Set Arg Arg Set His Atg Leu Pro 275 280 285 Thi- Arg Set Leu Gly Thr Val Leu Gin Gin Ala Leu Arg Giu Giu Pro 290 295 300 Gin Len Gin Gly Gly Gin Tht Pro Tht Val Gly Thr Asn Gin Met Gly 305 310 315 320 Al a <210> 11 <211> 1356 <212> DNA <213> Homo sapiens <400> 11 atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct Page 11 WO 01/36471 WO 0136471PCTIUSOO/3 1509 caaaccccag tcggaatctg gccgctgtga cacctctgcc agctctgccc ctgagcgtg t tactattacg tctgtgctgg agggtctcct cacagtgcct ctgctcctca cagcacgggc agccgctcca gggggaggga ttgccctact caggtggaga tatggatgtc aagccagcte ttcctgcagt cccageecca gagacctctg ctccgtcctg gtccctctac tgccagtggg tggccctctt cttcatgctc tggccgtgat cgceaagacg tggtggacct gctggctgcc tctttgacca cgccctcttt gctttgtcag cctggccate tagtccaecc catgcgctac tgggtgtgtg ggtgaaggcc gggaggaagg agctcecagt actgccagct ttttgtggtg tacttgtggt etactgcagc cgctgcccac gtggatggag cgatggtcac cagctcgggg aagcaqcagt ggttcteetg tctctttcca cctctatgtt gtgtggtcac ctggattggc teaaccggca qatccggggq eagaggagga gctgaggctg tccttcaggg gactggetgt agcaggagcc aectgctgtt agttcctgga gcagcaactc ccgcctcacc ccggctgqag gtcceggagg ctgctggact cctgccctcc ctgaccctca ggggaggtgg etc tcgg tg t gaggtgcgca ttggccatgg gtccccccag gtctttgctg atgttccgag aeaccccggc geccccaga getg tggggg gccctgagtg tacttttgct gagctcaqca cctageeggg ccttctgagt gactttcgaa accagcgaca teat ga tggggetaeg tgactgctgt gaaaatttgt tgccetggc cctgccgcct cagceatcaa tgaegctggg ettctgtgcc gctgttcaet teetttactt tggeccgegt aacgctcega ccacceaca gacagttcet eteagcecat tcacttccaa ageagtttgt agggcteeat eetgggttte tcccaggcca tcatcatgtc ggatgttgct ggctggcaat cttegtcttc catgctctcc etacttgttt tgtggagcgc getggtggcc agtgttggga ceagtgqage tctgttgece ggctgccatg atctetcagc ceggaegttt gctctgttgg tteaaetggg ccctttcttc ctgettettc tgaggagaac cegacceta gatagctgag agacagetac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1356 <210> 12 <211> 451 <212> PRT <213> Homo sapiens <400> 12 Met Glu Ser 1 Gly Arg Val Glu Val Gly Met Leu Leu Ser Pro 5 Pro Gin Leu Arg Ile Pro Gin Ser Thr Pro Gly Pro 25 Asp Val Ala Ser 40 Ser Gly Asn Ser Ser Thr Leu 10 Ser Thr Ala Ser Gly Val Pro Glu Ser Val Ala Leu Phe Phe Ala Gly Asn Ala Ala Val Met Leu Asp Leu Thr Ala Val 55 Page 12 WO 01/36471 WO 0136471PCT/USOO/3 1509 Ala Lys Leu Val Ser Ser 100 Arg Leu Ser Val Met Arg Val Gly 165 Gly Arg 180 Ser Leu Phe Ala Tyr Cys Pro Leu 245 Ser Ser 260 Pro His Val Gly Leu Tyr Ser Val 325 Phe Tyr 340 Phe Val Ser Arg Thr Gly Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe 75 Asp Leu Ser Ala Tyr Leu Ser Ala 135 Tyr Giu 150 Val Trp Vai Ser Gin Trp Val Leu 215 Ser Met 230 Pro Thr Arg Ser Arg Thr Gly Gin 295 Val Ala 310 Val Thr Gly Cys Cys Phe Glu Gly 375 Cys Pro 390 Leu Ala Ala Leu Phe Asp 105 Phe Leu Ser 120 Ile Asn Val Val Arg Met Val Lys Ala 170 Trp Glu Giu 185 Ser His Ser 200 Tyr Phe Leu Phe Arg Val Trp Met Glu 250 Thr Met Vai 265 Phe Gly Giy 280 Phe Leu Leu Leu Ser Ala Trp Ile Gly 330 Leu Asn Arg 345 Phe Lys Pro 360 Ser Ile Giu Ser Giu Ser Leu Tbr Leu His Ala Leu Val Cys Phe 125 Giu Arg Tyr 140 Thr Leu Gly 155 Leu Ala Met Gly Ala Pro Ala Tyr Cys 205 Leu Pro Leu 220 Ala Arg Val 235 Thr Pro Arg Thr Ser Ser Gly Lys Ala 285 Cys Trp Leu 300 Gin Pro Ile 315 Tyr Phe Cys Gin Ile Arg Ala Pro Giu 365 Giu Asn Phe 380 Trp Val Ser 395 Pro Ser Pro Lys Gin 405 Glu Pro Pro Ala Val 410 Asp Phe Arg Ile Pro Gly 415 Page 13 WO 01/3647 1 PCTIUSOO/31509 Gin Ile Ala Giu Glu Thr Ser Glu Phe Leu Giu Gin Gin Leu Thr Ser 420 425 430 Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu Giu Ser 450 <210> 13 <211> 1041 <212> DNA <213> Homo sapiens <400> 13 atggagagaa aatttatgtc cttqcaacca tccatctccg tatcagaaat ggaaccaaat ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120 ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180 tatgttttcc tgcagcctta taagaagtcc acatCtgtga acgttttcat gctaaatctg 240 gccatttcag atctcctgtt cataagcacq cttcccttca gggctgacta ttatcttaga 300 ggctccaatt ggatatttgg agacctggcc tgcaggatta tgtcttattc cttgtatgtc 360 aacatgtaca gcagtattta tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420 gttcacccct ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480 atcatatgga tccttatcat ggcttrctca ataatgctcc tggacagtgg ctctgagcag 540 aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc 600 atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact cagcatctgt 660 tatctgctga tcattcgggt tctgttaaaa gtggaggtcc cagaatcggg gctgcgggtt 720 tctcacagga aggcactgac caccatcatc atcaccttga tcatcttctt cttgtgtttc 780 ctgccctatc acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840 gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900 aatcctctgc tctattactt tgctggggag aatittaagg acagactaaa gtctgcactc 960 agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag tgtgtggttg 1020 agaaaggaaa caagagtata a 1041 <210> 14 <211> 346 <212> PRT <213> Homo sapiens <400> 14 Met Giu Arg Lys Phe Met Ser Leu Gin Pro Ser Ile Ser Val Ser Giu 1 5 10 Met Giu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser Arg Asn Cys Thr 25 Page 14 WO 01/36471 PCT/US00/31509 Asn Phe Trp Gly Tyr Lys Ser Asp Leu Arg 100 Ser Tyr 115 Val Leu Leu His Trp lie Glu Gin 180 Ile Ala 195 Leu Leu Val Leu Arg Lys Cys Phe 260 Lys Val 275 Leu Ala Phe Ala Gly His Trp Leu 340 Lys Arg Glu Phe Val Leu Gly Asn Lys Ser Thr Ser Leu Leu Phe Ile Gly Ser Asn Trp Ser Leu Tyr Val 120 Ser Val Val Arg 135 Val Thr Ser lie 150 Leu Ile Met Ala 165 Asn Gly Ser Val Lys Leu Gin Thr 200 Pro Phe Phe Thr 215 Leu Lys Val Glu 230 Ala Leu Thr Thr 245 Leu Pro Tyr His Gly Leu Cys Lys 280 Leu Ala Ala Ala 295 Gly Glu Asn Phe 310 Pro Gin Lys Ala 325 Ara Lvs Glu Thr Phe Pro Ile Val Leu Ile Ile Tyr Val Phe Leu Leu Asn Arg Ala Ala Cys 110 Ile Tyr His Pro Leu Cys Leu Asp 175 Leu Asn 190 Leu Val Leu Leu Leu Arg Ile Ile 255 His Leu 270 Ala Leu Pro Leu Ser Ala Phe Pro 335 <210> <211> 1527 <212> DNA <213> Homo sapiens Page WO 01/36471 PCTJUSOO/31509 <400> atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120 ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300 agccacttct qcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360 accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420 tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480 ctgcagagca ctcctccact ctacggctgg ggcCaggctg cctttgatga gcgcaatgct 540 ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600 atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctq tgcagcccgg 660 aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720 tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780 gagtttcgcc qccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840 gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900 aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960 aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080 aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140 cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200 atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320 tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380 attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440 gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500 tcctacgatt ctgctacttt tccttga 1527 <210> 16 <211> 508 <212> PRT <213> Homo sapiens <400> 16 Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5 10 Page 16 WO 01/36471 PCT/US00/31509 Pro Leu Thr Val Leu Ala Arg Phe Val Ala Asn Ser 100 Phe Ala Ser Ile Gly Tyr Thr Pro 165 Ala Leu 180 Ser Val Tyr Ser Asn Val Asn Glu 245 Ser Glu 260 Arg Met Gly Thr Glu Ser Ser Thr 325 Glu Val 340 Lys Met Val Ile Val Leu Phe Asn Trp Val Phe Cys Val Asn 120 His Pro 135 Leu Leu Leu Tyr Ser Met Ser Phe 200 Val Phe 215 Arg His Glu Glu Arg Arg Ala Lys 280 Glu Ser 295 Thr Val Val Glu Gin Cys Pro Ile Phe Leu Gin Arg Asp Leu Val Pro Ser Leu 110 Val Ser 125 Ser Lys Ile Val Ala Ala Ser Pro 190 Leu Ile 205 Arg Gin Arg Val Lys Glu Glu Val 270 Lys Ala 285 Arg Gly Ser Met Lys Ala Gly Glu 350 Met Glu Phe Gly Glu Asp Asp Ile 355 360 Asn Phe Ser Glu Asp Asp Val Glu 365 Page 17 WO 01/36471 WO 0136471PCTUSOO/31509 Ala Val 370 Ser Asn Asn Ile Pro Pro Pro Leu Giu Ser 3.75 Pro Arg 390 Phe Ser Leu Pro Pro Ser Arg Arg 380 Asn Ser Asn Cys Tyr Gin Lys Ala Ala Lys Phe Ile Ile Tyr Val Leu Gly Phe Leu Ala Gin Trp Vai 435 Ile His Pro Ala Val Trp Thr Ile Ile Val 425 Trp Met Asp Val Glu Thr Leu Phe Phe Leu 445 His Lys Thr Ile 460 Cys Lys Glu Lys Pro Tyr Cys 415 Gin Val Pro 430 Gin Cys Cys Lys Lys Glu Pro Pro Lys 480 Thr Giu Gly Tyr Val Tyr Asp Met Leu Phe Phe Giu Asp Ser His Pro 485 Lys Ile Val Pro Ser 500 <210> 17 <211> 1068 <212> DNA <213> HOMO sapiens <400> 17 atgcccttga cggacggci atatttgtct gggttatac agatctttca ttaaagctc gctgattgcc tgatgggtc gggcagtatc agaagtatg ttcctggcca tgctgtcca aagttcctgg tcattgtct gtcatcctca tttgcatct aagqattatt ttggaaact caaacagaeg atattggaa ctggcttttc tcatcattg gccttgcaga ccacagaag ttctttttta tagtgttct ctttccctct tccgggtgc cttccagtta acagtgctt Leu Pro Gly Gly Gly Tyr Asp Ser Ala Thr Phe Pro 505 t c a t c c t 9 t 9 t t c a t ttcttcattt tttcattacc aaatacaact ttacttgttc cttgctgtgg cgaagtctct ccccttcagt gatggcggga ttatgggaaa caaagggtat gttttcctat aaggaattgt tgatgccatc aataccagac gaatccaatc gaggacctct tgctttggaa cacgctatgt tttgttggca atggagagcg gttctgctac aacattcgac tttt taatag aatggagtat tctcttggaa attactatgt tttggaagag tgctggattc acaatgactt ctctatactc tggctaacaa atctttttgt ccatcaaaat ttttcgatat tgcagtgccg tgacctactt ctggaaaacg ctgtaattcc gtttcccact ttttcctagg tctgttccat aggtggctgt ctgtatttgt cctgqatagt tcacaaccaa tatcctcaga cattggcatg cctttgttgc aaaataccga Cctcatgggg gactttggag gcagacctca attttggaat ttattatgac tgtgaacttg tcaaaaaacc tgcaaatcgt agttaaaatc gatttttttc cttttttaag gacaagttga aacagctgct gcacaaacat cagaggaaat caattttcaa aattaaaaaa Page 18 WO 01/36471 WO 0136471PCTIUSOO/31509 aaaagtttat ctacatccat tgtgtggata gaggactoct cttccctgaa actrggggtt 1020 ttgaacaaaa taacacttgg agacagtata atgaaaccag tttcctag 1068 <210> 18 <211> 35.

<212> PR <213> Hoi <400> 18 Met Pro Li 1 Ann Ilie Li Gly Asn Li Thr Thr H.

Met Gly V.

Gly Gin T Arg Leu Mi Leu Leu TI Phe Ser A 130 Cys Ile T~ 145 Lys Asp T' Leu Tyr Ti Gly Ile P1 Ii Ser Tyr I2 210 Thr Giu V 225 Phe Phe P~ Val Val L

T

no sapi eu Thr eu Arg eu Phe is Ala al Tyr jr Gin et Giy 100 hr Tyr sn ile rp Met yr Phe yr Asp 180 ie Leu 95 le Thr ii Arg he Ile js Ile 260 iens Asp Ile Val Met Leu Lys Phe Leu Ser Ser Trp Val 25 Met Arg 40 Lys Ile Val Gly Leu Leu Met Leu J05 Giu Lys 120 Lys Arg Leu Ile Tyr Gly Asp Ile 185 Leu Leu 200 Ser Ile Gly Arg Asp Ala Phe Arg 265 Asp Leu Leu ['he Ile Thr Ile Lys Ala Cys Ala Asp Asp Ile Lys Giu Ser Val Glu Val Ser 110 Val Ile Val 125 Ser Val Ile 140 Ile Pro Phe Gly Val Cys Lys Giy Tyr 190 Leu Ile Ile 205 Thr Ala Leu 220 Ala Val Ala Trp Ile Pro Ile Pro Asp 270 Cys Leu Tyr Arg Gin Cys Val Leu Phe Pro Leu Ile Trp Asn 160 ['he Pro 175 Ser Leu Val Phe Gin Thr Asn Arg 240 Val ['he 255 Thr Met Thr Ser Trp Ile Val Ile ['he ['he Leu Pro Vai Asn Ser Ala Leu Asn Page 19 WO 01/36471 WO 0136471PCTUSOO/31509 275 Pro Ile Leu Tyr Thr Leu 290 Gin Leu Leu His Lys His 305 310 Lys Ser Leu Ser Thr Ser 280 Thr Thr 295 Gin Arg Ile Val Asn Phe Phe Lys Ser Ile 315 Trp Ile Giu Lys Leu Lys Lys Ile Lys Asp Ser Ser Ser Leu 335 Met Lys Lys Leu Giy Pro Vai Ser 355 Asn Lys Ile Giy Asp Ser <210> 19 <211> 969 <212> DNA <213> Homo sapiens <400> 19 atggatccaa ccatctcaa actctttgct acaagcagi gggctgacag gaaacgcac ttctccatct acatcctcz atatattccc tgttaagct gtgatgatgt tttcctact tgcctgtccg tcctgtggc gtggtgtgtg tcctgctct tgtggcttcc tgttcagtg gtcgcgtggc tgatttttt aggattctct gtggatccc acagtactgg tcttcctcc tggatccacg tggacaggg tccgctctta acagcagtg cgtcaaaata ggcagaacc gtqgatgaaqg tggagggc gagcagtga <210> <211> 322 <212> PRT <213> Homo sapiens <400> Ic 18

IC

a8 cttggacaca cttgagcctc tgtgCtctgg cttggccgca catcagtatc tgcaggcctg catctggtac gCcctgt~c tgctgattct atgtgtggtt gaagataccg ctgtggcctg agtcttattt caaccccatc gaagctggtt gcttcctgag gaactgacac acggtgctga ctcctgggct gcagacttcc ccccatacca agctttctga cgctgccacc ctgctgcgga gcttggtgtc ctctgtgggt ctgaccaggc ccctttggca tgtcatgttc atttacttct ctccagaggg gaaatcctgg caatcaacgg cgtgcatcgt gccgcatgcg tcttcctcag tctctaaaat gtgccgtgag gccccacaca gcatcctgga aaacatcaga ccagcctggt tgtacgtgac ttcagttttt atctagtttc tcgtgggCtc ctctgcagga agctqtcggg aactgaggag ttCccttgtc caggaacgcc cggccgcctt cctctatcct caccgagcgc cctgtcagcg gtggatgtta tttcatcaca cctgctgatc catcctgctc cctattttta tattttcctg ctttaggcag cgcgtctgag aagcagattg Page WO 01/36471 PCT/USOO/31509 Met Asp Pro Thr Ile Ser Thr Leu Asp Thr Giu Leu Thr Pro Ile Asn 1 5 10 Gly Thr Glu Giu Thr Leu Cys Tyr Lys Gin Thr Leu Ser Leu Thr Val 25 Leu Thr Cys Ile Val Ser Leu Val Gly Leu Thr Gly Asn Ala Val Val 40 Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Phe Ser Ile Tyr 55 Ile Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser Gly Arg Leu 70 75 Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile Pro His Thr Ile Ser Lys 90 Ile Leu Tyr Pro Val Met Met Phe Ser Tyr Phe Ala Gly Leu Ser Phe 100 105 110 Leu Ser Ala Val Ser Thr Glu Arg Cys Leu Ser Val Leu Txp Pro Ile 115 120 125 Trp Tyr Arg Cys His Arg Pro Thr His Leu Ser Ala Val Val Cys Val 130 135 140 Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Giu Trp Met Leu 145 150 155 160 Cys Gly Phe Leu Phe Ser Gly Ala Asp Ser Ala Trp Cys Gin Thr Ser 165 170 175 Asp Phe Ile Thr Val Ala Trp Leu Ile Phe Leu Cys Val Val Leu Cys 180 185 190 Gly Ser Ser Leu Val Leu Leu Ile Arg Ile Leu Cys Gly Ser Arg Lys 195 200 -205 Ile Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu Val 210 215 220 Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gin Phe Phe Leu Phe Leu 225 230 235 240 Trp Ile His Val Asp Arg Giu Val Leu Phe Cys His Val His Leu Val 245 250 255 Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr 260 265 270 Phe Phe Val Gly Ser Phe Arg Gin Arg Gin Asn Arg Gin Asn Leu Lys 275 280 285 Leu Val Leu Gin Arg Ala Leu Gin Asp Ala Ser Giu Val Asp Giu Gly 290 295 300 Gly Gly Gin Leu Pro Glu Giu Ile Leu Giu Leu Ser Gly Ser Arg Leu 305 310 315 320 Glu Gin <210> 21 <211> 1305 Page 21 WO 01/36471 WO 0136471PCT[USOO/31509 <212> DNA <213> Homo sapiens <400> 21 atggaggatc tctttagccc ttgctgggct ggggtctcaa agccgccgcg tccgcctggt accacagtgc tgtgccgcct aaqatggact tcctgctggt gcgctgtcac agctggcctg gcgtgccgct tcctgcagct gtgctcatcg ccctcgagcg gcgcgtgccc tcgccgccct ttcgtggtgc gcggggactc cagccaggcg cgcccccggc ttcgcgcccc tgccgcgctg ttcgtcgcgc ctgttacggt cggcaccggc cgcaggcccc cctqcgccca gcgcgctgcc gcgctgctgt tcgtgggctg tcgtccgggc ccgcgggaga gcgatggcca acagcgctct cggctccggc gacagctgcg gcggaggacg aggaggggcc cctcattatc accatgctcg ccgcgcccca gacccctgcc <210> 22 <211> 434 <212> PRT <213> Homo sapiens <400> 22 ctcaattctg cctgaccttg gttcctgggg gtgcggcggc gcagctggcc ggaactgctg gctgcaggca ccggcgcgcg gggctggctg cccctcgccg cgcccgcgcc gcacctgcag cctgggcgtc cgcggctgca ccgcgccaag cgagctgccc ctgggaggga caatcccttc gaagcggctg ccggggccac gcgggaaccg ttgctcctgc ccgccggcgc gggcaaggag gtcatcctgg ggcgggCcct ctggcggacc ggcgagcccc tccgggcggg gtgcgtcttc ctggcactgc ctgccgccqc tggccggggg gtctacgcgt gcttgcggcc gcgcciggt gtgcagagcc tactttgccg gagggcctgt gtctacctct ggctctctgt caggcgctct ctggacgagg gaaagtgcct ccaacatttc cccctgcctc tggtggcggt gggcgggccc tgtacgcgtg gcgcggccac gcgcctcggc cgCacggccg tgctggcgct cgccgccgcc agcgtcgctg tctacgaggc acctactctc cggcgagccc tgaagatgag cccggc tggc cggcggcgct tcttccaggc gctgcgcgcc accgccaacg gcggcttgcg tctag cgtgcccatc tgggccgccc ggcaggcaac caagcgtcgc cgggggcacg gggggacctg ccacctcgtg gccgctgcc gcCcccggcc aacgtccctg ccacgggatc cgtcgcgggc cgtctggtgg aggtcgagcc cctgctgctg ggccgcgtgg gcgcgtggtg gggcgactgc gcagggaggc ctggccccac cccaccccct 120 180 240 300 360 420 480 540 600 660 720 '780 840 900 960 1020 1080 1140 1200 1260 1305 Met Glu Asp Leu Phe Ser Pro Ser Ile 1 5 Ser Val Pro Ile Leu Leu Gly Trp, Gly 25 Gly Ala Pro Ala Ser Gly Pro Pro Ser 40 Leu Pro Pro Ala Pro Asn Ile 10 Leu Asn Leu Thr Leu Gly Gin Arg Arg Val Arg Leu Val Phe Page 22 WO 01/36471 PCTUSOO/3 1509 Leu Gly Val Ile Leu Val Val Ala Val Ala Giy Asn Thr Thr Val Leu 55 Cys Arg Leu Cys Gly Gly Gly Gly Pro Trp Ala Gly Pro Lys Arg Arg 70 75 Lys Met Asp Phe Leu Leu Val Gin Leu Ala Leu Ala Asp Leu Tyr Ala 90 Cys Gly Gly Thr Ala Leu Ser Gin Leu Ala Trp Glu Leu Leu Gly Glu 100 105 110 Pro Arg Ala Ala Tbr Gly Asp Leu Ala Cys Arg Pbe Leu Gin Leu Leu 115 120 125 Gin Ala Ser Gly Arg Gly Ala Ser Ala His Leu Val Val Le Ile Ala 130 135 140 Leu Glu Arg Arg Arg Ala Val Arg Leu Pro His Giy Arg Pro Leu Pro 145 150 155 160 Ala Arg Ala Leu Ala Ala Leu Gly Trp Leu Leu Ala Leu Leu Leu Ala 165 170 175 Leu Pro Pro Ala Phe Val Val Arg Gly Asp Ser Pro Ser Pro Leo Pro 180 185 190 Pro Pro Pro Pro Pro Thr Ser Leo Gin Pro Gly Aia Pro Pro Ala Ala 195 200 205 Arg Ala Trp Pro Gly Giu Arg Arg Cys His Gly Ile Phe Ala Pro Leu 210 215 220 Pro Arg Trp His Leo Gin Val Tyr Ala Phe Tyr Glu Ala Val Ala Gly 225 230 235 240 Phe Val Ala Pro Val Thr Val Leu Gly Val Ala Cys Gly His Leu Leo 245 250 255 Ser Val Trp Trp Arg His Arg Pro Gin Ala Pro Ala Ala Ala Ala Pro 260 265 270 Trp Ser Ala Ser Pro Gly Arg Ala Pro Ala Pro Ser Ala Leu Pro Arg 275 280 285 Ala Lys Val Gin Ser Leu Lys Met Ser Leu Leo Leu Ala Leu Leu Phe 290 .295 300 Val Gly Cys Giu Leu Pro Tyr Phe Ala Ala Arg Leo Ala Ala Ala Trp 305 310 315 320 Ser Ser Gly Pro Ala Gly Asp Trp Glu Gly Glu Gly Leo Ser Ala Ala 325 330 335 Leu Arg Val Val Ala Met Ala Asn Ser Ala Leu Asn Pro Phe Val Tyr 340 345 350 Leu Phe Phe Gin Ala Gly Asp Cys Arg Leu Arg Arg Gin Leu Arg Lys 355 360 365 Arg Leo Gly Ser Leu Cys Cys Ala Pro Gin Gly Gly Ala Glu Asp Glu 370 375 380 Gbu Gly Pro Arg Gly His Gin Ala Leu Tyr Arg Gin Arg Trp Pro His 385 390 395 400 Page 23 WO 01/36471 WO 01/647 1PCTIUSOO/31509 Pro His Tyr His His Ala Arg Arg Glu Pro Leu Asp Glu Gly. Gly Leu 405 410 415 Arg Pro Pro Pro Pro Arg Pro Arg Pro Leu Pro Cys Ser Cys Glii Ser 420 425 430 Ala Phe <210> 23 <211> 1041 <212> DNA <213> HOMO sapiens <400> 23 atgtacaacg ggtcgtgctg ctgctcattg tggcctttgt tgcttccaca tgaagacctg gatttcctcc ttatgatctg tgggcttttg gggacattcc gggagcatcg tgttccttac caccacgcgg tgaacactat gccctggtca tcctgggaac acggccgtct cctgtgagag ttccagctgg agttctttat tggagcctga ggcggaggca ttcatcatgg tggtggcaat ctctatttcc tctggacggt cacataaccc tcagcttcac tcaagcccct cctttcccaa caqccaggac actcaaaaac aggagttgca tcagtgtggc cacattgttg agtggcactg ccgcatcgag ggggacacca tctcccaggt gatgccgccg gctgggcgca gaagcccagc cctgcctttt ctgccgagtg ggtggtggct ctccacccgg aqtgtatctt cttcatcatg gcCcctcggc gcagctggcct tgtgttcatc gccctcgagt ctacatgaac attctacaac acaaaggccg ctaggcaatg actgtttacc cggacagact gggctcttca gcggacaggt gtggcggctg ttgctgaga gagtcggcca atcatcttat agacaggctc acatgctacc gcctgcgatc agcatgctgg aagctcoaaaa gaagagatgc gggtcgccct ttttcaattt attacctcag cgttggccat atttcaaagt gcatcgtctg accatctctg atggctggca tttgCtcctt ggatgaagaa tgcccagcgt cctctgtcca atcccctggt tctgcagtct caatttcgaa gtgtggtttc ggccgtggct acgtacjacac gaacagggcc ggtccacccc caccctgtgg cqtgcaagag tgacatcatg caagattgtt ggcgacccgg gtctgctaga tggggccctg gtattatttt gaaacccaag cctcggtcgc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 aaatagtttc caaagccagt ctgatgggca atgggatccc <210> 24 <211> 346 <212> PRT <213> Homo sapiens <400> 24 Met Tyr Asn Gly Ser Cys Cys Arg Ile Glu Gly Asp Thr Ile Ser Gin 1 5 10 Val Met Pro Pro Leu Leu Ile Val Ala Phe Val Leu Gly Ala Leu Gly 25 Page 24 WO 01/36471 WO 0136471PCT/USOO/31509 Gly Val Ser Thr Ile Cys Ala Phe Leu Cys Gly Phe Cys Phe His Met 40 Lys Thr Trp Lys Met Asn Arg Ala Gly 100 Arg Tyr Phe Lys Val 115 Thr Arg Val Ala Ala 130 Leu Gly Thr Val Tyr 145 Thr Ala Val Ser Cys 165 His Asp Ile Met Phe 180 Leu Phe Cys Ser Phe 195 Leu Ala Arg Gin Ala 210 Val Ala Ile Val Phe 225 Leu Tyr Phe Leu Trp 245 His Gly Ala Leo His 260 Leu Asp Pro Leu Val 275 Tyr Asn Lys Leo Lys 290 Ser Lys Thr Gin Arg 1 305 Arg Ser Cys Ile Ser 1 325 Gin Trp Asp Pro His*] 340 <210> <211> 1011 <212> DNA <213> Homo sapiens Asn Leu Thr Asp Cys Arg Val Phe 105 Pro His 120 Val Cys Leu Glu Phe Ile Glu Phe 185 Val Trp 200 Lys Lys Cys Tyr Pro Ser Leo Ser 265 Phe Ser 280 Ser Leu Glu Met Asn Ser Glu Trp 345 Ala Asp Leu Arg Leo Phe Vai Val Val Asn 125 Trp Ala 140 Leu Cys Phe Leu Leu Arg Arg His Thr Leu Ala Ala Ala Asp 110 Thr Ile Ser Leo Val Ile Val Gin Glu 160 Asn Gly Trp 175 Gly Ile Ile 190 Arg Gin Gin Ile Met Val Ser Ala Arg 240 Pro Ser Val 255 Asn Ser Met 270 Pro Lys Phe Pro Gly His Leu Gly Arg 320 Ser Asp Gly 335 Page WO 01/36471 WO 01/647 1PCT/USOO/3 1509 <400> atgaacaaca atttacatcc ttaacaaaaa aacttacttg tgggaatatc gcaagtatgt ttaatgcaaa catttactga ggagttgtac gaaggagaag attgcaggtc tactactctt gatttgactt tgcttccttc tgtcagcaat agaagtaqca tataatctct atacaacatg tcctttgtat taggtaaaaa tgtgcagtgc aatctgctca ttgtcagtct aggattcctc aaaaatttcg tgggcataat agagcctatg tcattggaac ttgtaagcca acagttctgt cttatagtat tgaattattt cagaccccat ttacaaagtc tattcaacca tgttggtgtt aacatcaacg catgcctttc atgcagagtg cttaatttta gcaagagact ccaqcccaac cattccagtt ctacaatcgg cacatttatt tctgagaaaa gaaaagacat ttttaaaccc aatagaaaca tatatttctt taattcagca tctatgatct tttggaaaca cacatctacc atgagtatct gtcaattttc agttggattg acttcatgct tttgctagaa accgta tact cagatggaac ggattttcct ataagaacct cttttggtca at t ttt tat g aaaaacattc ttattagata catatgcaat cttcCatggc ctctctctca tgtcacacct atttcctgaa tgggaactct cca ta agccg atgaqaaaat aactatgcat actcagtcat taggagccat ttttagtagt gtacgtccat tccagattct ttctacacca tcacct gt ct aaacattcaa catatggttg tttaccaatc atggatattt tgtgactgca aggtttccaa atccatgcat ctatgctacc attttatggc ttacatatgg agaggctaca gatctctcag actaacatca tatggagaaa actaatagtt aagagataac tgcttcggcc gaagacacta a 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1011 <210> 26 <211> 336 <212> PRT <213> Homo sapiens <400> 26 Met Asn Asn Asn Thr 1 5 Thr Cys Ile Gin Ser Met Ile Ser Ser Met Ala Leu Pro Asn Thr Leu Ser Thr His Cys Ser Ala Ile Tyr Ile Leu Cys Ile Val Gly Val Phe Gly Lys Lys Thr Gin Trp Ile Leu Tbr Lys Ile Ile Tyr Leu Met Pro Phe His Leu Val Thr Asn Leu Leu Val Ser Ile Tyr Leu Lys Gly Phe Trp Giu Tyr Gin Ser Ala Gin Cys Arg Met Phe Vai 105 Val Asn Phe Leu Gly Thr Ser Trp Leu Ser Met His Aia Ser 100 Ser Leu Leu Ile Page 26 WO 01/36471 WO 0136471PCTUSOO/31509 Ile Ala Ilie 115 Glu Thr Thr Ser Arg Tyr Ala Thr Leu Met Gin Lys Asp 125 130 Lys Phe Arg 145 Ser Cys Tyr Giu Lys 135 Gin Pro Asn Phe Ala Ile Phe Tyr Arg Lys Leu 155 Pro Val Thr Gly His 140 Cys Ile Val Tyr Ser Ser Gin Leu Leu Lys Tyr Ile Trp 160 Tyr Ser Val Gly Ile Glu Vai Val Leu Giu Ala Thr 180 Leu Gly Ala Gly 165 Ile Ile 175 195 Phe Ilie Gly Glu Gly Giu Giu Met Ile Ser Gin 200 Ser Phe Leu Val 215 Ala Gly Leu Cys Tyr Asn Arg Gin Met 190 Gly Thr Thr Tyr Ser Phe Phe Val Leu Thr 210 Val Ser His Leu Arg Ile Arg Thr Cys Ile Met Giu Leu Thr Tyr Val Lys Arg Leu Val Ile Gin Ile 255 Ile Phe Leu Leu Ilie Tyr Val Leu 275 Giu Thr Lys 290 Aso Pro Ile Phe Leu Pro Ser Ile Phe Lys Gin Arg Asp Gin Gin Leu Tyr Leu Ile Ser Ser Thr Asn Ile Leu Ile Phe Leu 310 Phe Thr Lys Leu Ala Ser Leu Leu Asp Lys Lys Lys Thr Asn Leu Ser Asn Ser Met Gin Ser <210> 27 <211> 1014 <2i2> DNA <213> Homo sapiens atgaatgagc tttggaaatt ggca ttate t aaaatgagac ctgtatctga tttggagatt atcctcttcc tgcttttcca t cac tgg tag cactagacta qcactgatga tcctcgtggg cttggaagag ccaqectec tcatgtgtaa tcacctgttt ttcacaaaac ctgtcattcc tttagcaaat aaacatccca atttccaggc cagcaccatc cttcctgatt gtttatccgc cagcatcttc tcgatgtgca gatgaccttc gcttctgatt ctcaagatgc aatgcagtag attatgctga cactactatg tagcttec cgctactgtg gttgtagcct ttgat cacat tccccgatta actacctccc tgatatccac acctggcctg ccagtggcga atttcaacct tgatcattca gtgctgtggt caaccaacag Page 27 tgcagctgct tgttatttat ttacattttc cacagatctg aaactggatc gtatagcagc cccaatgagc gtggatcatt gaccaacaga WO 01/36471 WO 0136471PCTUSOO/31509 tcagcctgtc attttgactg attatccaca aggctaacca agggtcattc catgaagctt ctatatgtgg gtaagcggga tcgacctcac caactacttt ctctgaccca ttctgctact ggatcgaatc acatcgtttc tggtcagcga accttgagca cagttcggat ctgcctcccc tggactgcaa ccttgcattt tcgcctgctt tagaccatta caactttcag agcaaagaaa gaactcaata ttggtgatag actgacagct tacgtatgtt tcaatcagtt gctgctctga caggctgtct attagttact ctattaagtg gtacaacctg tgacactttg ctataccacg gccttaagca gaaagcacga ttttaccctt ccatatcttg gttccattga gaatcagatc acacctttgg taacctgtta gctcaacagt gagatgcaaa caaacaaccc ttga 600 660 720 780 840 900 960 1014 <210> 28 <211> 337 c212> PRT <213> Homo sapiens <400> 28 Met Asn Glu Pro Leu Asp Tyr Leu Ala Asn Ala Ser Asp Phe Pro Asp Tyr Ala Ala Met His Tyr Pro Gly Asn Phe Gly Asn Cys Thr Asp Glu Asn Ile Pro Leu Lys Val Gly Phe Pro Val Ile Gly Ile Ile Phe Leu Ala Val Val Tin Tyr Ile Lys Met Arg Pro Lys Ser Ser Thr Met Leu Asn Ala Cys Thr Asp Leu Tyr Leu Tin Glu Asn Trp Ile 100 Phe His Pho Asn Pro Phe Leu His Tyr Tyr Ala Ser Gly Gly Asp Phe Lys Phe Ile 110 Cys Phe Ser Leu Tyr Ser Leu Phe Leu 115 Ile Phe Arg Tyr Cys Val His Pro Met Phe Ser Ile 130 His Lys Thr Arg Cys Val Ala Cys Val Val Trp Ile Leu Val Ala Pro Met Thn Ile Thr Ser Thr Asn 175 Arg Thr Asn Asn Thr Ile 195 Ser Ala Cys Leu Thr Ser Ser Asp Glu Leu 190 Thr Phe Cys Lys Trp Tyr Asn Leu Thr Ala Leu Pro Leu Val Ile Val Thr Leu Cys Tyr Thr Thr Ile Ile His Thr Page 28 WO 01/36471 WO 0136471PCTfUSOO/31509 His Gly Leu Asp Ser Cys Lys Gin Val Cys Leu Thr Ile Leu Leu Ala Phe His Ile Leu 260 Ser Cys Ser Ile 275 Val Ile Arg Ser Arg Leu Lys Ala Arg 240 Phe Leu Pro 255 Leu Ser Ile 270 Val Ser Arg Tyr Val Val GiU Asn Gin His Giu Ala Tyr Ile 285 Gly Asn Leu Leu Leu 300 290 Val Scr Ala Ala Leu Asn Asp Asn Phe Ala Val Cys Thr Val Arg Cys Ser Gly Asn Gin Ala Lys Ser Tyr Ser <210> 29 <211> 993 <212> DNA <213> Homo sapiens <400> 29 atggatccaa ccaccccggc gcccttcttc tgctttgtgg gccctggtcg ggctggtagg aggaacqcct ictctgtcta ttccagatta taaattgcct ttcectagct tcttcaccac agcaccgtca gcaccgagcg cgccccagac acctgtcagc agcatcttgg aagggaagtt cagacatttg atttcatcac tccagtctgg ccctgctggt ctgtacctga ccatcctgCt attcagtggt tcctaatatt catccagttt cagttgtcct ttcgtgggct cttttaggaa cagagggctc tgcaggacat ctggggaaca caagqagacc aaacgggttt cgtcctcagc ggtgtacctc tgtgatgacc ctgcctgtcc ggtcgtgtgt ctgtggcttc tqcagcgtgg caggatcctc cacagtgctg atggatctgg gtcatctctt gcagtggcgg tgctgaggtg gaaagtacaa ctgatcccgg gtgCtctggc ctggccgggg agtaacttct tgtgcctacc gtcctgtggc gtcctgCtct ttatttagtg ctgatttttt tgtggctcca gtgttcctcc aaggattctg aacagcagtg ctgcagcagc ga tcac ag tg cagtgaatgg tcttcctgat tcctgggctt ccgacttcct tctgttccat ttgcaggcct ccatctggta gggCcctgtc atggtgactc tattcatggt ggggtctgcc tctgcggcct atgtcttatt ccaaccccat cgatcctcaa aaggatgctt aaatgaccaa ccttttcatt Cccatgcgc cttcCtctgc ctccatcaat gagcatgctg tcgctgccgc cctactgctg tggttggtgt tctctgtggg actgaccagg gCcctttggc ttgtcatatt catttacttc gctggCtctc ccgtcagggc accccggaga tgtcgagaag cagtctggtg tag 993 Page 29 WO 01/36471 WO 0136471PCT/IJSOO/31509 <210> <211> 330 <212> PRT <213> Homo sapiens <400> Gly Thr Leu Cys Ala Leu Phe Arg Gly Ala Tyr Leu Phe Thr 105 Ser Thr Tyr Arg Leu Trp Gly Phe 170 Phe Ile 185 Ser Ser Pro Leu Leu Leu Ile Trp 250 Val Val 265 Phe Val Val Asn Leu Ile Gly Asn Ala Phe Leu Cys Cys Ser Cys Ala Arg Cys Arg His Leo Leo 160 Gly Asp 175 Leu Ile Val Arg Leu Thr Phe Gly 240 Val Leo 255 Asn Ser Lys Gin Trp Arg 290 Leu Gin Gin Pro Ile Leu Lys Leu Ala Leo Gin Arg Ala Leo 295 300 Page WO 01/36471 PCTIJSOO/31509 Gin Asp Ile Ala Gio Val Asp His Ser Glu Gly Cys Phe Arg Gin Gly 305 310 315 320 Thr Pro Glu Met Ser Arg Ser Ser Leu Val 325 330 <210> 31 <211> 1092 <212> DNA <213> Homo sapiens <400> 31 atgqgccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120 tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc tgctggcggc gctggacatg 180 cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa 240 gtcattggct tcctggacac cttcctggq tccaacgcgg cgctgagcqt ggcggcgctg 300 agcgcagacc agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360 cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420 gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480 ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540 ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc 600 agccactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660 caccccagtg tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720 aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc 780 aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agtggggcat cctcagcaag 840 tgcctgacct acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900 ttccgccaag tcctggccqg catggtqcac cggctgctga agagaacccc gcgcccagca 960 tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020 ccgcgcccag cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080 cagacacact ga 1092 <210> 32 <211> 363 <212> PRT <213> Homo sapiens <400> 32 Met Gly Pro Gly Giu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu 1 5 10 Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr 25 Page 31 WO 01/36471 WO 0136471PCTIUSOO/31509 Ala Glu Leu Gly Gly Val Ile Gly Ala Ala Tyr Ala 115 Ala Trp 130 Trp Leu Pro Glu Ala Val Gin Val 195 Thr Met 210 Gin Arg Lys Ile Val Met Gin Trp 275 Asp Pro 290 Ala Gly Thr His Lys Arg Glu Asn 355 Leu Arg Thr Arg Leu Leu Arg Gly Leu Asp Ser Ala Arg Leu Gin Ser Tyr Ser 150 Glu Arg 165 Phe Val Arg Vai Ala Leu Leu Ile 230 Ile Ala 245 Arg Leu Ile Leu Thr Tyr Val His 310 Ser Ser 325 Pro Arg Ser Cys Gly Val Leu Asp Ser Ala Ala Ser 90 Leu Ala Tyr Ala Ser Gly Ala Ser 155 Ala Ala 170 Ala Val His Cys Ala Asp Arg Arg 235 Phe Leu 250 Val Pro Leu Thr Arg Arg Leu Val Asn Pro Phe Thr Gly Ala Cys Ala Ala Leu Gly Phe Pro 110 Leu Leu Leu 125 Ala Leu Gly Ser Leu Arg Thr Ala Thr 175 Cys Leu Thr 190 Arg Met Asp 205 His Pro Ser His Arg Ala Cys Phe Ala 255 Val Thr Val 270 Ser Lys Ala Phe Arg Gin Pro Arg Pro Val His Gin 335 Gly Ser Val 350 <210> 33 <211> 1125 Page 32 WO 01/36471 WO 0136471PCTUSOO/3 1509 <212> DNA <213> Homo sapiens <400> 33 atgcccacac tcaatacttc ggcagtgtgc tgagtgctga atggttgccc tggcctatgg ctgtgggtac tgagtaactg ttcaacctgg ctctggcgga tcggcactgg actttcactg actgtcctca acgtctatgc tgggtggtgg ccatggctgc gccaccctgg cagtgtgggc gtggagggtg aggtgtgtgg ctgggggcct accaqctgca accaccagct acctgctgct agcagggtcg tggcccgctc cccaaccatg tggtcactct agtactttct atactatcca aatagctgcc tcaaccctgt gcaggcacct tcagggatct caggtggccc taaagcaggt ccttctaccc tgctcaccaa <210> 34 <211> 374 <212> PRT <213> Homo sapiens <400> 34 tgcctctcca tgatgctccg gcttgtgggg tgcccggaga cctgggactg gcccttcgga cagcaicttc ggggccaggc ggcggctgcc tgtgcgcctt qagggtqqtg gctggccttc tgtccgcatc ctggggtgtc gacgtatgtc qctgtactgt gcggtcgagg aggcaggcgg cctggacaga eccacattct atgcctgtca.

gccattggct gcccctggcc gcactcactc ggtgCcctct Ctcatcacag acccacctct ctggtgacgg tgcctgctgc ctggctttca ctgcagcggc ctggtggctt ctggtgaagt ttccctgtca ctcctgaggc ctgtggccc tgggtcgcaa gggacacccg tctgggccaa aattcctagc tgctgggaaa caccttcaga tcCccttttg gcaagatggt cgctgagcgt cacicttctg tgcccacagc gtttccccag tggtgccctt ggcaacggcg ccttcttcct ttgacctggt ctacttgctt qqgagccccg agggcggagg gcaacccccg ggtga tgcctccgga cctgaggctc tttggcggtq caccttcgtc ggcagccgag tctgacggcc tgctcgctac ggcccgaata tgtcttcggg caggtactgg gggcgtcatc gcggcaggac ctgctggttt gccctggaac ggcacacagc gcaggctctg ctgggtgcaa ggagagccgc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1125 Met Pro Thr Leu Asn 1 5 Thr Ser Ala Ser Pro Pro Thr Phe Asn Ala Ser Val Lys Phe Gly Gly Ser Val Leu Ser 25 Ala Asp Asp Ala Val Ala Leu Ala Phe Trp Ala Pro Met Pro Tyr Gly Leu Leu Ala Leu Arg Leu Met 40 Val Gly Ala Ile Gly Leu Ser Asn Cys Ala Arg Arg Gly Asri Leu Ala Leu Trp Val Leu Ala Pro Gly Pro Ser Asp Thr Phe Page 33 WO 01/36471 WO 0136471PCT/USOO/31509 Phe Asn Leu Ala Leu Trp Ala Ala Giu Ser 100 Leu Cys Lys Met Val 115 Ile Phe Leu Ile Thr 130 Met Ala Ala Gly Pro 145 Ala Thr Leu Ala Val 165 Ala Val Phe Gly Val 180 Leu Arg Phe Pro Ser 195 Val Val Leu Ala Phe 210 Leu Leu Leu Leu Ala 225 Ser Arg Val Val Ala 245 Leu Cys Trp Phe Pro 260 Lys Phe Asp Leu Val 275 Tyr Val Phe Pro Val 290 Asn Pro Val Leu Tyr 305 Ala Gly Thr Phe Arg 325 Gly Trp Val Gin Gin 340 Ala Ser Asn Pro Arg 355 Asp Arg Gly Thr Pro 370 <210> <211> 1092 <212> DNA <213> Homo sapiens Ala Asp Leu Gly Leu Ala Leu Thr Leu Pro Phe 90 Pro Phe Asn Val 125 Tyr Trp 140 Phe Trp Val Thr Val Arg Tyr Gin 205 Ile Thr 220 Arg Arg Val Ala Trp Gly Tyr Thr 285 Ser Asn 300 Pro Arg Trp Pro Gly Arg Leu Leu 365 atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg Page 34 WO 01/36471 WO 0136471PCTUSOO/31509 ttccgagatg acttcattgt gggcttctgg gcaatggcct tccagccgga ttttcctgtt cccttcctga tggacaacta cggctgatgc tcttcatgtt gtggcggtag acaggtattt aatcggacag cagccatcat cacctcctga agaagaagat agcatctgcc ataccttcca ctgggcatca tcctgttctg gaccggcatg ccaagatcaa gtcatctgct tccttcccag tcgggcacgc agaattgtga agcttcacct acatgaacag tttcccaact tcttctccac ccagataata accgcagcac gctccagagg cgttaatggc acctctcctt aa <210> 36 <211> 363 <212> PRT <213> Homo sapiens caaggtg ttg tgCcctgtgg caacctggca tgtgaggcgt ggctatgaac ccgggtggtc ctcttgcctt gccgatccag gtggcacgaa ctcagccaga gagagccatc cgtggttgtg agtgtaccgc catgctggac tttgatcaac gagcgtcgag caactccggt ccgccggtgt attttctgtt gtggctgact tgggactgga cgccagggca catccccacc ctgtggggca aatggcggtg gcCatgttcc attatctgga accttcatca cggatccgca tcggtggacc cccgtggtgt cgctgcCtcc ctcacagggg gagccatgga tggggctgga tccacctcaa ttctactgat agtttgggga gcatcatctt acgccctgaa tcactattgg caaatttgtg tcctggagtt gcctgcggca tggtggtggc tcttctggct tggcgttctt actacttctc agaggaagat accccaacaa gcccctctta gtttatcttc gtcctggaaa catctgcctg catcccttgc cctcacggtg caagatctcc cctgacagtc cagcagcttc cttcctgcc gagacaaatg catcgtcttt cctgcacact tatcactctc cagcccatcc gacaggtgag aaccagaggc tctggqccca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1092 <400> 36 Met Asn Arg 1 Asn Cys Cys Val Leu Gly Leu Trp Ile His His Leu Gin Asp His Phe Leu Glu Ile Asp Lys Lys Phe Arg Asp Asp Ile Val Lys Vai Glu Phe Ile Phe Giy LeU Leu 40 Gly Asn Lys Ser Leu Pro Pro Gly Leu Ala Ser Arg Ile Phe Cys Phe Leu Lys Ser Trp Leu Phe Asn Leu Val Ala Asp Phe Leu Leu Ile Ile Trp, Asp Trp Lys Cys Phe Leu Met Asn Tyr Val Arg Arg .90 Phe Met 105 Phe Giy Arg Gin Asp Ile Pro Arg Leu Met Leu Leu Ala Met Page WO 01/36471 WO 0136471PCTLJSOO/31509 Giy Ser Ile Ile 115 Val Val His Pro Phe Leu Thr His His Ala 135 Cys Leu Leu Val Ala Val Asp Tyr Phe Arg Arg Thr Ala Asn Lys Ile 130 Ala Ile Ile Ser Trp Gly Ile Ile Gly Leu Thr Vai 160 Gly Gly Ala Asn Leu 175 Leu Leu Lys Met Pro Ile Cys Ser Ser Phe 180 Phe Leu Leu Giu Ile Cys His Phe Gin Trp His Phe Phe Leu Leu Gly Ile Ile Glu Ala Met 190 Phe Cys Ser Arg His Ala 195 Ala Arg Ile Ile Trp Ser Gin Arg Gin 210 Lys Ile Lys Arg Ala Ile Thr Phe 230 Ile Met Ala Ile Val Ile Cys Phe Pro Giy Leu Leu His Asp Leu Ala 275 Leu Asp Pro Ser Val Val Val 250 Thr Gin Asn Cys 265 Thr Leu Scr ?he Ile Arg Ile Phe Trp 255 Giu Val Tyr Phe Ile Thr Tyr Arg Ser Vai 270 Asni Ser Met Pro Asn Phe 290 Phe Ser Vai Val Tyr Tyr 295 Leu Ile Asn Arg 310 Ser Ser Pro Thr Cys Leu Gin Met Thr Gly Asp Asn Asn Ser Thr Ser Val Thr Gly Asp Pro Asn 335 Giu Pro Lys Thr Arg Trp Scr Pro 355 Pro Giu Ala Leu Met Aia Asn Ser 345 Thr Ser Pro Tyr Leu Gly Pro 360 <210> 37 <211> 1044 <212> DNA <213> Homo sapiens <400> 37 atgggggatg agctggcacc ctcatcagca agacaccctg gacctcaggg tgcccagctc gcagccacac tggctgtcag cgacaggagc cccactacct ctcctccaca tgctcatctc ttgCcctgtg catgccccaa catgctgtac ccccctgctg gctcccggct ctccagcagc ggcactacag gcagccagca tggcttttcc ctggtgacca aacatcctgc ctgggtggct cttggccggc acacttcctt ttccctcaag tcctgcggaa tctcagacct gggagctggg Page 36 cctgatccag gggcctgggg cctgctggct ccaacggctg ggcctacatt ccgcatqgcc WO 01/36471 WO 0136471PCTIUSOO/3 1509 tgtggcattc gccattgtgc tcccatgggg acattcctta tacatcctac tacacctcca aggatctatg ggcaccctgc tccctggaca ctggcagcta ctgctccgct caggccafct tcactgatgc tgcacaccta ctgcctggaa tttggctcag caccaagcat ttctgtgcgt cagaggccaa tgatccactc tggtgctgac acagtgaggt accggcagct ttaccatttc tgtctttgCC cctggcagtc ggcagtggcc caagtggcag gggcacccag tctgttcctc gacttcaggc agtgctgatc caggtaccac actcatgatg gttgggcatg ctag gcct gcacca atccatccac ctcatctggc gatgcccagc ccgggatgtg tgcacagctc atctgggggc acattgtacg cacattgact cttccccgtg gtccggggcc gcaccatcct tgcgctacct tggtggcctg tggaggagca gcctcctggt tcattgccaa agggctattc tgagcacagg ctgggactca ccatgctccc acctcccatc gtccttcacc ctccttcatg ctgcttcccc aggagcttca cattgttacc ctgtttctgg ccgggccagg ggtggtgttc cacatggctc atacctgtac caggaggcac 420 480 540 600 660 720 780 840 900 960 1020 1044 <210> <211> <212> <213> 38 347

PRT

Homo sapiens <400> 38 Met Gly Asp 1 Ala Leu Ile Ser Asn Thr Glu Leu Ala Pro Cys Pro 5 Gin Leu Ile Ser Lys Thr Gly Thr Thr Cys Met Pro Ala Prp Pro Gin Ala Ala Ser Ser Met Ser Leu Gly Leu Asp Leu Arg Val Leu Tyr Trp Leu Phe Leu Ser Ser Leu Leu Ala Ala Thr Leu Val Ser Pro Leu Leu Val Thr Ile Arg Asn Gin Gin Glu Pro Leu Leu Pro Asn Ile Leu Leu Arg Leu Ser Asp Leu Gly Leu Ala Tyr Gly Trp Glu 115 Phe Ala Ala Leu Leu His Met Ser Ser Ser Ser Gly Arg Met Gly Ile Leu 110 Asp Ala Val Ile Val Leu 130 His Thr 145 Ser His Cys Thr Ser Thr 135 Leu Ala Val Ile 150 Leu Ser Phe Tyr His Pro Gly Ala Ala 165 Trp Lys Ala Val Leu Arg Tyr Leu Ser Phe Met 155 160 Ala Leu Ile Trp Leu Val Ala 170 175 Page 37 WO 01/36471 WO 01/647 1PCTIUSOO/31509 Cys Cys Phe Gin Leu Giu 195 Thr Gin Pro Thr Phe Leu Ile Trp Leu Ser Lys Trp Gin Asp Ala 185 190 Gin Gly Ala Tyr Ile Leu Val Ile Val Gly Cys Gly 210 Leu Cys Pro Pro 205 Thr Tyr 220 Ala Asn Trp Gly Ser Met Gly Thr Ser Ile Cys Phe Trp 240 Gin Gly Tyr Val Leu Phe Thr Ala Leu Ile Tyr Ala Lys Thr Ser 255- Ser Arg Ala Tyr Val Ser 275 Tyr His His Thr Leu Leu Ser Val Leu Giy Vai Val Leu Asp Met Ile Thr Leu 270 Leu Thr Arg Ala Ala Asn Ile Asp Se 290 Ser Giu 305 Leu Leu Ser Arg r Gly 295 t Leu His Thr Trp Val Leu Met Arg Tyr Arg 325 Arg His Gin 340 Pro Arg Ala Pro Tyr Leu Leu Leu Giy Met Val 330 Ile Ser Arg Gly His Ala Ile Phe <210> 39 <211> 1023 <212> DNA <213> Homo sapiens <400> 39 atgaatccat ttcatgcatc aataaagagt ttgcttatca attgggatta tctgttcaac agatccagga aaaaaacagt gtccacatag ttggaatgcc tttggggggc ctctctgcac agtgccatca tgactgtaat ctgacacgtt ggagaacaag tcctttatcc tggcattgcc gttgagagtt gtgcttttga ttgacgataa caactttttt ttatgctata cttgggagat ttgttggaac aactgccagt agctggtt ccctgacatc ttttcttattcatcatcaca gagtgtggac gtacaagacc tgtctgggtc tttgacatcc tttccctcta gtatcaacag acctctgccg gtggtagata ggcaacatcc tatatctgca caccaatggg tccctggata aggtactttg atccggatca tactcgaagg cctgacgatg cccttgattt aataaggatg aacttttaaa cagtcatcct tcattgtatt acctggctgt cccgaggggg cttgtaacca ccctcgtcca atttgggcct tcatcaaatt tactctggta tggtgtgcta ccagatgctg caaatcctgg cccttccatj cactataata ggctgatttg agagtgggtg atttgcctgt accatttcga ttgggcagct taaagacggt tacactttat tattttaatt caatcccagt gtaccaaaac agagagtgat gaagttgaca aagatggtgc tggtgctggt ggtagtcttt Page 38 WO 01/36471 PCT/US00/31509 atcctgagtg ctgcccctta tcatgtgata caactggtga acttacagat ggaacagccc acactggcct tctatgtggg ttattacctc tccatctgtc tcagctatgc cagcagcagc attaaccctt ttctctacat cctgctgagt ggaaatttcc agaaacgtct gcctcaaatc caaagaagag cgactgagaa ggaaatcaac aatatgggaa acactctgaa atcacacttt tag <210> <211> 340 <212> PRT <213> Homo sapiens 840 900 960 1020 1023 <400> Pro Phe Ser Trp Val Ile Gly Asn Val Pro Ile Val Trp Val 100 Cys Asn 115 Arg Tyr Arg Tyr Ile Leu Asp Gly 180 Leu Trp 195 Pro Leu Met Tyr Ser Cys Trp Asn Thr Ser Ala Glu Leu Leu Glu Phe Ser Met Ile Val Tyr Ile Pro Phe Gly Pro Ala Cys 120 Leu Val 135 Ile Arg Pro Val Ser Cys Leu Tyr 200 Val Cys 215 Asn Lys Val Val Thr Gly Arg Lys Asp Leu Arg Gly Ser Leu Met Ser Arg Trp Ala Ala 160 Ile Lys 175 Pro Asp Phe Phe Tyr Thr Pro Ser 240 Val Pro Lys Gin Arg Val Met Lys Leu Thr Lys Met Val Leu Val Leu Page 39 WO 01/36471 WO 0136471PCT/USOO/31509 245 Val Val Val Phe Ile Leu Ser Ala Ala 260 265 Val Asn Leu Gin met Glu Gin Pro Thr 275 280 Tyr Leu Ser Ile CyS Leu Ser Tyr Ala 290 295 Leu Tyr Ile Leu Leu Ser Gly Asn Phe 305 310 Gin Arg Arg Ala Thr Glu Lys Glu Ile 325 Lys Ser His Phe 340 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 41 cttgcagaca tcaccatggc agcc <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 42 gtgatgctct gagtactgga ctgg <210> 43 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 43 gaagctgtga agagtgatgc <210> 44 <211> 24 <212> DNA Tyr His Val Ile 270 Ala Phe Tyr Val 285 Ser Ser Ile Asn 300 Lys Arg Leu Pro 315 Asn Met Gly Asn 255 Gin Leu Gly Tyr Pro Phe Gin Ile 320 Thr Leu 335 Page WO 01/36471 PCT/USOO/31509 <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 44 gtcagcaata ttgataagca gcag 24 <210> <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> ccatggggaa cgattctgtc agctacg 27 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 46 gctatgcctg aagccagtct tgtg 24 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 47 ccaggatgtt gtgtcaccgt ggtggc 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 48 cacagcgctg cagccctgca gctggc 26 Page 41 WO 01/36471 PCT/US00/31509 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 49 cttcctctcg tagggatgaa ccagac 26 <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> ctcgcacagg tgggaagcac ctgtgg 26 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 51 gcctgtgaca ggaggtaccc tgg 23 <210> 52 <211> <212> DNA <213>- Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 52 catatccctc cgagtgtcca gcggc <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <221> misc feature Page 42 WO 01/36471 PCT/US00/31509 <223> Novel Sequence <400> 53 gcatggagag aaaatttatg tccttgcaac c 31 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 54 caagaacagg tctcatctaa gagctcc 27 <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> gctgttgcca tgacgtccac ctgcac 26 <210> 56 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 56 ggacagttca aggtttgcct tagaac 26 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 57 ctttcgatac tgctcctatg ctc 23 <210> 58 <211> 26 Page 43 WO 01/36471 PCT/USOO/31509 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 58 gtagtccact gaaagtccag tgatcc 26 <210> 59 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 59 tttctgagca tggatccaac catctc 26 <210> <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> ctgtctgaca gggcagaggc tcttc <210> 61 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 61 ggaactcgta tagacccagc gtcgctcc 28 <210> 62 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 62 Page 44 WO 01/36471 PCTIUSOO/31509 ggaggttgcg ccttagcgac agatgacc 28 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 63 ctgcacccgg acacttgctc tg 22 <210> 64 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 64 gtctgcttgt tcagtgccac tcaac <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> tatctgcaat tctattctag ctcctg 26 <210> 66 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 66 tgtccctaat aaagtcecat gaatgc 26 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> Page WO 01/36471 <221> misc feature <223> Novel Sequence <400> 67 ggagacaacc atgaatgagc cac <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 68 tatttcaagg gttgtttgag taac <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 69 ggcaccagtg gaggttttct gagcatg <210> <211> 27 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> ctgatggaag tagaggctgt ccatctc <210> 71 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 71 cctggcgagc cgctagcgcc atg <210> 72 PCTIUS00/31509 23 24 Page 46 WO 01/36471 PCT/US00/31509 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 72 atgagccctg ccaggccctc agt 23 <210> 73 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 73 ctgcgatgcc cacactcaat acttctg 27 <210> 74 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223>, Novel Sequence <400> 74 aaggatccta cacttggtgg atctcag 27 <210> <211> 22 <212> DNA <213> Artificial Sequence <400> gctggagcat tcactaggcg ag 22 <210> 76 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 76 agatcctggt tcttggtgac aatg 24 <210> 77 Page 47 WO 01/36471 PCT/US00/31509 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 77 agccatccct gccaggaagc atgg 24 <210> 78 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 78 ccagactgtg gactcaagaa ctctagg 27 <210> 79 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 79 agtccacgaa caatgaatcc atttcatg 28 <210> <211> <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> atcatgtcta gactcatggt gatcc <210> 81 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence Page 48 WO 01/36471 WO 0136471PCTUSOO/3 1509 <400> 81 ggggagggaa agcaaaggtg gtcctcctgg <210> 82 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 82 ccaggagaac cacctttgct ttccctcccc <210> 83 <211> 1356 <212> DNA <213> Homo sapiens <400> 83 atggagtcct cacccatccc caaaccccag gtccctctac tcggaatctg tggccctctt gccgctgtga tggccgtgat cacctctgcc tggtggacct agctctgccc tctttgacca ctgagcgtgt gctttgtcag tactattacg tagtccaccc tctgtgctgg tgggtgtgtg agggtctcct gggaggaagg cacagtgcct actgccagct ctgctcctca tacttgtggt cagcacgggc cgctgcccac agccgCtcca cgatggiCac gggggaggga aagcaaaggt ttgccctact tctctttcca caggtggaga gtgtggtcac tatggatgtc tcaaccggca aagccagctc cagaggagga ttcctgcagt tccttcaggg ccagtcatca tgccagtggg cttcatgctc cgccaagacg gCtggctgcc cgccot ct tt cctggccatc catgcgctac ggtgaaggcc agctcccagt ttttgtggtg ctactgcagc gtggatggag cagctcgggg ggttctcCtg cctctatgtt ctggattggu gatccggggg gctgaggctg gactggctgt gggaactctt ccactttggg gtcccggagg tggggctacg ctgctggact tgactgctgt cctqccctcc gaaaatttgt ctgaccctca tgcccctggc ggggaggtgg cctgccgcct ctctcggtgt cagccatcaa gaggtgcgca tgacgctggg ttggccatgg cttctgtgcc gtccccccag gctgttcact gtctttgctg tcctttactt atgttccgag tggcccgqt acaccccggc aacgctccga gccccccaga ccaccccaca gctgtggggg gacagttcct gccctgagtg ctcagcccat tacttttgct tcacttccaa gagctcagca agcagtttgt cctagccggg agggctccat ccttctgagt cctgggtttc gagggtccct ggatgttgct ggctggcaat cttcgtcttc catgctctcc ctacttgttt tgtggagcgc gctggtggcc agtgt tggga ccagtggagc tctgttgccc ggctgccatg atctctcagc ccggacgttt gCtctgttgg ttcaactggg ccctttcttC ctgcttcttC tgaggagaac ccgaccccta 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag Page 49 WO 01/36471 PCT/US00/31509 gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320 ctccgtcctg ccgcctcacc ccggctggag tcatga 1356 <210> 84 <211> 451 <212> PRT <213> Homo sapiens <400> 84 Met Glu Ser Ser Pro Ile Pro Gin Ser Ser Gly Asn Ser Ser Thr Leu 1 5 10 Gly Arg Val Pro Gin Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro 25 Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe 40 Met Leu L LeLeu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met 55 Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe 70 75 His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 90 Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu 100 105 110 Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu 115 120 125 Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val 130 135 140 Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala 145 150 155 160 Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val 165 170 175 Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro 180 185 190 Pro Gly Cys Ser Leu Gin Trp Ser His Ser Ala Tyr Cys Gin Leu Phe 195 200 205 Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile 210 215 220 Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met 225 230 235 240 Gin His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gin Arg Ser 245 250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro 260 265 270 Gin Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Lys Val Val Page WOO01/36471 PCT/UJSOO/31509 275 280 285 Leo Leu Ala Val Gly Gly Gin Phe Leu Leo Cys Trp Leo Pro Tyr Phe 290 295 300 Ser Phe His Leo Tyr Val Ala Leo Ser Ala Gin Pro Ile Ser Thr Gly 305 310 315 320 Gin Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330 335 Asn Pro Phe Phe Tyr Gly Cys Leo Asn Arg Gin Ile Arg Gly Glu Leo 340 345 350 Ser Lys Gin Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Gl Leo 355 360 365 Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gin Phe 370 375 380 Leu Gin Gly Thr Giy Cys Pro Ser Giu Ser Trp Val Ser Arg Pro Lou 385 390 395 400 Pro Ser Pro Lys Gin Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly 405 410 415 Gin Ile Ala Glu Glu Thr Ser Glu Phe Leo Gio Gin Gin Leu Thr Ser 420 425 430 Asp Ilie Ile Met Ser Asp Ser Tyr Leo Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu Gbu Ser 450 <210> <211> 28 <212> DNA <213> Homo sapiens <400> caggaaggca aagaccacca tcatcatc 28 <210> 86 <211> 28 <212> DNA <213> Homo sapiens <400> 86 gatgatgatg gtggtctttg ccttcctg 28 <210> 87 <211> i041 <212> DNA <213> Homo sapiens <400> 87 atggagagaa aatttatgtc cttgcaacca tccatctccg tatcagaaat ggaaccaaat ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120 ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180 Page 51 WO 01/36471 WO 0136471PCTIUSOO/31509 tatgttttcc gccatttcag ggctccaatt aacatgtaca gttcacccct atcatatgga aacggcagtg atgaactata tatCt gctg a tctcacagga ctgccctatc gacagactgc aatcctctgc agaaaaggcc agaaaggaaa tgcagcctta atctcctgtt ggatatttgg gcagtattta ttcggcttct tccttatcat tcacatcatg ttgccttggt tcattcgggt aggcaaagac acacactgag ataaagcttt tctattactt atccacagaa caagagtata taagaagtcc cataagcacg agacctggcc t tt cctga cc gcatgtcacc ggcttcctca cttagagctg ggtgggctgc tctgttaaaa caccatcatc gaccgtccac ggttatcaca tgctggggag ggcaaagaca aca tctgtga cttcccttca tgcaggatta gtgctgagtg agcatcagga ataatgctcc aatctctata ctgctgccat gtggaggtcc atcaccttga ttgacgacat ctggccttgg aattttaagg aagtgtgttt acgttttcat gggctgacta tgtcttattc ttgtgcgttt gtgcctggat tggacagtgg aaattgctaa ttttcacact cagaatcggg tcatcttctt ggaaagtggg cagcagccaa acagactaaa tccctgttag gctaaatctg ttatcttaga cttgtatgtc cctggcaatg cctctgtggg ctctgagcag gctgcagacc cagcatctgt gctgcgggtt cttgtgtttc tttatgcaaa tgcctgcttc gtctgcactc tgtqtggttg 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1041 <210> <211> <212> <213> 88 346

PRT

Homo sapiens <400> 88 Met Glu Arg Lys Phe Met Ser Leu Gin Pro Ser Ile Ser Val Ser Glu is Met Glu Pro Ile Glu Asn Phe Phe Trp Gly Thr Phe Ser Lys Arg Giu Phe Asn Asn Ser Arg Asn Cys Thr Leu Ile Ile P~ro Ile Val Gly Val Leu Gin Pro Giy Asn 55 Thr Ser Gly Leu Ser Ile Tyr Val Phe Leu Tyr Lys Lys Vai Asn Val Phe Met Pro Phe Ile Ser Asp Leu Phe Ile Ser Thr Leu Tyr Tyr Leu Arg 100 Ile Met Ser Tyr 115 Leu Thr Val Leu 130 Gly Ser Asn Trp Ser Leu Tyr Vai 120 Ser Val Val Arg 135 Phe Gly Asp Leu Leu Asn Leu s0 Arg Ala Asp Ala Cys Arg 110 Ile Tyr Phe His Pro Phe Met Tyr Ser Phe Leu Ala Met 140 Page 52 WO 01/36471 PCTIUSOO/31 509 Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile Leu Cys Gly 145 150 155 160 Ile Ile Trp Ile Leu Ile Met Ala Ser Ser Ile Met Leu Leu Asp Ser 165 170 175 Gly Ser Giu Gin Asn Gly Ser Val Thr Ser Cys Leu Giu Leu Asn Leu 180 185 190 Tyr Lys Ile Ala Lys Leu Gin Thr Met Asn Tyr Ile Ala Leu Val Val 195 200 205 Gly Cys Leu Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile 210 215 220 Ile Arg Val Leu Leu Lys Val Glu Val Pro Giu Ser Gly Leu Arg Val 225 230 235 240 Ser His Arg Lys Ala Lys Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe 245 250 255 Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His Leu Thr 260 265 270 Thr Trp Lys Val Giy Leu Cys Lys Asp Arg Leu His Lys Ala Leu Val 275 280 285 Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala Cys Phe Asn Pro Leu Leu 290 295 300 Tyr Tyr Phe Ala Gly Glu Asn Phe Lys Asp Arg Leu Lys Ser Ala Leu 305 310 315 320 Arg Lys Gly His Pro Gin Lys Ala Lys Thr Lys Cys Val Phe Pro Val 325 330 335 Ser Val Trp Leu Arg Lys Glu Thr Arg Vai 340 345 <210> 89 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 89 ccagtgcaaa gctaagaaag tgatcttc 28 <210> <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> gaagatcact ttcttagctt tgcactgg 28 Page 53 WO 01/36471 WO 0136471PCT/USOO/31 509 <210> 91 <211> 1527 <212> DNA <213> Homo sapiens <400> 91 atgacgtcca cctgcaccaa.

ctctccaaaa tgcccatcag ttcctcgccg cctctttcgt cagctgctgc aggtgaccaa atttcgctcg tggccccctg agccacttct gcacggccct accattgtcg tggtgtcagt tccaagatga cccagcgccg ctgcagagca cicctccact ctctgctcca tgatctgggg atcgtcattc cactgattgt aggcagcatg ctctgctgta tgtgtggaga atgaggatga.

gagtttcgcc gccagcatga gacggcagcc tgaaggccaa aggggcagcg aggaggtcag aaggaaggca qcaccaaagt gtcaaccagt gcagcattga aatttcagtg aggatgacgt cgtaacagca acagcaaccc atcttcatca tcattttctc ctggccgtgt gggtggatgt tggcttttct tcctgcagtg attaagaagg aaatccagga qaagatagcc acccagacct tcctacgatt ctgctacttt <210> 92 <211> 508 <212> PRT <213> Homo sapiens cagcacgcgc cctggcccac cggcaacata ccgttttatc ggtggtggcc ggttagcctc ggatcgctac cggttacctg ctacggctgg ggccagcccc catgattgcc caatgtcaag agagggagca aggtgaggtc ggaaggaagc agagagcagc tgaggaqaac cttgggtgaa cgaggcagtg tcctctgccc ctatgtgcta cgaaacccag ctgcatccac catgctgaag gcccggaaca tccttga gagagtaaca ggcatcatcc gtgctggcgc tttaacctcc acctctgtgc acccacctgt ttgtccatca ctcctctatg ggccaggctg agctacacta tgctactccg agacacagct gagaagaagg aaggccaagg acggggacca acggtggcca agcatgaagg gatgacatgg aacatcccgg aggtgctacc tccctggggc gtaccccagt ccctatgtct aagttcttct gagqgtggga gcagccacac gctcaaccgt tagtgttgca tcgtcaccga ctctcttctg tcgccttcgc tccaccctct gcacctggat cctttgatga ttctcagcgt tggtgttctg iggaagtgcg aggagttcca agggcagaat gtgagagtag gcgacggcag cagacaaggg agtttggtga agagcctccc agtgcaaagc cctactgctt gggtgatcac atggctacat gcaaggaaaa ctgaaggcaa gtgcatgccc gctggttatc gcgcaagccg cctgctgcag gcccctcaac cagcgtcaac ctcctacccg tgtggccatc gcgcaatgct ggtgtccttc tgcagcccgg agtcaaggac ggatgagagt ggaagccaag tgtagaggcc catggagggt tcgcacagag agacgacatc acccagtcgt taagaaagtg tttagcagtc cataatcatc gcacaagacc gcccccgaaa gattgtccct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1~440 1500 1527 Page 54 WO 01/36471 PCT/US00/31509 <400> 92 Met Thr Si 1 Thr Cys Mi Ile Arg Si 3! Asn Ile V, Val Thr A: TIe Ser LT Trp Leu Arg Gin 145 Leu Glu Thr Ile Leu 225 Cys Gin Lys Gly Glu 305 Ser Thr Met Pro Ile Phe 40 Leu Gin Asn Leu Val Val Cys Thr 105 Asn Thr 120 Pro Leu Leu Tyr Tyr Gly Met Ile 185 Phe Ile 200 Phe Cys His Ser Glu Gly Arg Gin 265 Lys Asp 280 Ser Ser Val Ala Arg Glu Ser 10 Ile Ser Leu Leu Ala Ala Arg Lys Pro Leu Val Thr 75 Ala Thr Ser Ala Leu Val Ile Val Val Ser Tyr Pro 140 Gly Thr Trp 155 Trp Gly Gin 170 Trp Gly Ala Val Ile Pro Ala Ala Arg 220 Leu Glu Val 235 Ala Glu Lys 250 His Glu Gly Gly Ser Leu Val Glu Ala 300 Ser Asp Gly 315 Asn Ser Ser His Ala His Gly Ile Ser Phe Val Gly Gin Leu Leu Gin Asp Leu Leu Gin Val Pro Leu Phe Ser Leu Thr His 110 Val Ser Val Asp 125 Ser Lys Met Thr Ile Val Ala Ile 160 Ala Ala Phe Asp 175 Ser Pro Ser Tyr 190 Leu Ile Val Met 205 Arg Gin His Ala Arg Val Lys Asp 240 Lys Glu Glu Phe 255 Glu Val Lys Ala 270 Lys Ala Lys Glu 285 Arg Gly Ser Glu Ser Met Glu Gly 320 Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Page WO 01/36471 WO 0136471PCTUSOO/31 509 Giu Val 340 Gly Giu Ile Pro Pro Leu Ile Ile 405 Val Leu 420 Ile Thr Tyr Val Met Leu His Pro 485 Pro Ser 500 Asp Asp Val Glu Ser Asn Lys Val 400 Tyr Cys 415 Val Pro Cys Cys Lys Giu Pro Lys 480 Giu Gly 495 <210> 93 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 93 gccgccaccg cgccaagagg aagattggc <210> 94 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 94 gccaatcttc ctcttggcgc ggtggcggc <210> <211> 1092 <212> DNA Page 56 WO 01/36471 WO 0136471PCT/USOO/31509 <213> Homo sapiens <400> atgggccccg gcgaggcgct ctatccaacg cactggtgct tcaggcgtcc tcctggtgaa cccttcacgc tgctcggtgt gtcattggct tcctggacac agcgcagacc agtggctggc cgctatgccg gcctgctgct gcacttggct qctcgtggct ccgcccgagc ctgagcgtcc ttcgtgctgc cgctggcggt agccactgcc agcgcatgga caccccagtg tgcggcagcg aggaagattg gcattgctat aggctggcgg agctcgtgcc tgcctgacct acagcaaggc ttccgccaag tcctggccgg tccacccatg acagctctct ccgcgcccag cgtccaccca cagacacact ga gctggcgggt.

gctttgttgc tctgtcgctg gatgcgcggg cttcctggcg agtgggcttc gggctgtgcc tggctacaqc gcgcttcgca gctctgcctc caccgtcacc ctgcctcatc tgcgaccttc cttcgtcacc ggtggccgac catgqtgcac ggatgtggcc caacggctct ctcctggtga gcctacagcg ggccacctgc cggacaccgt tccaacgcgg ccactgcgct tggggacagt agcgccttcg gccttcaccg acctcgctcc atgaaggcgc cagcagaagc ctcatctgct gtgaacgccc ccgttcacgt cggctgctga ggcatggtgc gtggacacag tggtactggc ctgagctccg tgctggcggc cggcgcccgg cgctgagcgt acgccggacg cgctggcctt cgtcctgttc ccacgctcca aggtgcaccg tcgcgctgct ggcgccgcca ttgccccgta agaaggqcat actctctgct agagaacccc accagctgct agaatgattc cqtggcgctg cactcgagcc gctggacatg cgcatgccaa ggcggcgctg cctgcgaccg ctcaggcgct gctgcgcctg tgccgtgggc ggtggcacgc cgccgacctg ccgcgccacc tgtcatgacc cctcagcaag ccgccggccg gcgcccagca gaagagaacc ctgcctgcag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 10B0 1092 <210> 96 <211> 363 <212> PRT <213> Homo sapiens <400> 96 Met Gly Pro Gly Glu Ala Leu Leu Ala Leu Leu Val Leu Leu Cys Ala Val Ala Leu Ser Ala Glu Leu Ser Leu Gly His Leu Gly Val Met Leu Ser Asn Ala Met Val Leu Cys Ala Tyr Val Asn Leu Arg Thir Arg Ser Gly Val Leu Leu Leu Leu Ala Ala Leu 55 Asp Met Arg Gly Arg Thr Pro Ser Ala Pro Val Ile Gly Phe Leu Asp Thr Phe Leu Ala 75 Ser Asn Page Pro Phe Thr Leu Gly Ala Cys Gin Ala Ala Leu Ser 57 WO 01/36471 WO 0136471PCTJUSOO/31509 Val Ala Ala Arg Tyr Ala 115 Cys Ala Trp 130 Ser Trp Leu 145 Pro Pro Glu His Ala Val Leu Gin Val 195 Val Thr Met 210 Arg Gin Arg 225 Arg Lys Ile Tyr Val Met Ala Gin Lys 275 Ala Asp Pro 290 Leu Ala Gly 305 Ser Thr His Leti Lys Arg Thr Glu Asn 355 <210> 97 <211> 34 Ser Ala Arg Leu Gin Ser Tyr Ser 150 Giu Arg 165 Phe Val Arg Val Ala Leu Leu Ile 230 Ile Ala 245 Arg Leu Ile Leu Thr Tyr Vai His 310 Ser Ser 325 Pro Arq Ser Cys Asp Gin Trp 105 Arg Pro Arg 120 Leu Ala Phe 135 Ser Ala Phe Pro Arg Phe Leu Pro Leu 185 Ala Arg Ser 200 Ala Leu Leu 215 Gin Gin Lys Ile Ala Thr Aia Giu Leu 265 Ser Lys Cys 280 Ser Leu Leu 295 Arg Leu Leu Leu Asp Val Pro Aia Ser 345 Leu Gin Gin 360 Ala Val Ala Gly Gly Ala 140 Ser Cys 155 Ala Phe Val Leu Cys Gin Asp Leu 220 Arg Arg 235 Leu Ile Pro Phe Thr Tyr Arg Pro 300 Arg Thr 315 Giy Met His Asn H is Phe Pro Leu 110 Leu Leu Gly Leu Gly Cys Leu Arg Leu 160 Ala Thr Leu 175 Leu Thr Ser 190 Met Asp Thr Pro Ser Val Arg Ala Thr 240 Phe Ala Pro 255 Thr Val Asn 270 Lys Ala Val Arg Gin Val Arg Pro Ala 320 His Gin Leu 335 Ser Val Asp 350 <212> DNA <213> Artificial Sequence <220> <221> misc-feature <223> Novel Sequence <400> 97 gatctctaga atggagtcct cacccatccc ccag Page 58 WO 01/36471 WO 0136471PCT/USOO/31509 <210> 98 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 98 gatcgatatc cgtgactcca gccggggtga ggcggc 36 <210> 99 <211> 2610 <212> DNA <213> Homo sapiens and Rat <400> 99 atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct caaaccccag gtccctctac tgccagtqgg gtcccggagg tggggctacg ggatgttgct 120 tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180 gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240 cacctctgcc tggtggdcct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300 agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360 ctgagcgtgt gctttqtcag cctgqccatc ctctcgqtgt. cagccatcaa tgtggagcgc 420 tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480 tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540 agggtctcct. gggaggaaqg agctcccagt gtccccccag gctgticact ccagtggagc 600 cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660 ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt. ggctgccatg 720 cagcacgggc: cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780 agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840 gggggaggga. aagcagcagt. ggttctcctg gctgtqgggg gacagttcct gctctgttgg 900 ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960 caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020 tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt. ctgcttcttc 1080 aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140 ttcctgcagt. tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200 cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260 gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320 Page 59 WO 01/36471 WO 0136471PCT/USOO/31509 ctccgtcctg ggactagtgq aacagtaaga atcgagaagc ctgggtgctg aatgggttta ggtgagaagg attgtggccg ttcagagtgg ttctatgagc tccaacgagt aagcaggccg ggaatctttg ggccagcgcg ttcgtggtgg ctgcaggagg tctgtgatcc tcgaagattg cccgagcccg ctgagaatca gccgtggaca atgcatcttc ccgcctcacc atccgagctc cgaggacca agctgcagaa gagagtctgg acggagaggg ccaccaaagt ccatgagcaa actacattct atgccaaggc accagctgat actacgtgcc agaccaagtt atgaacgccg ccagcagcag ctctgaacct tcttcctcaa aggactactt gagaggaccc gcactgctag ctgagaacat gccaatacga ccggctggag ggtaccaagc gcgcaacgag ggacaagcag caaaagcacc cggcgaagag gcaggacatc cctggtgcc gagcgtgatg tctgtgggag cgactgtgcc aag Lgaccag ccaggtggac caagtggatc ctacaacatg cttcaagagc caagcaagat tccagagttc acgcgtgacc tggagatgga ccgccgtgtc gctgctctaa tcagcgatat ttgggctgca gagaaggcgc gtctaccggg attgtgaagc gacccgcagg aaaaacaacc cccgtggagc aacgtgccaa gatgagggag cagtacttcc gacctgcttc aaagtcaact cagtgcttca gtcatccggg atctggaaca ctgcttgctg gctcgctaca cgggccaagt cgtcactact ttcaacgact ctgcagaatt ggtcgatggg agcgcgaggc ccacgcaccg agatgaggat ctgcaaggag tgaaggaggc tggccaaccc actttgactt ttcgtgcctg tggacaagat gctgccgcgt tccacatgtt atgatgtgac aggacaacca acagatggct agaaggtcct ccactcctga acttcatccg gctaccctca gccgtgacat ccaccacact ctgcctcggc caacaaaaag cctgctgctg cctacatgtt caacagcgat cattgaaacc tgagaaccag cccacctgaa ctacgagcgc tgatgtgatc cctgacctct cgatgtgggc tgccatcatc gaccaaccgt gcgtaccatc cgctgggaaa ggatgcgact ggatgagttt ctttacctgc catccagcgc 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2610 <210> 100 <211> 869 <212> PRT <213> Homo sapiens <400> 100 Met Glu Ser Ser Pro 1 5 and Rat Ilie Pro Gin Ser Gly Asn Ser Ser Thr Leu Gly Arg Val Pro Gin Thr Pro Gly Pro 25 Ser Thr Ala Glu Ser Val Glu Val Gly Leu Arg Asp Vai Ala Ser 40 Ser Gly Val Pro Ala Leu Phe Phe Ala Ala Val Met Met Leu Leu Leu Asp Leu Ala Val Ala Gly Page WO 01/36471 PCT/US00/31509 Ala Val lie Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe 70 75 His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 90 Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu 100 105 110 Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu 115 120 125 Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val 130 135 140 Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala 145 150 155 160 Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val 165 170 175 Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro 180 185 190 Pro Gly Cys Ser Leu Gin Trp Ser His Ser Ala Tyr Cys Gin Leu Phe 195 200 205 Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile 210 215 220 Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met 225 230 235 240 Gin His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gin Arg Ser 245 250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro 260 265 270 Gin Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val 275 280 285 Leu Leu Ala Val Gly Gly Gin Phe Leu Leu Cys Trp Leu Pro Tyr Phe 290 295 300 Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly 305 310 315 320 Gin Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330 335 Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gin Ile Arg Gly Glu Leu 340 345 350 Ser Lys Gin Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu 355 360 365 Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gin Phe 370 375 380 Leu Gin Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu 385 390 395 400 Pro Ser Pro Lys Gin Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly 405 410 415 Page 61 WO 01/3647 1 PCT/USOO/3 1509 Gin Ile Ala Giu Glu Thr Ser Gin Phe Leu Giu Gli Gin Leu Thr Ser 420 425 430 Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu Giu Ser Ala Ile Ser Ala Gin Phe His His Thr Gly Leu Val Asp 450 455 460 Pro Ser Ser Val Pro Ser Len Gly Cys Arg Ser Met Gly Cys Len Gly 465 470 475 480 Asn Ser Lys Thr Giu Asp Gin Arg Asn Giu Gin Lys Ala Gin Arg Gin 485 490 495 Ala Asn Lys Lys Ile Gin Lys Gin Len Gin Lys Asp Lys Gin Val Tyr 500 505 510 Arg Ala Thr His Arg Leu Len Leu Leu Gly Ala Giy Giu Ser Gly Lys 515 520 525 Ser Thr Ile Val Lys Gin Met Arg Ile Leu His Val Asn Gly Phe Asn 530 535 540 Gly Gin Gly Gly Gin Gin Asp Pro Gin Ala Ala Arg Ser Asn Ser Asp 545 550 555 560 Giy Giu Lys Ala Thr Lys Val Gin Asp Ile Lys Asn Asn Leu Lys Giu 565 570 575 Ala Ile Gin Thr Ile Val Ala Ala Met Ser Asn Leu Val Pro Pro Val 580 585 590 Gin Leu Ala Asn Pro Gin Asn Gin Phe Arg Val Asp Tyr Ile Leu Ser 595 600 605 Val Met Asn Val Pro Asn Phe Asp Phe Pro Pro Giu Phe Tyr Giu His 610 615 620 Ala Lys Ala Leu Trp Gin Asp Gin Giy Val Arg Ala Cys Tyr Glu Arg 625 630 635 640 Ser Asn Gin Tyr Gin Len Ile Asp Cys Ala Gin Tyr Phe Len Asp Lys 645 650 655 Ile Asp Val Ile Lys Gin Ala Asp Tyr Vai Pro Ser Asp Gin Asp Len 660 665 670 Len Arg Cys Arg Val Leu Thr Ser Giy Ile Phe Giu Thr Lys Phe Gin 675 680 685 Val Asp Lys Val Asn Phe His Met Phe Asp Val Gly Giy Gin Arg Asp 690 695 700 Giu Arg Arg Lys Trp Ile Gin Cys Phe Asn Asp Vai Thr Ala Ile Ile 705 710 715 720 Phe Val Val Ala Ser Ser Ser Tyr Asn Met Val Ile Arg Giu Asp Asn 725 730 735 Gin Thr Asn Arg Leu Gin Gin Aia Leu Asn Len Phe Lys Ser Ile Trp 740 745 750 Asn Asn Arg Trp Len Arg Thr Ile Ser Val Ile Leu Phe Len Asn Lys Page 62 WO 01136471 WO 0136471PCTUSOO/31509 755 Gin Asp Leu 770 Asp Tyr Phe 785 Pro Glu Pro Arg Asp Giu Tyr Cys Tyr 835 765 Ser Lys Ile Giu Leu Ala Glu Val Leu Ala Gly Lys 780 Pro Glu Gly Glu 805 Phe Leu 820 Pro His Ala Arg Tyr Thr Pro Glu Asp Ala Asp Pro Arg Arg Ile Ser Phe Thr Cys 840 Val Thr 810 Arg Ala Lys Tyr Phe Ile 815 Thr Ala Ser Gly Asp Gly Arg His 825 830 Ala Val Asp Thr Glu Asn Ile Arg 845 Ile Ile Gin Arg Met His Leu Arg Arg Val Phe Asn Asp Cys Arq 850 855 Gin Tyr Glu Leu Leu 865 <210> i0i <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence Asp <400> 101 tctagaatga cgtccacctg caccaacagc <210> 102 <211> 34 <212> DNA <213> Artificial Sequence <220> <221> misc: feature <223> Novel Sequence <400> 102 gatatcgcag gaaaagtagc agaatcgtag gaag <210> 103 <211> 2781 <212> DNA <213> Homo Sapiens and Rat <400> 103 atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtqcatgccc: ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg cagctgctgc: aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag Page 63 WO 01/36471 WO 0136471PCTUSOO/31 509 atttcgctcg agccacttct acca ttgtcg tccaagatga ctgcagagca ctctgctcca atcgtcattc aggcagcatg tgtgtggaga gagtttcgcc gacggcagcc aggggcagcg aaggaaggca gtcaaccagt aatt tcagtg cgtaacagca atcttcatca ctggccgtgt tggcttttct attaagaagg gaagatagcc tcctacgatt gatccgagct accgaggacc cagctgcaga ggagagtctg aacggagagg gccaccaaag gccatgagca gactacattc catgccaagg taccagctga tggccccctg gcacggccct tggtgtcagt cccagcgccg ctcctccact tgatctgggg cactgattgt ctctgctgta atgaggatga gccagcatga tgaaggccaa aggaggtcag gcaccaaagt gcagcattga aggatgacgt acagcaaccc tcatittctc gggtggatgt tcctgcagtg aaatccagga acccagacct ctgctacttt cggtaccaag agcgcaacga aggacaagca gcaaaagcac gcggcgaaga tgcaggacat acctggtgcc tgagcgtgat ctctgtggga tcgactgtgc ggtggtggcc ggttagcctc ggatcgctac cgqttacctg ctacggctgg ggccagcccc catgattgcc caatgtcaag agagggagca aggtgaggtc ggaaggaagc agagagcagc tgaggagaac cttgggtgaa cgaggcagtg tcctctgccc ctatgtgcta cgaaacccag ctgcatccac catgctgaag gcccggaaca tcctgcgata cttgggctgc ggagaaggcg ggtctaccgg cattgtgaag ggacccgcag caaaaacaac ccccgtggag gaacgtgcca ggatgaggga ccagtacttc acctctgtgc acccacctgt ttgtccatca ctcctctatg ggccaggctg agctacacta.

tgctactccg agacacagct gagaagaagg aaggccaagg acggggacca acggtggcca agcatgaagg gatgacatgg aacatcccgg aggtgctacc tccctqqqgc gtaccccagt ccctatgtct aagttcttct gagggtggga 'tctgcaqaat aggtcgatgg cagcgcgagg gccacgcacc caga tgagga gctgcaagga ctgaaggagg ctggccaacc aactttgact gttcgtgcct ctggacaaga ctctcttctg tcgccttcgc tccaccctct gcacctggat cctttgatga ttctcagcgt tgqtgttctg tggaagtgcg aggagttcca agggcagaat gtgagagtag gcgacggcag cagacaaggg agtttggtga agagcctccc agtgcaaagc cctactqctt gggtgatcac atggctacat gcaaggaaaa ctgaaggcaa tccaccacac gctgcCtcgg ccaacaaaaa gcctgctqct tcctacatgt gcaacagcga ccattgaaac ctgagaacca tcccacctga gctacgagcg ttgatgtgat gcccctcaac cagcgtCaac ctccracccg tgtggccatc gcgca atgc t ggtgtccttc tgcagcccgg agtcaaggac ggatgagagt ggaagccaag tgtagaggcc catggagggt tcgcacagag agacgacatc acccagtcgt tgctaaagtg tttagcagtc cataatcatc gcacaagacc gcccccgaaa gattgtccct tggactagtg caacagtaag gatcgagaag gctgqqtgct taatgggttt tggtgagaag cattgtggcc gttcagagtg attctatgag ctccaacgag caagcaggcc 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 gactacgtgc caagtgacca ggacctgctt cgctgccgcg tcctgacctc tggaatcttt Page 64 WO 01/36471 WO 0136471PCT[USOO/31509 gagaccaagt gatgaacgcc gccagcagca gctctgaacc ctcttcctca gaggactact ggagaggacc agcactgcta actgagaaca tccaggtgga gcaagtggat gctacaacat tcttcaagag acaagcaaga ttccagagtt cacgcgtgac gtggagatgg tccgccgtgt caaagtcaac ccagtgcttc ggtcatccgg catctggaac tctgcttgct cgctcgctac ccgggccaag acgtcactac cttcaacgac ttccacatgt aatgatgtga gaggacaacc aacagatggc gagaaggtcc accactcctg tacttcatcc tgctaccctc tgccgtgaca tcgatgtggg ctgccatcat agaccaaccg tgcgtaccat tcgctgggaa aggatgcgac gggatgagtt actt tacctg tcatccagcg cggccagcgc cttcgtggtg tctgcaggaq ctctgtgatc atcgaagatt tcccgagccc tctgagaatc cgccg tggac catgcatctt 2280 2340 2400 2460 2520 2580 2640 2700 2760 2781 cgccaatacg agctgctcta a <210> 104 <211> 926 <212> PRT <213> Homo <400> 104 Met Thr Ser 1 Thr Cys Met Ile Arg Ser Asn Ile Val sapiens and Rat Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His Leu Ser Lys Met Ile Ser Leu Ala Val Leo Val Phe Leu Ala Ala His Gly Ile Phe Val Gly Leu Leo Gin Leu Ala Leo Gin Arg Lys Thr Asn Arg Phe Phe Asn Leo Leo Thr Asp Leu Leo Ile Ser Leo Val Pro Trp Val Val Thr Ser Val Pro Leo Phe Trp Pro Leu Leo Phe Ala 115 Arcj Tyr Leo His Phe Cys Ala Leu Val Ser Ala Ser Val Asn Thr Ile 120 Val Val Leo Thr His 110 Ser Val Asp Lys Met Thr 130 Gin Arg 145 Leu Gin Glu Ara Ser Ile Ile His 135 Gly Tyr Leo Leu 150 Pro Leo Arg Leo Ser Tyr Pro 140 Tyr Gly Thr Trp 155 Gly Trp Gly Gin 170 Ile Val Ala Ile 160 Ala Ala Phe Asp 175 Ser Thr Pro 165 Asn Ala Leu Pro Leu Tyr Cys Ser Met 180 Ile Trp Gly Ala Ser Pro Ser Tyr 185 190 Page WO 01/36471 PCT/US00/31509 Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met 195 200 205 Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala 210 215 220 Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe 245 250 255 Gin Asp Glu Ser Glu Phe Arg Arg Gin His Glu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp 340 345 350 Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380 Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gin Cys Lys Ala Ala Lys Val 385 390 395 400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys 405 410 415 Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gin Val Pro 420 425 430 Gin Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gin Cys Cys 435 440 445 Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu 450 455 460 Ile Gin Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys 465 470 475 480 Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly 485 490 495 Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro Ala Ile Ser Ala 500 505 510 Glu Phe His His Thr Gly Leu Val Asp Pro Ser Ser Val Pro Ser Leu 515 520 525 Gly Cys Arg Ser Met Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gin 530 535 540 Page 66 WO 01/36471 WO 0136471PCTIUS00131509 Met Ser Asn Phe Arg Val 660 Phe Pro Pro 675 Gly Val Arg 690 Cys Ala Gin Tyr Val Pro Gly Ile Phe 740 Phe Asp Val 755 ?he Asn Asp 770 Asn Met Val Leu Asn Leu Ser Val Ilie 820 Leu Ala Gly 835 Tyr Thr Thr 850 Val Thr Arg Ala Gin Arg 550 Lys Gin Val Glu Ser Gly Asn Gly Phe 600 Ser Asn Ser 615 Asn Leu Lys 630 Val Pro Pro Tyr Ile Leu Phe Tyr Glu 680 Cys Tyr Glu 695 Phe Leu Asp 710 Asp Gin Asp Thr Lys Phe Gly Gin Arg 760 Thr Ala Ile 775 Arg Glu Asp 790 Lys Ser Ile Phe Leu Asn Ser Lys Ile 840 Giu Asp Ala 855 Lys Tyr Phe 870 Glu Ala Tyr Arg 570 Lys Ser 585 Asn Gly Asp Giy Giu Ala Val Giu 650 Ser Val 665 His Ala Arg Ser Lys Ile Leu Leu 730 Gin Vai 745 Asp Giu Ile Phe Asn Gin Trp Asn 810 Lys Gin 825 Glu Asp Thr Pro Ile Arg Lys Lys Thr His Ile Val Gly Gly 605 Lys Ala 620 Glu Thr Ala Asn Asn Val Ala Leu 685 Glu Tyr 700 Val Ile Cys Arg Lys Val Arg Lys 765 Val Ala 780 Asn Arg Arg Trp Leu Leu Phe Pro 845 Pro Gly 860 Giu Phe Glu Lys 560 Leu Leu 575 Gin Met Giu Asp Lys Val Val Ala 640 Giu Asn 655 Asn Phe Giu Asp Leu Ile Gin Ala 720 Leu Thr 735 Phe His Ile Gin Ser Ser Gin Giu 800 Arg Thr 815 Giu Lys Phe Ala Asp Pro Arg Ile 880 Ser Thr Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr Page 67 WO 01/36471 PCT/US00/31509 885 890 895 Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg 900 905 910 Asp lie Ile Gin Arg Met His Leu Arg Gin Tyr Glu Leu Leu 915 920 925 <210> 105 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 105 catgtatgcc agcgtcctgc tcc 23 <210> 106 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 106 gctatgcctg aagccagtct tgtg 24 <210> 107 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 107 gcacctgctc ctgagcacct tctcc <210> 108 <211> 26 <212> DNA <213> Artificial Seqeunce <220> <221> misc feature <223> Novel Sequence <400> 108 cacagcgctg cagccctgca gctggc 26 <210> 109 Page 68 WO 01/36471 PCT/US00/31509 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 109 ccagtgatga ctctgtccag cctg 24 <210> 110 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 110 cagacacttg gcagggacga ggtg 24 <210> 111 <211> 26 <212> DNA <213> Artficial Sequence <220> <221> misc feature <223> Novel Sequence <400> 111 cttgtggtct actgcagcat gttccg 26 <210> 112 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 112 catatccctc cgagtgtcca gcggc <210> 113 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence Page 69 WO 01/36471 PCT/US00/31509 <400> 113 atggatcctt atcatggctt cctc 24 <210> 114 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 114 caagaacagg tctcatctaa gagctcc 27 <210> 115 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 115 ctctgatgcc atctgctgga ttcctg 26 <210> 116 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 116 gtagtccact gaaagtccag tgatcc 26 <210> 117 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 117 tggtggcgat ggccaacagc gctc 24 <210> 118 <211> 24 <212> DNA <213> Artificial Sequence Page WO 01/36471 PCT/US00/31509 <220> <221> miscfeature <223> Novel Sequence <400> 118 gttgcgcctt agcgacagat gacc 24 <210> 119 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 119 tcaacctgta tagcagcatc ctc 23 <210> 120 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 120 aaggagtagc agaatggtta gcc 23 <210> 121 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 121 gacacctgtc agcggtcgtg tgtg 24 <210> 122 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 122 ctgatggaag tagaggctgt ccatctc 27 Page 71 WO 01/36471 PCT/US00/31509 <210> 123 <211> 24 <212> DNA <213> Articial Sequence <220> <221> misc feature <223> Novel Sequence <400> 123 gcgctgagcg cagaccagtg gctg 24 <210> 124 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 124 cacggtgacg aagggcacga gctc 24 <210> 125 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 125 agccatccct gccaggaagc atgg 24 <210> 126 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 126 ccaggtaggt gtgcagcaca atggc <210> 127 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence Page 72 WO 01/36471 PCT/US00/31509 <400> 127 ctgttcaaca gggctggttg gcaac <210> 128 <211> <212> DNA <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> .128 atcatgtcta gactcatggt gatcc <210> 129 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 129 Thr Leu Glu Ser Ile Met 1 <210> 130 <211> <212> PRT <213> Artificial Sequence <220> <221> miscfeature <223> Novel Sequence <400> 130 Glu Tyr Asn I.eu Val 1 <210> 131 <211> <212> PRT <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 131 Asp Cys Gly Leu Phe 1 <210> 132 Page 73 WO 01/36471 PCT/US00/31509 <211> 36 <212> PRT <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 132 Gly Ala Thr Cys Ala Ala Gly Cys Thr Thr Cys Cys Ala Thr Gly Gly 1 5 10 Cys Gly Thr Gly Cys Thr Gly Cys Cys Thr Gly Ala Gly Cys Gly Ala 25 Gly Gly Ala Gly <210> 133 <211> 53 <212> PRT <213> Artificial Sequence <220> <221> misc feature <223> Novel Sequence <400> 133 Gly Ala Thr Cys Gly Gly Ala Thr Cys Cys Thr Thr Ala Gly Ala Ala 1 5 10 Cys Ala Gly Gly Cys Cys Gly Cys Ala Gly Thr Cys Cys Thr Thr Cys 25 Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly Ala 40 Thr Gly Gly Thr Gly Page 74

Claims (49)

1. An isolated polynucleotide encoding a G protein-coupled receptor polypeptide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 22; the sequence set forth in SEQ ID NO: 21; the sequence of a polynucleotide that hybridizes under stringent conditions to or or a complementary sequence thereto: the sequence of a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 61 and/or SEQ ID NO: 62; a sequence encoding a constitutively-activated variant of SEQ ID NO: 22 wherein said constitutively-activated variant comprises a mutation positioned 16 amino residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22 and a variant of SEQ ID NO: 21 comprising a mutation positioned at a codon encoding an amino acid positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22.

2. An isolated polynucleotide encoding a G protein-coupled receptor polypeptide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of: a sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; the nucleotide sequence set forth in SEQ ID NO: 21; and a variant of comprising a substitution of one or two or three nucleotides within the codon at positions 880 to 882 of SEQ ID NO: 21 for other nucleotide residue(s) thereby producing a codon encoding a lysine residue.

3. The isolated polynucleotide of claim 1 or 2 of human origin.

4. The isolated polynucleotide according to any one of claims 1 to 3 when obtained from a pancreas tissue or cell. The isolated polynucleotide according to any one of claims 1 to 4, wherein said nucleotide sequence is selectively expressed endogenously by human pancreas.

6. The isolated polynucleotide of claim 5 wherein the expression is determined using a process that comprises performing RT-PCR.

7. An isolated polynucleotide encoding a GPCR fusion protein, wherein said polynucleotide comprises a nucleotide sequence of the isolated polynucleotide according to any one of claims 1 to 6.

8. The isolated polynucleotide of claim 7 comprising nucleic acid encoding a G protein. i 9. The isolated polynucleotide of claim 8 wherein the G protein is Gs. A vector comprising the polynucleotide according to any one of claims 1 to 9. 0

11. The vector of claim 10, wherein said vector is an expression vector and wherein the polynucleotide according to any one of claims 1 to 9 is operably linked to a promoter.

12. A recombinant host cell comprising the vector of claim

13. A recombinant host cell comprising the vector of claim 11.

14. An isolated membrane of the recombinant host cell of claim 13 wherein said membrane comprises a G protein-coupled receptor or GPCR fusion protein encoded by the isolated polynucleotide according to any one of claims 1 to 9. A method of producing a G protein-coupled receptor or a GPCR fusion protein comprising the steps of: transfecting the expression vector according to claim 11 into a host cell thereby producing a transfected host cell; and culturing the transfected host cell under conditions sufficient to express a human G protein-coupled receptor of GPCR fusion protein from the expression vector.

16. The method of claim 15 further comprising obtaining the transfected host cell.

17. The method of claim 15 or 16 wherein the host cell is a mammalian cell.

18. The method according to claim 17 wherein the mammalian cell is selected from the group consisting of a COS-7 cell, a 293 cell, and a 293T cell.

19. The method according to any one of claims 15 to 18 wherein the host cell is a pancreatic cell.

20. An isolated or recombinant G protein-coupled receptor polypeptide comprising an amino acid sequence selected from the group consisting of: a sequence comprising the amino acid sequence set forth in SEQ ID NO: 22; a sequence encoded by a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 21 or a complementary sequence thereto; a sequence encoded by a polynucleotide that is amplifiable by polymerase chain reaction (PCR) using a primer that comprises the nucleotide sequence set forth in SEQ ID NO: 61 and/or SEQ ID NO: 62; and the sequence of a constitutively-activated variant of SEQ ID NO: 22 wherein said constitutively-activated variant comprises a mutation positioned 16 amino acid residues N-terminal from the conserved proline residue within the TM6 domain of SEQ ID NO: 22.

21. The isolated or recombinant G protein-coupled receptor polypeptide of claim wherein said polypeptide is of human origin.

22. An isolated or recombinant G protein-coupled receptor polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22.

23. An isolated or recombinant G protein-coupled receptor polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22 wherein the leucine residue at position 294 of SEQ ID NO: 22 is substituted for a lysine residue.

24. An isolated or recombinant GPCR fusion protein comprising an amino acid sequence of the isolated or recombinant human G protein-coupled receptor polypeptide according to any one of claims 20 to 23. The isolated or recombinant GPCR fusion protein according to claim 24 comprising a G protein. 0*

26. The isolated or recombinant GPCR fusion protein according to claim wherein the G protein is Gs. 001 27. Use of the isolated or recombinant G protein-coupled receptor according to any one of claims 20 to 23 or the GPCR fusion protein according to any one of claims 24 to 26 in drug screening.

28. The use of claim 27 wherein the drug screening is to identify a pharmaceutical agent for the treatment of a disease or disorder related to the pancreas. too o*t 000 .OO0 00O0e0 .00 0, 0:

29. The use of claim 27 wherein the drug screening is to identify a pharmaceutical agent for the treatment of a disease or disorder related to a function selective to the pancreas. A method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the G protein-coupled receptor polypeptide according to any one of claims 20 to 23 or an isolated membrane comprising said G protein- coupled receptor polypeptide; and measuring the ability of the compound to inhibit or stimulate functionality of the G protein coupled receptor polypeptide wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide.

31. The method according to claim 30 further comprising providing the host cell or membrane.

32. The method according to claim 30 or 31 wherein the host cell comprises the expression vector of claim 1 1.

33. The method according to any one of claims 30 to 32 wherein the G protein- coupled receptor polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22.

34. The method according to any one of claims 30 to 32 wherein the G protein- coupled receptor polypeptide comprises a non-endogenous variant of SEQ ID NO: 22 wherein the leucine residue at position 294 of SEQ ID NO: 22 is substituted for a lysine residue. 8@ S b S. S S *5 *5 S S 5.55 S S S *5*t The method according to any one of claims 30 to 34 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor comprises determining intracellular inositol triphosphate (IP 3 diacylglycerol (DAG), cyclic AMP (cAMP) or cyclic GMP (cGMP).

36. The method according to any one of claims 30 to 34 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor comprises determining intracellular cyclic AMP (cAMP) level.

37. The method according to any one of claims 30 to 34 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor comprises determining intracellular inositol triphosphate (IP 3 accumulation in the presence of a Gq/Gi fusion protein or coupled to a Gq/Gi fusion protein.

38. The method according to any one of claims 30 to 37 wherein the modulator is selected from an agonist, a partial agonist and an inverse agonist

39. A method of identifying a modulator of a G protein-coupled receptor comprising the steps of: contacting a candidate compound with a recombinant host cell that expresses the GPCR fusion protein according to any one of claims 24 to 26 or an isolated membrane comprising said GPCR fusion protein; and measuring the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide portion of said GPCR fusion protein wherein inhibition or stimulation of said functionality indicates that the candidate compound is a modulator of the G protein-coupled receptor polypeptide. The method according to claim 39 further comprising providing the host cell or membrane.

41. The method of according to claim 39 or 40 wherein the host cell comprises the expression vector of claim 11.

42. The method according to any one of claims 39 to 41 wherein the GPCR fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 22 and the amino acid sequence of a G protein.

43. The method according to any one of claims 39 to 41 wherein the GPCR fusion protein comprises the amino acid sequence of a non-endogenous variant of SEQ ID NO: 22 and the amino acid sequence of a G protein, wherein said non-endogenous variant substantially comprises the sequence of SEQ ID NO: 22 wherein the leucine residue at position 294 of SEQ ID NO: 22 is substituted for a lysine residue.

44. The method of claim 42 or 43 wherein the G protein is Gs. The method according to any one of claims 39 to 44 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide of said GPCR fusion protein comprises determining intracellular inositol triphosphate (IP 3 diacylglycerol (DAG), cyclic AMP (cAMP) or cyclic GMP (cGMP).

46. The method according to any one of claims 39 to 44 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide of said GPCR fusion protein comprises determining intracellular cyclic AMP (cAMP) level.

47. The method according to any one of claims 39 to 44 wherein the ability of the compound to inhibit or stimulate functionality of the G protein-coupled receptor polypeptide of said GPCR fusion protein comprises determining intracellular inositol triphosphate (IP 3 accumulation in the presence of a Gq/Gi fusion protein or coupled to a Gq/Gi fusion protein.

48. The method according to any one of claims 39 to 47 wherein the modulator is selected from an agonist, a partial agonist and an inverse agonist

49. A process for identifying a compound that modulates signal transduction mediated by a G protein-coupled receptor in the pancreas, said process comprising performing the method according to any one of claims 30 to 48. The process according to claim 49 wherein the signal transduction mediated by a G protein-coupled receptor in the pancreas modulates blood concentration of glucose, glucagon or insulin.

51. A process for identifying a compound for the treatment of diabetes or a o condition associated with diabetes said process comprising performing the method according to any one of claims 30 to 48.

52. The process according to claim 51 wherein the condition associated with diabetes is a defect in glucagon regulation or insulin regulation.

53. The isolated polynucleotide according to any one of claims 1 to 9 substantially ooo* as described herein with reference to the examples and/or drawings.

54. The vector according to claim 10 or 11 substantially as described herein with reference to the examples and/or drawings. The recombinant host cell according to claim 12 or 13 substantially as described herein with reference to the examples and/or drawings.

56. The membrane according to claim 14 substantially as described herein with reference to the examples and/or drawings. 74

57. The method according to any one of claims 15 to 19 substantially as described herein with reference to the examples and/or drawings.

58. The isolated or recombinant human G protein-coupled receptor polypeptide or GPCR fusion protein according to any one of claims 20 to 26 substantially as described herein with reference to the examples and/or drawings.

59. The use according to any one of claims 27 to 29 substantially as described herein with reference to the examples and/or drawings.

60. The method according to any one of claims 30 to 48 substantially as described herein with reference to the examples and/or drawings.

61. The process according to any one of claims 49 to 52 substantially as described S• herein with reference to the examples and/or drawings. S61. The processArena Pharmaceuticals, Inc. Patent hereinAttorneys for the Applicant: F B RICE CO