Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, human orphan g protein-coupled receptors.

Description

This application is a continuation of 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 (a) the level of constitutive activation of the subsequent mutated form of the receptor is substantially the same as that evidenced by the first mutation of the receptor; and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 80%, more preferably at least about 90% and most preferably at least 95%. Ideally, and owing to the fact that the most preferred mutation disclosed herein for achieving constitutive activation includes a single amino acid and/or codon change between the endogenous and the non-endogenous forms of the GPCR, the percent sequence homology should be at least 98%.

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.

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.

VECTOR sin 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.

Identification of Human GPCRs

The efforts of the Human Genome project have 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 GPCRs, disclosed herein, were discovered by reviewing the GenBank™ database, while other GPCRs were discovered by utilizing a nucleic acid sequence of a GPCR, previously sequenced, to conduct a BLAST™ search of the EST database. Table A, below, lists the disclosed endogenous orphan GPCRs along with a GPCR's respective homologous GPCR:

TABLE A
		Open		Reference To
Disclosed		Reading	Percent	Homologous
Human	Accession	Frame	Homology To	GPCR
Orphan	Number	(Base	Designated	(Accession
GPCRs	Identified	Pairs)	GPCR	No.)
hARE-3	AL033379	1,260 bp	52.3% LPA-R	U92642
hARE-4	AC006087	1,119 bp	36% P2Y5	AF000546
hARE-5	AC006255	1,104 bp	32% Oryzias	D43633
			latipes
hGPR27	AA775870	1,128 bp
hARE-1	AI090920	999 bp	43%	D13626
			KIAA0001
hARE-2	AA359504	1,122 bp	53% GPR27
hPPR1	H67224	1,053 bp	39% EBI1	L31581
hG2A	AA754702	1,113 bp	31% GPR4	L36148
hRUP3	AI035423	1,005 bp	30%	2133653
			Drosophila
			melanogaster
hRUP4	AI307658	1,296 bp	32% pNPGPR	NP_004876
			28% and 29%	AAC41276
			Zebra fish	and
			Ya and Yb,	AAB94616
			respectively
hRUP5	AC005849	1,413 bp	25% DEZ	Q99788
			23% FMLPR	P21462
hRUP6	AC005871	1,245 bp	48% GPR66	NP_006047
hRUP7	AC007922	1,173 bp	43% H3R	AF140538
hCHN3	EST 36581	1,113 bp	53% GPR27
hCHN4	AA804531	1,077 bp	32% thrombin	4503637
hCHN6	EST 2134670	1,503 bp	36% edg-1	NP_001391
hCHN8	EST 764455	1,029 bp	47%	D13626
			KIAA0001
hCHN9	EST 1541536	1,077 bp	41% LTB4R	NM_000752
hCHN10	EST 1365839	1,055 bp	35% P2Y	NM_002563

Receptor homology is useful in terms of gaining an appreciation of a role of the disclosed receptors within the human body. Additionally, such homology can provide insight as to possible endogenous ligand(s) that may be natural activators for the disclosed orphan GPCRs.

B. Receptor Screening

Techniques have become more readily available over the past few years for endogenous-ligand identification (this, primarily, for the purpose of providing a means of conducting receptor-binding assays that require a receptor's endogenous ligand) because the traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the receptor. Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been heretofore recognized is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists, and inverse agonists of the receptor. For those diseases which result from an overly active receptor or an under-active receptor, what is desired in a therapeutic drug is a compound which acts to diminish the active state of a receptor or enhance the activity of the receptor, respectively, not necessarily a drug which is an antagonist to the endogenous ligand. This is because a compound that reduces or enhances the activity of the active receptor state need not bind at the same site as the endogenous ligand. Thus, as taught by a method of this invention, any search for therapeutic compounds should start by screening compounds against the ligand-independent active state.

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

Screening candidate compounds against an endogenous or non-endogenous, constitutively activated version of the human orphan GPCRs disclosed herein can provide for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand. By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed, it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document.

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

C. Disease/Disorder Identification and/or Selection

Preferably, the DNA sequence of the human orphan GPCR can be used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue 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.

As the data below indicate, RUP3 is expressed within the human pancreas, suggesting that RUP3 may play a role in insulin regulation and/or glucagon regulation. Accordingly, candidate compounds identified using a constitutively activated form of RUP3 may be useful for understanding the role of RUP3 in diabetes and/or as therapeutics for diabetes.

D. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes constitutively active (i.e., active in the absence of endogenous ligand binding thereto), it binds to a G protein (e.g., Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [³⁵S] GTPγS 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 (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the 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 and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the other hand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple the Gi (or Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3^rdEd.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) which then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., β-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein. The reporter protein such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).

Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP₂, releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP₃). Increased accumulation of IP₃ is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3^rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP₃ accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-dependent 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 activated orphan GPCR, or a non-endogenous, constitutively activated orphan GPCR, for screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provides a unique challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, it is often useful that an approach be utilized that can enhance the signal obtained by the activated receptor. A preferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a GPCR is or has been constitutively activated, using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the constitutively activated orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques, although the GPCR Fusion Protein can also be (and preferably is) used with an endogenous, constitutively activated GPCR. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). We have a preference (based upon convenience) of use of a spacer in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., 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.

E. Other Utility

Although a preferred use of the human orphan 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 human orphan GPCRs is that its utility as a research tool is enhanced in that by determining the location(s) of such receptors within the body, the GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.

Although a preferred use of the non-endogenous versions of the human RUP3 disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), this version of human RUP3 can also be utilized in research settings. For example, in vitro and in vivo systems incorporating RUP3 can be utilized to further elucidate the roles RUP3 plays in the human condition, particularly with respect to the human pancreas, both nonnal and diseased (and in particular, diseases involving regulation of insulin or glucagon, e.g., diabetes), as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. A value in non-endogenous human RUP3 is that its utility as a research tool is enhanced in that, because of its unique features, non-endogenous RUP3 can be used to understand the role of RUP3 in the human body before the endogenous ligand therefor is identified. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of the patent document.

EXAMPLES

The following examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with the ability to make minor modifications to these sequences while achieving the same or substantially similar results reported below. Unless otherwise indicated below, all nucleic acid sequences for the disclosed endogenous orphan human GPCRs have been sequenced and verified. For purposes of equivalent receptors, those of ordinary skill in the art will readily appreciate that conservative substitutions can be made to the disclosed sequences to obtain a functionally equivalent receptor.

Example 1

Endogenous Human GPCRs

1. Identification of Human GPCRs

Several of the disclosed endogenous human GPCRs were identified based upon a review of the GenBank database information. While searching the database, the following cDNA clones were identified as evidenced below.

			Open
Disclosed		Complete	Reading	Nucleic	Amino
Human		DNA	Frame	Acid	Acid
Orphan	Accession	Sequence	(Base	SEQ ID.	SEQ ID.
GPCRs	Number	(Base Pairs)	Pairs)	NO.	NO.
hARE-3	AL033379	111,389 bp	1,260 bp	1	16
hARE-4	AC006087	226,925 bp	1,119 bp	3	4
hARE-5	AC006255	127,605 bp	1,104 bp	5	6
hRUP3	AL035423	140,094 bp	1,005 bp	7	8
hRUP5	AC005849	169,144 bp	1,413 bp	9	10
hRUP6	AC005871	218,807 bp	1,245 bp	11	12
hRUP7	AC007922	158,858 bp	1,173 bp	13	14

Other disclosed endogenous human GPCRs were identified by conducting a BLAST search of EST database (dbest) using the following EST clones as query sequences. The following EST clones identified were then used as a probe to screen a human genomic library.

			Open	Nucleic	Amino
Disclosed			Reading	Acid	Acid
Human		EST Clone/	Frame	SEQ	SEQ
Orphan	Query	Accession No.	(Base	ID.	ID.
GPCRs	(Sequence)	Identified	Pairs)	NO.	NO.
hGPCR27	Mouse	AA775870	1,125 bp	15	16
	GPCR27
hARE-1	TDAG	1689643	999 bp	17	18
		AI090920
hARE-2	GPCR27	68530	1,122 bp	19	20
		AA359504
hPPR1	Bovine	238667	1,053 bp	21	22
	PPR1	H67224
hG2A	Mouse	See Example	1,113 bp	23	24
	1179426	2(a) below
hCHN3	N.A.	EST 36581	1,113 bp	25	26
		(full length)
hCHN4	TDAG	1184934	1,077 bp	27	28
		AA804531
hCHN6	N.A.	EST 2134670	1,503 bp	29	30
		(full length)
hCHN8	KIAA0001	EST 76445	1,029 bp	31	32
hCHN9	1365839	EST 1541536	1,077 bp	33	34
hCHN10	Mouse EST	Human	1,005 bp	35	36
	1365839	1365839
hRUP4	N.A.	AI307658	1,296 bp	37	39
N.A. = “not applicable”

2. Full Length Cloning

a. hG2A (Seq. Id. Nos. 23 & 24)

Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid hG2A coding sequences. The 5′end of this coding sequence was obtained by using 5′RACE™, and the template for PCR was Clontech's Human Spleen Marathon-ready™ cDNA. The disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ. ID. NO.: 39 and SEQ. ID. NO.: 40 as follows:

• • 5′-CTGTGTACAGCAGTTCGCAGAGTG-3′(SEQ. ID. NO.: 39; 1^st round PCR)
  • 5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′(SEQ. ID. NO.: 40; second round PCR).

PCR was performed using Advantage™ GC Polymerase Kit (Clontech; manufacturing instructions will be followed), at 94° C. for 30 sec followed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30 cycles of 94° for 5 sec and 70° for 4 min. An approximate 1.3 Kb PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen). The cloned-insert was sequenced using the T7 Sequenase™ kit (USB Amersham; manufacturer instructions will be followed) and the sequence was compared with the presented sequence. Expression of the human G2A will be detected by probing an RNA dot blot (Clontech; manufacturer instructions will be followed) with the P³²-labeled fragment.

b. hCHN9 (Seq. Id. Nos. 33 & 34)

Sequencing of the EST clone 1541536 indicated that hCHN9 is a partial cDNA clone having only an initiation codon; ie., the termination codon was missing. When hCHN9 was used to “blast” against the data base (nr), the 3′ sequence of hCHN9 was 100% homologous to the 5′ untranslated region of the leukotriene B4 receptor cDNA, which contained a termination codon in the frame with hCHN9 coding sequence. To determine whether the 5′ untranslated region of LTB4R cDNA was the 3′ sequence of hCHN9, PCR was performed using primers based upon the 5′ sequence flanking the initiation codon found in hCHN9 and the 3′ sequence around the termination codon found in the LTB4R 5′ untranslated region. The 5′ primer sequence utilized was as follows:

• • 5′-CCCGAATTCCTGCTFGCTCCCAGCTTGGCCC-3′ SEQ. ID. NO.: 41; sense) and
  • 5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′ (SEQ. ID. NO.: 42; antisense).
    PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 10 sec. A 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV (see below) and sequenced (see, SEQ. ID. NO.: 33).

c. hRUP4 (Seq. Id. Nos. 37 & 38)

The full length hRUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates:

• • 5′-TCACAATGCTAGGTGTGGTC-3′ (SEQ. ID. NO.: 43; sense) and
  • 5′-TGCATAGACAATGGGATTACAG-3′ (SEQ. ID. NO.: 44; antisense).
    PCR was performed using TaqPlus™ Precision™ polymerase (Stratagene; manufacturing instructions will be followed) by the following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for 45 sec, and 72° C. for 10 min. Cycles 2 through 4 were repeated 30 times.

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

5′-TCACAATGCTAGGTGTGGTCTGGCTGGTG (SEQ. ID. NO.: 45)
GCAGTCATAGTAGGATCACCATGTGGCACGTG
CAACAACTTGAGATCAAATCTGACTTCCTATA
TGAAAAGGAACACATCTGCTGCTTAGAAGAGT
GGACCAGCCCTGTGCACCAGAAGATCTACACC
ACCTTCATCCTTGTCATCCTCTTCCTCCTGCC
TCTTATGGTGATGCTTATTCTGTACGTAAAAT
TGGTTATGAACTTTGGATAAAGAAAAGAGTTG
GGGATGGTTCAGTGCTTCGAACTATTCATGGA
AAAGAAATGTCCAAAATAGCCAGGAAGAAGAA
ACGAGCTGTCATTATGATGGTGACAGTGGTGG
CTCTCTTTGCTGTGTGCTGGGCACCATTCCAT
GTTGTCCATATGATGATTGAATACAGTAATTT
TGAAAAGGAATATGATGATGTCACAATCAAGA
TGATTTTTGATATCGTGCAAATTATTGGATTT
TCCAACTCCATCTGTAATCCCATTGTCTATGC
A-3′

Based on the above sequence, two sense oligonucleotide primer sets:

(SEQ. ID. NO.: 46; oligo 1)
5′-CTGCTTAGAAGAGTGGACCAG-3′
(SEQ. ID. NO.: 47; oligo 7)
5′-CTGTGCACGAGAAGATCTACAC-3′
and two antisense oligonucleotide primer sets:
(SEQ. ID. NO.: 48; oligo 3)
5′-CAAGGATGAAGGTGGTGTAGA-3′
(SEQ. ID. NO.: 49; oligo 4)
5′-GTGTAGATCTTCTGGTGCACAGG-3′

were used for 3′-and 5′-race PCR with a human brain Marathon-Ready™ cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions. DNA fragments generated by the RACE PCR were cloned into the pCRII-TOPO™ vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers. The 3′ RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon. The 5′ RACE product contained an incomplete 5′ end; i.e., the ATG initiation codon was not present.

Based on the new 5′ sequence, oligo 3 and the following primer:

• • 5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ. ID. NO.: 50; oligo 5)
    were used for the second round of 5′ RACE PCR and the PCR products were analyzed as above. A third round of 5′ RACE PCR was carried out utilizing antisense primers:
  • 5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ (SEQ. ID. NO.: 51; oligo 6) and
  • 5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′ (SEQ. ID. NO.: 52; oligo7).
    The sequence of the 5′ RACE PCR products revealed the presence of the initiation codon ATG, and further round of 5′ RACE PCR did not generate any more 5′ sequence. The completed 5′ sequence was confirmed by RT-PCR using sense primer 5′-GCAATGCAGGCGCTTAACATFAC-3′ (SEQ. ID. NO.: 53; oligo 8)
    and oligo 4 as primers and sequence analysis of the 650 bp PCR product generated from human brain and heart cDNA templates (Clontech, Cat# 7404-1). The completed 3′ sequence was confirmed by RT-PCR using oligo 2 and the following antisense primer:
  • 5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ. ID. NO.: 54; oligo 9)
    and sequence analysis of the 670 bp PCR product generated from human brain and heart cDNA templates. (Clontech, Cat# 7404-1).

d. hRUP5 (Seq. Id. Nos. 9 & 10)

The full length hRUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ. ID. NO.: 55), and an antisense primer containing TCA as the stop codon (SEQ. ID. NO.: 56), which had the following sequences:

5′-ACTCCGTGTCCAGCAGGACTCTG-3′  (SEQ. ID. NO.: 55)
5′-TGCGTGTTCCTGGACCCTCACGTG-3′ (SEQ. ID. NO.: 56)

and human peripheral leukocyte cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 through step 4 repeated 30 times: 94° C. for 30 sec: 94° for 15 sec; 69° for 40 sec; 72° C. for 3 min; and 72° C. from 6 min. A 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPO™ vector (Invitrogen) and completely sequenced using the T7 DNA Sequenase™ kit (Amsham). See, SEQ. ID. NO.: 9.

e. hRUP6 (Seq. Id. Nos. 11 & 12)

The full length hRUP6 was cloned by RT-PCR using primers:

(SEQ. ID. NO.: 57)
5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ and
(SEQ. ID. NO.: 58)
5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′;

and human thymus Marathon-Ready™ cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech, according to manufacturer's instructions) was used for the amplification in a 50 ul reaction by the following cycle: 94° C. for 30sec; 94° C. for 5 sec; 66° C. for 40sec; 72° C. for 2.5 sec and 72° C. for 7 min. Cycles 2 through 4 were repeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (see, SEQ. ID. NO.: 11) using the ABI Big Dye Terminator™ kit (P.E. Biosystem).

f. hRUP7 (Seq. Id. Nos. 13 & 14)

The full length RUP7 was cloned by RT-PCR using primers:

(SEQ. ID. NO.: 59; sense)
5′-TGATGTGATGCCAGATACTAATAGCAC-3′
and
(SEQ. ID. NO.: 60; antisense)
5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′

and human peripheral leukocyte cDNA (Clontech) as a template. Advantage™ cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 to step 4 repeated 30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 2 minutes; 72° C. for 10 minutes. A 1.25 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.

g. hARE-5 (Seq. Id. Nos. 5 & 6)

The full length hARE-5 was cloned by PCR using the hARE5 specific primers 5′-CAGCGCAGGGTGAAGCCTGAGAGC-3′ SEQ. ID. NO.: 69 (sense, 5′ of initiation codon ATG) and 5′-GGCACCTGCTGTGACCTGTGCAGG-3′ SEQ. ID. NO.: 70 (antisense, 3′ of stop codon TGA) and human genomic DNA as template. TaqPlus Precision™ DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 96° C., 2 minutes; 96° C., 20 seconds; 58° C., 30 seconds; 72° C, 2 minutes; and 72° C., 10 minutes

A 1.1 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ. ID. NO.: 5) using the T7 DNA Sequenase™ kit (Amsham).

h. hARE-4 (Seq. Id. Nos.: 3 & 4)

The full length hARE-4 was cloned by PCR using the hARE-4 specific primers 5′-CTGGTGTGCTCCATGGCATCCC-3′ SEQ.ID.NO.:67 (sense, 5′ of initiation condon ATG) and 5′-GTAAGCCTCCCAGAACAGAGG-3′ SEQ. ID. NO.: 68 (antisense, 3′ of stop codon TGA) and human genomic DNA as template. Taq DNA polymerase (Stratagene) and 5% DMSO was used for the amplification by the following cycle with step 2 to step 3 repeated 35 times: 94° C., 3 minutes; 94° C., 30 seconds; 59° C., 2 minutes; 72° C., 10 minute

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

i. hARE-3 (Seq. Id. Nos.: 1 & 2)

The full length hARE-3 was cloned by PCR using the hARE-3 specific primers 5′-gatcaagcttCCATCCTACTGAAACCATGGTC-3′ SEQ.ID.NO65 (sense, lower case nucleotides represent Hind III overhang, ATG as initiation codon) and 5′-gatcagatctCAGTT CCAATATTCACACCACCGTC-3′ SEQ. ID. NO.: 66 (antisense, lower case nucleotides represent Xba I overhang, TCA as stop codon) and human genomic DNA 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., 3 minutes; 94° C., 1 minute; 55° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes.

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

j. hRUP3 (Seq. Id. Nos.: 7 & 8)

The full length hRUP3 was cloned by PCR using the hRUP3 specific primers 5′-GTCCTGCCACTTCGAGACATGG-3′ SEQ. ID.NO.:71 (sense, ATG as intiation codon) and 5′-GAAACTTCTCTCTGCCCTTACCGTC-3′

SEQ.ID.NO.:72 (antisense, 3′ of stop codon TAA) and human genomic DNA 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., 3 minutes; 94° C., 1 minute; 58° C., 1 minute; 72° C., 2 minutes: 72° C., 10 minutes

A 1.0 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ. ID. NO.: 7)using the T7 DNA sequenase kit (Amsham).

Example 2

Receptor Expression

Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of 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. The general procedure for expression of the disclosed GPCRs is as follows.

On day one, 1×10⁷293T cells per 150 mm plate were 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 20 μg DNA (e.g., pCMV vector, pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B will be prepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B are admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture can be referred to as the “transfection mixture”. Plated 293T cells are washed with 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture will then be added to the cells, followed by incubation for 4 hrs at 37° C./5% CO₂. The transfection mixture was then be removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37° C./5% CO₂. After 72hr incubation, cells can then be harvested and utilized for analysis.

Example 3

Tissue Distribution of the Disclosed Human GPCRs

Several approaches can be used for determination of the tissue distribution of the GPCRs disclosed herein.

1. Dot-Blot Analysis

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

TABLE B

            Tissue Distribution
ORPHAN GPCR (highest levels, relative to other tissues in the dot-blot

hGPCR27     Fetal brain, Putamen, Pituitary gland, Caudate nucleus
hARE-1      Spleen, Peripheral leukocytes, Fetal spleen
hPPR1       Pituitary gland, Heart, salivary gland, Small intestine,
            Testis
hRUP3       Pancreas
hCHN3       Fetal brain, Putamen, Occipital cortex
hCHN9       Pancreas, Small intestine, Liver
hCHN10      Kidney, Thyroid

TABLE C

            Tissue Distribution
ORPHAN GPCR (highest levels, relative to other tissues in the dot-blot

hARE-3      Cerebellum left, Cerebellum right, Testis, Accumbens
hGPCR3      Corpus collusum, Caudate nucleus, Liver, Heart, Inter-
            Ventricular Septum
hARE-2      Cerebellum left, Cerebellum right, Substantia
hCHN8       Cerebellum left, Cerebellum right, Kidney, Lung

To ascertain the tissue distribution of hRUP3 mRNA, RT-PCR was performed using hRUP3-specific primers and human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was utilized for the PCR reaction, using the following reaction cycles in a 40 ul reaction: 94° C. for 2 min; 94° C. for 15 sec; 55° C. for 30 sec; 72° C. for 1 min: 72° C., for 10 min. Primers were as follows:

(SEQ. ID. NO.: 61; sense)
5′-GACAGGTACCTTGCCATCAAG-3′
(SEQ. ID. NO.: 62; antisense)
5′-CTGCACAATGCCAGTGATAAGG-3′.

20 ul of the reaction was loaded onto a 1% agarose gel: results are set forth in FIG. 3.

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

b. hRUP4

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

20 ul of the reaction were loaded on a 1% agarose gel to analyze the RT-PCR products, and hRUP4 mRNA was found expressed in many human tissues, with the strongest expression in heart and kidney. (see, FIG. 4). To confirm the authenticity of the PCR fragments, a 300 bp fragment derived from the 5′ end of hRUP4 was used as a probe for the Southern Blot analysis. The probe was labeled with ³²P-dCTP using the Prime-It II™ Random Primer Labeling Kit (Stratagene) and purified using the ProbeQuant™ G-50 micro columns (Amersham). Hybridization was done overnight at 42° C. following a 12 hr pre-hybridization. The blot was finally washed at 65° C. with 0.1×SSC. The Southern blot did confirm the PCR fragments as hRUP4.

c. hRUP5

RT-PCR was performed using the following hRUP5 specific primers:

(SEQ. ID. NO.: 63; sense)
5′-CTGACTTCTTGTTCCTGGCAGCAGCGG-3′
(SEQ. ID. NO.: 64; antisense)
5′-AGACCAGCCAGGGCACGCTGAAGAGTG-3′

and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was used for the amplification in a 40 ul reaction by the following cycles: 94° C. for 30 sec, 94° C. for 10 sec, 62° C. for 1.5 min, 72° C. for 5 min, and with cycles 2 through 3 repeated 30 times. 20 ul of the reaction were loaded on a 1.5% agarose gel to analyze the RT-PCR products, and hRUP5 mRNA was found expressed only in the peripheral blood leukocytes (data not shown).

d. hRUP6

RT-PCR was applied to confirm the expression and to determine the tissue distribution of hRUP6. Oligonucleotides used, based on an alignment of AC005871 and GPR66 segments, had the following sequences:

(SEQ. ID. NO.: 73; sense)
5′-CCAACACCAGCATCCATGGCATCAAG-3′,
(SEQ. ID. NO.: 74; antisense)
5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′

and the human multiple tissue cDNA panels (MTC, Clontech) were used as templates. PCR was performed using TaqPlus Precision™ polymerase (Stratagene; manufacturing instructions will be followed) in a 40 ul reaction by the following cycles: 94° C. for 30 sec; 94° C. 5 sec; 66° C. for 40 sec, 72° C. for 2.5 min, and 72° C. for 7 min. Cycles 2 through 4 were repeated 30 times.

20 ul of the reaction were loaded on a 1.2% agarose gel to analyze the RT-PCR products, and a specific 760 bp DNA fragment representing hRUP6 was expressed predominantly in the thymus and with less expression in the heart, kidney, lung, prostate small intestine and testis. (see, FIG. 5).

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

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

Although a variety of Vectors are available to those in the art, for purposes of utilization for both 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 Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

Claims

1. A method for identifying a compound for regulating insulin concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of seq id NO: 8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating insulin concentration in the blood of a mammal.

2. The method of claim 1 wherein said compound for regulating insulin concentration in the blood of a mammal is a therapeutic for treating diabetes.

3. The method of claim 1 wherein the compound for regulating insulin concentration in the blood of a mammal is selected from agonist, partial agonist, and inverse agonist of the receptor.

4. The method of claim 1 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8.

5. The method of claim 1 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of seq id NO: 8.

6. A method for identifying a compound for regulating glucose concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of seq id NO: 8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucose concentration in the blood of a mammal.

7. The method of claim 6 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8.

8. The method of claim 6 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of seq id NO: 8.

9. A method for identifying a compound for regulating glucagon concentration in the blood of a mammal comprising the steps of:

contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of seq id NO: 8; and

measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucagon concentration in the blood of a mammal.

10. The method of claim 9 wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8.

11. The method of claim 9 where said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8;

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of seq id NO: 8.

12. A method for identifying a compound for inhibiting or stimulating a receptor comprising:

a) the amino acid sequence of seq id NO: 8;

b) a mutant of seq id NO: 8, wherein lysine is substituted for leucine at amino acid residue 224;

c) an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of seq id NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.;

d) an amino sequence encoded by the nucleotide sequence of seq id NO: 7;

e) a g protein-coupled receptor having at least 95% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

f) a g protein-coupled receptor encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of seq id NO:7, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels,

comprising the steps of:

i) contacting one or more candidate compounds with a host cell or membrane thereof,

13. The method of claim 12, wherein the compound is selected from agonist, partial agonist, and inverse agonist of the receptor.

14. The method of claim 13, wherein the compound is an agonist of the receptor.

15. The method of claim 13, wherein the compound is a partial agonist of the receptor.

16. The method of claim 13, wherein the compound is an inverse agonist of the receptor.

17. The method of claim 12, wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8;

b) a nucleotide sequence encoding a polypeptide comprising a mutant of seq id NO: 8, wherein lysine is substituted for leucine at amino acid residue 224;

c) a nucleotide sequence that hybridizes to the complete complement of seq id NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.;

d) the nucleotide sequence of seq id NO: 7;

e) a nucleotide sequence encoding a g protein-coupled receptor having at least 95% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

f) a nucleotide sequence having at least 95% identity to the nucleotide sequence of seq id NO: 7, wherein said nucleotide sequence encodes a g protein-coupled receptor capable of modulating insulin or glucagon levels.

18. The method of claim 12, wherein said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of seq id NO: 8;

b) a nucleotide sequence encoding a polypeptide comprising a mutant of seq id NO: 8, wherein lysine is substituted for leucine at amino acid residue 224;

c) a nucleotide sequence that hybridizes to the complete complement of seq id NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.;

d) the nucleotide sequence of seq id NO: 7;

e) a nucleotide sequence encoding a g protein-coupled receptor having at least 95% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

f) a nucleotide sequence having at least 95% identity to the nucleotide sequence of seq id NO: 7, wherein said nucleotide sequence encodes a g protein-coupled receptor capable of modulating insulin or glucagon levels,

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising:

a) the amino acid sequence of seq id NO: 8;

b) a mutant of seq id NO: 8, wherein lysine is substituted for leucine at amino acid residue 224;

c) an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of seq id NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.;

d) an amino sequence encoded by the nucleotide sequence of seq id NO: 7;

e) a g protein-coupled receptor having at least 95% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

f) a g protein-coupled receptor encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of seq id NO:7, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels.

19. The method of claim 12, wherein the receptor comprises the amino acid sequence of seq id NO: 8.

20. The method of claim 12, wherein the receptor is a mutant of seq id NO: 8, wherein lysine is substituted for leucine at amino acid residue 224.

21. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the activity of a second messenger.

22. The method of claim 21, wherein the second messenger is selected from the group consisting of adenyl cyclase and phospholipase C.

23. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the level of a second messenger.

24. The method of claim 23, wherein the second messenger is selected from the group consisting of cAMP, diacyl glycerol, and inositol 1,4,5-triphosphate.

25. The method of claim 12, wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the binding of GTPγS to a membrane comprising said g protein-coupled receptor.

26. The method of claim 12, wherein the host cell is a mammalian host cell.

27. The method of claim 12, wherein the host cell is a yeast host cell.

28. The method of claim 12, wherein the host cell comprises a reporter system comprising multiple cAMP responsive elements operably linked to a reporter gene.

29. The method of claim 12, wherein said receptor is a constitutively activated receptor.

30. The method according to claim 12, wherein said method comprises identifying a compound for inhibiting or stimulating a receptor comprising:

a) a g protein-coupled receptor having at least 98% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

b) a g protein-coupled receptor encoded by a nucleotide sequence having at least 98% identity to the nucleotide sequence of seq id NO:7, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels, comprising the steps of:

contacting one or more candidate compounds with a host cell or membrane thereof,

31. The method of claim 17, wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a g protein-coupled receptor having at least 98% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

b) a nucleotide sequence having at least 98% identity to the nucleotide sequence of seq id NO: 7, wherein said nucleotide sequence encodes a g protein-coupled receptor capable of modulating insulin or glucagon levels.

32. The method of claim 18, wherein said host cell is produced by a method comprising:

transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:

a) a nucleotide sequence encoding a g protein-coupled receptor having at least 98% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

b) a nucleotide sequence having at least 98% identity to the nucleotide sequence of seq id NO: 7, wherein said nucleotide sequence encodes a g protein-coupled receptor capable of modulating insulin or glucagon levels,

wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising:

a) a g protein-coupled receptor having at least 98% identity to the amino acid sequence of seq id NO: 8, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels; or

b) a g protein-coupled receptor encoded by a nucleotide sequence having at least 98% identity to the nucleotide sequence of seq id NO:7, wherein said g protein-coupled receptor is capable of modulating insulin or glucagon levels.