WO2007149807A1 - Methods for identifying modifiers of gpr1 activity - Google Patents

Abstract

The invention relates, in part, to methods for determining if a molecule interacts with a receptor. In some aspects, the invention involves, in part, the identification of a receptor binding partner for GPRl, as well as methods relating to assaying receptor activity and compositions used in such assays. The invention further provides examples of ligands for GPRl. The invention also describes various assays and methods.

Description

METHODS FOR IDENTIFYING MODIFIERS OF GPRl ACTIVITY

RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S. C. 119(e) of U.S. Provisional Patent Application Nos. 60/815,062, filed June 20, 2006, and 60/885,076, filed January 16, 2007, the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates, in part, to methods for determining if a molecule interacts with a receptor. In some aspects, the invention involves, in part, the identification of a receptor binding partner for GPRl, as well as methods relating to assaying receptor activity and compositions used in such assays. The invention also relates, in part, to modulating activity of a GPRl or a GPRl ligand.

BACKGROUND

[0003] Cells recognize and respond to a great variety of extracellular stimuli using specific receptor molecules located on the cell surface. The largest class of cell surface receptors are the G-protein coupled receptors (GPCRs), an exceedingly diverse group of protein containing molecules, involved in the recognition of endogenous ligands such as hormones, neurotransmitters, peptides, glycoproteins, lipids, nucleotides and ions, as well as exogenous stimuli, including light, odors, pheromones and tastes, e.g., see Strader, et al, Ann. Rev. Biochem., 65:101-32(1994); Bockaert, et al, EMBO J., 750:1723-9(1999); and Mombarets, et al, Science, 286(5440):707-711(1999). As a result of their central role in many signaling events, GPCRs are the targets of an increasingly large number of therapeutic agents (Howard et al, Trends Pharmacol ScL, 22(3): 132-40(2001), and mutations in GPCRs have been linked to numerous diseases and disorders, e.g., see Spiegel, Ann. Rev. Physiol, 58: 143-70(1996); and Rana, et al, Ann. Rev. Pharmacol Toxicol, 47:593-624(2001).

[0004] The discovery of candidate therapeutic agents that act on GPCR-mediated pathways requires the development of assays to monitor the activity of specific GPCR targets. Different GPCRs are coupled to distinct G-protein-regulated signal transduction pathways, and thus assays that measure G protein-regulated signaling pathways depend on knowledge of the G-protein specificity of the target receptor, or require engineering of the cellular system to force coupling of the target receptor to a measurable pathway. In contrast, one apparently common feature of GPCRs is that ligand binding triggers receptor desensitization, a process that is mediated by the recruitment of intracellular arrestin proteins to the activated receptor. Thus, the ligand-induced activation of GPCRs may be assayed by monitoring the interaction of arrestin with the test GPCR. A major advantage of this approach is that no knowledge of G protein pathways is required.

[0005] As part of a broad effort to identify receptors that mediate the activity of heretofore "orphan" signaling factors and elucidate the biological function of these ligand- receptor pairs, cell-based assays have been developed to measure the ligand-mediated activation of a large fraction of the GPCRs found in the human genome. See, e.g., U.S. Patent Application No. 10/888,313. Among these is an assay to measure the activity of GPRl (mRNA accession no. NM 005279 (SEQ ID NO:1) or GenBank Accession No. NP 005270), e.g., as described in Marchese et al {Genomics 25:609-18 (1994)). GPRl, an orphan GPCR distantly related to chemokine receptors, has been reported to act as a potential co-receptor to allow entry of HIV and related viruses into brain-derived cells (e.g., Gabuzda and Wang, J Neuroviro. 5:643-58 (1999); Shimizu et al, J Virol. 75:5231-9 (1999); Croitoru- Lamoury et al, GHa. 41:354-70 (2003)), mesangial cells (e.g., Tokizawa et al., Kidney Int. 58:607-17) and other cell types (e.g., Edinger et al., Proc Natl Acad Sd U S A £4:14742-7 (1997); Farzan et al, J Exp Med 756:405-11 (1997); Edinger et al, Virology 249:367-7% (1998); Zhang et al, J Virol. 72:9337-44 (1998); or Singh et al, J Virol. 75:6680-90 (1999)). Peptide analogues corresponding to the N-terminal domain of GPRl inhibit infection of a broad range of HIV strains (Ikeda et al, BioorgMed Chem Lett 11:2607-9 (2001)).

[0006] A close GPCR relative of GPRl, CMKLRl, also known as ChemR23 or DEZ, was reported to be a receptor for chemerin (also known as Tig2), a polypeptide ligand found in inflammatory fluids. See, e.g., Genbank Accession No. Q99969; Wittamer et al, J Exp Med 198:977-85 (2003); Meder et al, FEBS Lett. 555:495-9 (2003); and Wittamer et al, J Biol Chem 279:9956-62 (2004). Chemerin has been shown to direct the chemotaxis of immune cells to sites of inflammation (Vermi et al, J Exp Med. 201:509-15 (2005)). In one of the studies (Wittamer et al, J Exp Med 198:977-85), the related orphan GPRl was reported to be unresponsive to chemerin.

[0007] U.S. Patent Application No. 10/888,313 and herein describes assays which are used to identify molecules which interact with other molecules, such as receptors in the GPCR family. This type of assay is ideal for "de-orphanizing" orphan receptors, as was done here and will be shown in the examples which follow. Briefly, the ligand-mediated activation of a target GPCR is measured by monitoring the interaction of an arrestin with target GPCRs in a cell based system. The GPCR of interest is fused at its C-terminal end to a non- endogenous transcription factor via a protease cleavage sequence. The resulting construct is expressed in a cell or cell line containing a quantifiable reporter gene, regulated by a tethered transcription factor, together with a chimeric protein consisting of an arrestin fused to a protease specific for the cleavage site discussed herein. The availability of a cell-based assay to measure GPRl activity provides a means of developing drugs that act on GPRl to modulate any of GPRl 's activities, e.g., in therapeutic areas.

[0008] Details of related assay methodology have been described previously. See, e.g., U.S. Patent No. 7,049,076, referred to herein. Briefly, the ligand-mediated activation of a target GPCR is measured by monitoring the interaction of an arrestin with target GPCRs intracellularly. The GPCR of interest is fused at its C-terminal end to a non-endogenous transcription factor via a protease cleavage sequence. This is expressed in a cell line containing a quantifiable reporter gene regulated by the tethered transcription factor, together with chimeric protein consisting of arrestin fused to the protease specific for the cleavage site above. The assay is then performed by adding a ligand to the growing cells for a defined period, and measuring the activity of the reporter gene. If the ligand binds to the target receptor, it stimulates the recruitment of the interacting arrestin fusion protein, which results in cleavage of the protease site and release of the transcription factor. The free transcription factor then enters the nucleus and stimulates expression of the reporter gene. Using this approach, quantification of the reporter gene activity affords a measurement of the degree of binding of the interacting arrestin protein to the test GPCR. This assay system has been validated for a diverse array of GPCRs, including receptors that couple to each of the major G protein pathways, and receptors activated by a variety of ligand types, such as hormones, neurotransmitters, peptides and chemokines.

[0009] As discussed below, this methodology has been applied to GPRl .

SUMMARY OF THE INVENTION

[0010] The invention is directed, in part, to biological assays and compounds which can be used in such assays.

[0011] In particular aspects, the invention is directed, in part, to methods for determining if a test compound interacts with a GPRl and/or modulates GPRl activity. In some embodiments, such methods comprise contacting (a) a test compound, and (b) (i) a GPRl ligand or (ii) a GPRl ligand derivative which interacts with GPRl or a functional derivative thereof, and comparing interaction of (b) with GPRl in the presence of (a), to its interaction with GPRl in the absence of (a) or at a different concentration of (a), as a determination of whether the test compound interacts with GPRl . In some embodiments, a GPRl ligand derivative may be labeled with a detectable moiety. Further, while assays of the invention may be performed using cell based or cell free systems, in some embodiments, GPRl may be presented on a cell surface. Examples of GPRl ligands are presented herein.

[0012] Further, in some embodiments, a downstream activity of a GPRl ligand and/or GPRl (e.g., calcium fluxes) may be measured as a determination of an interaction of a GPRl with a GPRl ligand or a test compound.

[0013] In many instances, cells used in the practice of the invention will express GPRl endogenously. In other instances, the cells will contain a GPRl which is not naturally associated with those cells. For example, cells may be transformed or transfected with an isolated nucleic acid molecule which encodes GPRl or a molecule that modulates activity or expression of a GPRl . In some embodiments, cells used in the practice of the invention may be eukaryotic (e.g., mammalian, yeast, etc.) or prokaryotic.

[0014] From a temporal perspective, with respect to assay steps, GPRl and/or cells may be contacted with one or more test compounds and a GPRl ligand or GPRl binding fragment of a GPRl ligand simultaneously or at different times (e.g., a known ligand first, followed by a test compound or a test compound first, followed by a known ligand). In some embodiments, the known ligand is labeled.

[0015] The invention also includes methods for determining if a test compound modulates GPRl activity. In some aspects, such methods comprise contacting a compound to a cell which has been transformed or transfected with (a) a nucleic acid molecule which comprises: (i) a nucleotide sequence which encodes GPRl or subportion thereof (e.g., a GPRl modified to increase interaction with the test protein), (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein (e.g., a transcription factor such as tTA or GAL4) which activates a reporter gene (e.g., an exogenous gene to the cell in which it is introduced (e.g., β- galactosidase, β-lactamase or luciferase when a cell is employed which does not normally express the particular reporter or expresses it at low levels)) in the cell, and (b) a nucleic acid molecule which comprises: (i) a nucleotide sequence which encodes a test protein (e.g., an inhibitory protein such as arrestin) whose interaction with the GPRl in the presence of the test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site. In many embodiments, these methods further include determining one or more activities of the reporter gene as a determination of whether the compound modulates GPRl activity. In particular embodiments, the methods include contacting the cell with a GPRl ligand or a GPRl binding fragment thereof.

[0016] GPRl ligands include, but are not limited to, chemerin and GPRl binding fragments thereof.

[0017] While a considerable number of proteases may be employed in the practice of the invention, one particularly useful protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

[0018] In some embodiments, GPRl may be modified to have particular structural and/or functional characteristics. As an example, GPRl may be modified by replacing all or part of the nucleotide sequence of the C-terminal region of GPRl with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for the second test protein than the original sequence. Specific modifications of GPRl include modifications where the nucleotide sequence of the C-terminal region is replaced by a nucleotide sequence encoding all or a part of the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B, CCR4, or GRPR.

[0019] In particular embodiments of the invention, methods may be employed where more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, etc., such as two to ten, two to five, etc.) compound is contacted with a plurality of samples of cells (e.g., two, three, four, eight, ten, fifteen, eighteen, fifty, one hundred, three hundred, etc., such as two to five hundred, four to three hundred, ten to three hundred, fifteen to three hundred, thirty to three hundred, fifty to five thousand, ninety to ten thousand, ninety to one thousand, etc.), each of the samples being contacted by one or more of the compounds. In particular embodiments, one or more (or all) of the cells in the cell samples may be transformed or transfected with one or more (e.g., two, three, four, etc.) nucleic acids encoding a GPRl (e.g., one or more nucleic acid described herein). In many instances, one or more activities of reporter genes in the plurality of the samples will be determined to determine if any of the compounds interacts with GPRl.

[0020] In some instances, samples with may be contacted with more than one compound, wherein each compounds differs from all others. In some instances, some of the compounds may be the same. As for example, assays can be run in duplicate or other replicates (e.g., triplicate, quadruplicate, etc.).

[0021] The invention is further directed to methods for determining if a test compound is a GPRl ligand inhibitor or GPRl inhibitor. In some instances, such methods may comprise contacting a test compound and a GPRl ligand with GPRl or a GPRl fragment, determining activity of the GPRl ligand or GPRl in the presence of the test compound and optionally comparing activity in absence of the test compound or at a different concentration of the test compound. Identification of a decrease in GPRl activity in the presence of the test compound is indicative of a GPRl ligand inhibitor or GPRl inhibitor. Identification of an increase in GPRl activity in the presence of the test compound is indicative of a GPRl ligand activator or GPRl activator.

[0022] In particular embodiments of the invention, GPRl ligands or GPRl ligand fragments that bind GPRl may be labeled. These labels may be used for any number of purposes including competitive binding assays and GPRl ligand/receptor interactions. A considerable number of labels are know in the art and may be used in the practice of the invention, including radiolabels, dyes, fluorescent labels, and luminescent labels. In many instances, labels will be associated with GPRl ligands or fragments thereof in a manner which does not alter the functional activity of the GPRl ligand or fragment thereof.

[0023] In some aspects, the invention includes methods which involve contacting a test compound and a GPRl ligand, to GPRl or a GPRl fragment. In specific aspects, at least one of a GPRl ligand and/or the test compound is labeled. Further, the amount of label bound to GPRl or a GPRl fragment, as compared to binding of the label in the absence of unlabelled substance may be determined.

[0024] In some embodiments, a GPRl or GPRl fragment may be expressed by a cell, in a manner such that one or more activity of a GPRl ligand is a downstream property following binding of the GPRl ligand thereto.

[0025] The invention further includes methods for identifying analogues of GPRl ligands, comprising contacting a test compound to GPRl or a fragment of GPRl, and determining if the test compound binds to the GPRl or fragment of GPRl and/or exhibits a property exhibited by a GPRl ligand upon binding to GPRl or the fragment of GPRl, wherein presence of the property indicates the test compound is a GPR ligand analogue.

[0026] In some embodiments, the test compound will exhibit greater activity than a GPRl ligand upon the binding. In some embodiments, a test compound will exhibit lower activity than a GPRl ligand upon the binding.

[0027] The invention additionally includes methods for modulating activity of a GPRl. In particular embodiments, such methods include contacting GPRl with a substance which binds to GPRl, in an amount sufficient to bind to GPRl and modulate its activity. In some embodiments, this substance will not be a chemerin molecule. Further, the substance may be a non-orthosteric binding partner for GPRl. Additionally, the substance may be an allosteric modifier of GPRl . In particular embodiments, the substance may be a chemerin or chemerin derivative (e.g., chemerin 145-157 as set out in SEQ ID NO:7).

[0028] The invention includes methods for determining if a test compound behaves as an analogue of a GPRl ligand. In some embodiments, such methods comprise contacting (a) the test compound, and (b) chemerin or (ii) another agonist of GPRl, and comparing interaction of (b) with GPRl in the presence of (a), to its interaction with GPRlin the absence of (a), as a determination of whether the test compound interacts with GPRl and modulates agonistic activity of GPRl. In some instances, the test compound may exhibit greater activity than chemerin upon interaction with (e.g., binding) to GPRl. In specific embodiments, methods of the invention will include those which involve contacting the test compound and a GPRl ligand, to GPRl or a GPRl fragment, wherein one of a GPRl ligand or the test compound are labeled, and determining label bound to GPR lor the GPRl fragment, to binding of the label in the absence of unlabelled substance. Further, the GPRl or fragment of GPRl may be expressed by a cell used in such methods.

[0029] In some embodiments of the invention, a GPRl ligand is a chemerin or a chemerin derivative, e.g., comprising chemerin 145-157 (SEQ ID NO:5) as set out in SEQ ID NO:7.

[0030] In some embodiments, a GPRl may be present as an endogenously expressed molecule or in the context of a transformed or transfected cell. In some embodiments, a chemerin and test material may be admixed in a test assay, or may be tested separately.

[0031] Some assays of the invention may be carried out extracellularly or on a cellular basis. In some embodiments, carrying out assays using cells which express GPRl permits determination of modulators via a downstream activity assay, which an extracellular assay may not allow.

[0032] In some embodiments, an interaction of a modulator or test compound with GPRl may be determined, for example, by comparing the binding of chemerin or a fragment of chemerin which binds to GPRl, in the presence and/or absence of the test compound, where the chemerin molecule and/or the test compound is labeled with a detectable label.

[0033] Labeled molecules can be used, but are not necessary, e.g., when a cell based assay is used. This is because the interaction of the molecules with GPRl leads to a chain of "downstream activities," which can be measured, e.g., changes in calcium or cAMP levels.

[0034] In some embodiments, a format for carrying out an assay is that which is described in application Serial No. 10/888,313. [0035] In some embodiments, the test compounds will be added in the presence of a GPRl ligand (e.g., chemerin), when the GPRl ligand is present at a suboptimal stimulatory concentration. In some embodiments, the test compound may stimulate GPRl activity in the presence of a GPRl ligand molecule, but not when a GPRl ligand (e.g., chemerin) is lacking in the test well.

[0036] The invention also includes contacting GPRl will suboptimal concentrations or amounts of chemerin. By suboptimal concentration is meant a concentration of a molecule which does not induce the full response in the system in which it is involved. For example, with respect to a molecule which activates a GPRl, a suboptimal concentration would be an amount which generates less than the full response of the receptor. Thus, a concentration over the "optimal" concentration will not generate an increased response because the response is already maximal.

[0037] Thus, in some instances, the invention includes methods which involve contacting GPRl with a molecule known to induce receptor activity, wherein the concentration of the molecule is such that a maximal (i.e., 100%) response is not elicited from the receptor. In some embodiments, the concentration of the molecule may be designed to elicit a response ranging from about 0.5% to about 100%, from about 25% to about 75%, from about 10% to about 90%, from about 25% to about 80%, from about 30% to about 75%, from about 0.5% to about 50%, from about 5% to about 50%, from about 10% to about 60%, from about 40% to about 100%, from about 40% to about 90%, from about 40% to about 80%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 80%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 85% of the maximal response under the conditions employed. Such methods are particularly useful for looking for mimetics of molecules such as chemerin. Thus, in specific embodiments, the invention includes methods where GPRl is exposed to a suboptimal concentration of chemerin and another compound which is to be tested for properties associated with a chemerin mimetic.

[0038] The invention also includes recombinant cells which are transformed or transfected with nucleic acid molecules discussed herein. In specific aspects, such recombinant cells include those which contain (a) a nucleic acid molecule which comprises (i) a nucleotide sequence which encodes GPRl or subportion thereof, (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell, and (b) a nucleic acid molecule which comprises (i) a nucleotide sequence which encodes a test protein whose interaction with GPRl in the presence of the test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site.

[0039] Nucleic acid molecules (e.g., reporter constructs) introduced into cells in the practice of the invention may be stably incorporated into the genome of the cell. In some embodiments, nucleic acid molecules will be introduced in a manner in which they are not designed for incorporation into the genome of the cell. In such instances, these nucleic acid molecules may be transiently retained by the cells.

[0040] The invention further includes nucleic acid molecules, such a nucleic acid molecule which may be used in the practice of assay methods discussed herein. Some embodiments of the invention include, but are not limited to, isolated nucleic acid molecules which contain, in 5' to 3' order, (i) a nucleotide sequence which encodes GPRl or a GPRl fragment, (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell. In many instances, such nucleic acid molecules will also be designed to express the above (e.g., the above construct will be operably connected to a promoter). In some embodiments, the protease or portion of a protease used in this aspect of the invention may be tobacco etch virus nuclear inclusion A protease.

[0041] The invention also includes protein expression products of the nucleic acid molecules discussed herein (e.g., a fusion protein produced by expression of the isolated nucleic acid molecule described above).

[0042] The invention also provides kits, such as kits for performing assays. In specific embodiments, such a kit is useful for determining if a test compound modulates GPRl activity. Test kits of this type may contain one or more of the following: (a) a nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GPRl (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell (b) a nucleic acid molecule which comprises: (i) a nucleotide sequence which encodes a test protein whose interaction with the GPRl in the presence of the test compound is to be measured, (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site, and (c) one or more containers (e.g., containers for holding each of (a) and (b) separately from each other). Other components which may be included in kits of the invention include items described herein (e.g., buffers, enzymes, substrates for reporters, such as CCF2, etc.) [0043] Some embodiments of the invention, provide an expression vector comprising an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes GPRl (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in said cell, and further being operably linked to a promoter.

[0044] An additional embodiment comprises a fusion protein produced by expression of: an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell, and further being operably linked to a promoter; or an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GPRl in the presence of a test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site.

[0045] The protease or portion of a protease may be tobacco etch virus nuclear inclusion A protease. The protein which activates the reporter gene may be a transcription factor, such as tTA or GAL4. The test protein may be an inhibitory protein, such as an arrestin. The kit may further comprise a separate portion of an isolated nucleic acid molecule which encodes a reporter gene. In some embodiments, a reporter gene may encode β- galactosidase, β-lactamase or luciferase. The nucleotide sequence encoding GPRl may be modified to increase interaction with the second test protein, such as by replacing all or part of the nucleotide sequence of the C-terminal region of the first test protein with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for the second test protein than the original sequence. The nucleotide sequence of the C-terminal region may be replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B, CCR4, or GRPR.

[0046] An aspect of the present invention relates to methods for determining if a substance of interest binds to GPRl and e.g., shares properties with chemerin, or interferes therewith. The methodology involves co-transforming or co-transfecting a cell, which may be prokaryotic or eukaryotic, with two constructs. The first construct includes, a sequence encoding (i) GPRl, such as a transmembrane receptor, (ii) a cleavage site for a protease, and (iii) a sequence encoding a protein which activates a reporter gene. The second construct includes, (i) a sequence which encodes a test protein whose interaction with GPRl is measured and/or determined, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease sufficient to act on the cleavage site that is part of the first construct. In some embodiments, these constructs become stably integrated into the cells.

[0047] In some embodiments, there is provided an isolated nucleic acid molecule which comprises, (i) a nucleotide sequence which encodes a test protein whose interaction with GPRl in the presence of a test compound is to be measured, and (ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for said cleavage site. The test protein may be an inhibitory protein, such as an arrestin.

[0048] The invention also includes methods for measuring the ability of a compound to alter GPRl activity. In some embodiments, such methods comprise (a) contacting a cell which expresses a GPRl and generates an intracellular signal normally associated with a GPRl with (1) a GPRl ligand molecule or fragment thereof and (2) a compound to be screened for modulation of GPRl activity with respect to signal generation, (b) measuring a signal generated in (a), and (c) comparing the signal in (b) with a signal generated in the absence of the compound or at a different concentration of the compound. In specific embodiments, the GPRl ligand molecule is a fragment of a naturally occurring GPRl ligand molecule which, upon interaction with a GPRl, retains the ability to generate an intracellular signal normally associated with a GPRl .

[0049] In some embodiments, methods to measure the ability of a test compound to alter GPRl activity in the presence of a suboptimal stimulatory concentration of a GPRl ligand binding molecule can include (a) intracellular calcium release assays (e.g., using calcium indicator dyes), (b) intracellular cAMP generation assays (e.g., using antibody or enzyme complementation probes for {cAMP} quantitation), (c) reporter gene assays using various transcriptional response elements (e.g., calcium and/or cAMP responsive) acted upon by GPCR signaling pathways, (d) GTP-gammaS recruitment assays using GPRl -containing membrane preparations, and/or (e) ligand binding competition assays using a labeled form of a GPRl ligand (such as chemerin or a fragment thereof) and a GPRl -containing membrane preparation.

BRIEF DESCRIPTION OF THE FIGURES

[0050] Figure 1 shows a dose-response of GPRl and CMKLRl in a cell-based arrestin assay to a recombinant chemerin protein (IA) and chemerin 145-157 peptide (IB). Response in each case is normalized to percent maximal response. [0051] Figure 2 shows a dose-response of GPRl (native c-terminal tail) in the cell- based arrestin assay to recombinant chemerin protein.

[0052] Figure 3A-C shows a calcium mobilization assay showing activation of CMKLRl and GPRl by chemerin peptide (chemerin 149-157) in the presence of the promiscuous G-protein Gα-15. Time of ligand addition is indicated by the arrow.

[0053] Figure 4 shows an exemplary schematic of functional properties of some assays of the invention. In this figure, a "Ligand" is shown binding to a GPCR (shown as a seven transmembrane spanning protein). Association of the GPCR with a g-protein is indicated. Further indicated is the production of phospholipase C (PLC), protein kinase C (PKC), map kinases (MAPK), and beta-lactamase coding region (bla), and calcineurin (CN).

[0054] Figure 5 is a schematic which shows various aspects of the beta-lactamase reporter system, including the CCF2 substrate which may be used in assays employing this system. The fluorescent CCF2 substrate is a sensitive reporter of gene expression in living mammalian cells. The membrane-permeant, esterified form of the substrate (CCF2-AM) readily enters the cell where endogenous esterases convert it into its negatively charged form, thereby trapping the substrate in the cytosol. Incorporating an efficient FRET pair into the substrate enables detection of bla activity. Cells with no bla expression (intact substrate) fluoresce green, while cells with bla expression (cleaved substrate) fluoresce blue.

BRIEF DESCRIPTION OF THE SEQUENCES

[0055] SEQ ID NO:1 is a Homo sapiens G-protein-coupled receptor 1 (GPRl) with the following 355 amino acid sequence:

MEDLEETLFEEFENYSYDLDYYSLESDLEEKVQLGVVHWVSLVLYCLAFVLGIPGNA

IVIWFTGFKWKKTVTTLWFLNLAIADFIFLLFLPLYISYVAMNFHWPFGIWLCKANSF

TAQLNMFASVFFLTVISLDHYIHLIHPVLSHRHRTLKNSLIVIIFIWLLASLIGGPALYFR

DTVEFNNHTLCYNNFQKHDPDLTLIRHHVLTWVKFIIGYLFPLLTMSICYLCLIFKVK

KRSILISSRHFWTILVVVVAFVVCWTPYHLFSIWELTIHHNSYSHHVMQAGIPLSTGL

AFLNSCLNPILYVLISKKFQARFRSSVAEILKYTL WEVSCSGTVSEQLRNSETKNLCLL

ETAQ.

[0056] SEQ ID NO:2 is a variant cleavage site for TEV NIa-Pro with the following amino acid sequence: GSENLYFQL.

[0057] SEQ ID NO: 3 is a Homo sapiens gastrin-releasing peptide receptor (GRPR) with the following 384 amino acid sequence: MALNDCFLLNLEVDHFMHCNissHSADLPVNDDWSHPGiL YVIP AVYGVIILIGLIGNI

TLIKIFCTVKSMRNVPNLFISSLALGDLLLLITCAP VD ASRYLADRWLFGRIGCKLIPFI QLTSVGVSVFTLTALSADRYKAIVRPMDIQASHALMKICLKAAFIWIISMLLAIPEAVF

SDLHPFHEESTNQTFiscAP YPHSNELHPKIHSMASFLVFYVIPLSIISVYYYFIAKNLIQ

SAYNLPVEGNIHVKKQIESRKRLAKTVLVFVGLFAFCWLPNHVIYLYRSYHYSEVDT SMLHFVTSICARLLAFTNSCVNPFALYLLSKSFRKQFNTQLLCCQPGLIIRSHSTGRST TCMTSLKSTNPSVATFSLINGNICHERYV.

[0058] SEQ ID NO:4 is a Homo sapiens chemokine-like receptor 1 (CMKLRl) with the following 371 amino acid sequence: MEDEDYNTSISYGDEYPDYLDSIVVLEDLSPLEARVTRIFL VVVYSIVCFLGILGNGLV

IIIATFKMKKTVNMVWFLNLAVADFLFNVFLPIHITY AAMD YHWVFGTAMCKISNFL

LIHNMFTSVFLLTIISSDRCISVLLPVWSQNHRSVRLAYMACMVIWVLAFFLSSPSLVF

RDTANLHGKISCFNNFSLSTPGSSSWPTHSQMDPVGYSRHMVVTVTRFLCGFLVPVLI

ITACYLTIVCKLQRNRLAKTKKPFKIIVTIIITFFLCWCPYHTLNLLELHHTAMPGSVFS

LGLPLATAL AIANSCMNPILYVFMGQDFKKFKVALFSRLVN ALSEDTGHSSYPSHRSF

TKMSSMNERTSMNERETGML.

[0059] SEQ ID NO: 5 is a 13 -amino acid C-terminal chemerin peptide with the following amino acid sequence: PHSFYFPGQFAFS.

[0060] SEQ ID NO: 6 is a cleavage site with the following amino acid sequence: ENLYFQL.

[0061] SEQ ID NO:7 is a chemerin (a.k.a. tig2) with the following 163 amino acid sequence:

MRRLLIPLALWLGAVGVGVAELTEAQRRGLQVALEEFHKHPPVQWAFQETSVESAV DTPFP AGIFVRLEFKLQQTSCRKRD WKKPECKVRPNGRKRKCLACIKLGSEDKVLGR LVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQF AFSKALPRS.

[0062] SEQ ID NO:8 codes for a Homo sapiens GPRl (SEQ ID NO:1) with the following sequence:

1 ataaaagtggaatgaggaatgcagccgttctgaacaccaccctccatttcattctggaac

61 cgggaaggtacacccaggcatgacaatagcttctctcctcacagaaatttaactgatttc

121 ttcattctccatttagcaaggtcatggaagatttggaggaaacattatttgaagaatttg

181 aaaactattcctatgacctagactattactctctggagtctgatttggaggagaaagtcc

241 agctgggagttgttcactgggtctccctggtgttatattgtttggcttttgttctgggaa

301 ttccaggaaatgccatcgtcatttggttcacggggttcaagtggaagaagacagtcaeca

361 ctctgtggttcctcaatctagccattgcggatttcatttttcttctctttctgcccctgt

421 acatctcctatgtggccatgaatttccactggccctttggcatctggctgtgcaaagcca

481 attccttcactgcccagttgaacatgtttgccagtgtttttttcctgacagtgatcagcc

541 tggaccactatatccacttgatccatcctgtcttatctcatcggcatcgaaccctcaaga

601 actctctgattgtcattatattcatctggcttttggcttctctaattggcggtcctgccc 661 tgtacttccgggacactgtggagttcaataatcatactctttgctataacaattttcaga

721 agcatgatcctgacctcactttgatcaggcaccatgttctgacttgggtgaaatttatca

781 ttggctatctcttccctttgctaacaatgagtatttgctacttgtgtctcatcttcaagg

841 tgaagaagcgaagcatcctgatctccagtaggcatttctggacaattctggttgtggttg

901 tggcctttgtggtttgctggactccttatcacctgtttagcatttgggagctcaccattc

961 accacaatagctattcccaccatgtgatgcaggctggaatccccctctccactggtttgg

1021 cattcctcaatagttgcttgaaccccatcctttatgtcctaattagtaagaagttccaag

1081 ctcgcttccggtcctcagttgctgagatactcaagtacacactgtgggaagtcagctgtt

1141 ctggcacagtgagtgaacagctcaggaactcagaaaccaagaatctgtgtctcctggaaa

1201 cagctcaataagttattacttttccacaaatcagtatatggctttttatgtgggtcctct

1261 gactgatgctttcagattaaaattgtttccaagatagagagccgactccactttcatagt

1321 tattgtttctggtcactatataggcatcacatttttgtgtggatatgaaacttaggaagg

1381 atcctcttgactccttgtgatgtggcaataaattttttttaaaaaactgaaaatacttag

1441 gaaggatccgcataatttttttctgcaacttaaatgaaatgcatcattcttgttaattat

1501 accatggtgaattaatcacttttgaagcaatatcagttattttttgaataataacttttc 1561 taaagccttaagtcttaatattaaatatatgattagccaggcacggtggctgacacctgt 1621 aatcccagcactttgggaggccaaggtggggggattacccgaggtcaggaattcgagacc 1681 agcctgaccaacatggagaaaccccgtctctactaaaaatacaaaattagccggtcatgg

1741 tggtgcatgtctgcaaacccagctactcgggaggctgaagcaggagaatcacttgaacct

1801 gggaggcagaggttgtggtgagccaacatcacaccattgcactccagcctgggcaacaag 1861 agtaaaactctgtctcaaaaataaataaataaaatagataaataaatatatgattaacta 1921 attttaaaaatgttaaaatgtattcttaaattcattttaattttgtacaataacctgcta 1981 gacacatttttaaaatgcaacatgtgtacttaatttctttatgtaatctatgtatataca 2041 tttatgaattaaagtaattgttggttatcttaaaaaaaaaaaaaaaaaa

[0063] SEQ ID NO:9 contains a coding sequence for a Homo sapiens CMKLRl (SEQ ID NO:4) with the following sequence:

1 gaattcggcacgagtcagggaagcagccccggcggccagcagggagctcaggacagagca

61 ggctccctgggaagcctccgggtgataggggtgttccagctgcggcgctctgggggttca

121 gagggggatcttgaatgaacaaatgaatgaactgctttctgggcaaacagccacagccag

181 aggagcctgtgattggcagaaagaagccagggtgtgcaagtctccccaacagcctcgagt

241 ggcctgcagtcacagggaaccctcaggaagaccttccgggcagagaccagagggaagccc

301 atctctccagcagaactgcttggatttttctaccaggaggctcagggctctgcaacaatg

361 atagcagaagctgatggcatctagagatctaggctgggactagcacagcatcacttctac

421 cactttctgttggtcacagcaactcaccatgccagtgcagattcaaggggaggagaaata

481 gagtccacttcttgatgggaggcgtgacatagaatggaggatgaagattacaacacttcc

541 atcagttacggtgatgaataccctgattatttagactccattgtggttttggaggactta

601 tcccccttggaagccagggtgaccaggatcttcctggtggtggtctacagcatcgtctgc

661 ttcctcgggattctgggcaatggtctggtgatcatcattgccaccttcaagatgaagaag

721 acagtgaacatggtctggttcctcaacctggcagtggcagatttcctgttcaacgtcttc

781 ctcccaatccatatcacctatgccgccatggactaccactgggttttcgggacagccatg

841 tgcaagatcagcaacttccttctcatccacaacatgttcaccagcgtcttcctgctgacc

901 atcatcagctctgaccgctgcatctctgtgctcctccctgtctggtcccagaaccaccgc

961 agcgttcgcctggcttacatggcctgcatggtcatctgggtcctggctttcttcttgagt

102 ltccccatctctcgtcttccgggacacagccaacctgcatgggaaaatatcctgcttcaac

1081 aacttcagcctgtccacacctgggtcttcctcgtggcccactcactcccaaatggaccct

1141 gtggggtatagccggcacatggtggtgactgtcacccgcttcctctgtggcttcctggtc

1201 ccagtcctcatcatcacagcttgctacctcaccatcgtgtgcaaactgcagcgcaaccgc

1261 ctggccaagaccaagaagcccttcaagattattgtgaccatcatcattaccttcttcctc

1321 tgctggtgcccctaccacacactcaacctcctagagctccaccacactgccatgcctggc

1381 tctgtcttcagcctgggtttgcccctggccactgcccttgccattgccaacagctgcatg

1441 aaccccattctgtatgttttcatgggtcaggacttcaagaagttcaaggtggccctcttc

1501 tctcgcctggtcaatgctctaagtgaagatacaggccactcttcctaccccagccataga 1561 agctttaccaagatgtcatcaatgaatgagaggacttctatgaatgagagggagaccggc 1621 atgctttgatcctcactgtggaacccctcaatggactctctcaacccagggacacccaag 1681 gatatgtcttctgaagatcaaggcaagaacctctttagcatccaccaattttcactgcat

1741 tttgcatgggatgaacagtgttttatgctgggaatctagggcctggaacccctttcttct

1801 agtggacagaacatgctgtgttccatacagccttggactagcaatttatgcttcttggga 1861 ggccagccttgactgactcaaagcaaaaaaggaagaattc

[0064] SEQ ID NO: 10 contains a coding sequence for a Homo sapiens CMKLRl (SEQ ID NO:3) with the following sequence:

1 aaaggtatctgttaagctaggtaggaactgcagtcggctggttgcttctcatctggagaa 61 agcaggcaactgggcagtgattgaagtgtccagcagggggctggcattctctgtctataa 121 gtaacactggttcctcttcagagcctcagctcagcggagctgccgtttgctggtgaagcc 181 cgtgacgtgcaaagcatcctgcctataggatttgaggatttctcagtgcagtttttttct 241 acccactttaaacctccagattctaaatatcaggaaagacgctgtgggaaaatagcaggc 301 caaaagttcttagtaaactgcagccagggagactcagactagaatggaggtagaaagaac 361 tgatgcagagtgggtttaattctaagcctttttgtggctaagttttgttgttgttaactt 421 attgaatttagagttgtattgcactggtcatgtgaaagccagagcagcaccagtgtcaaa 481 atagtgacagagagttttgaataccatagttagtatatatgtactcagagtatttttatt 541 aaagaaggcaaagagcccggcatagatcttatcttcatcttcactcggttgcaaaatcaa 601 tagttaagaaatagcatctaagggaacttttaggtgggaaaaaaaatctagagatggctc 661 taaatgactgtttccttctgaacttggaggtggaccatttcatgcactgcaacatctcca 721 gtcacagtgcggatctccccgtgaacgatgactggtcccacccggggatcctctatgtca 781 tccctgcagtttatggggttatcattctgataggcctcattggcaacatcactttgatca 841 agatcttctgtacagtcaagtccatgcgaaacgttccaaacctgttcatttccagtctgg 901 ctttgggagacctgctcctcctaataacgtgtgctccagtggatgccagcaggtacctgg 961 ctgacagatggctatttggcaggattggctgcaaactgatcccctttatacagcttacct 1021 ctgttggggtgtctgtcttcacactcacggcgctctcggcagacagatacaaagccattg 1081 tccggccaatggatatccaggcctctcatgccctgatgaagatctgcctcaaagccgcct 1141 ttatctggatcatctccatgctgctggccattccagaggccgtgttttctgacctccatc 1201 ccttccatgaggaaagcaccaaccagaccttcattagctgtgccccatacccacactcta 1261 atgagcttcaccccaaaatccattctatggcttcctttctggtcttctacgtcattccac 1321 tgtcgatcatctctgtttactactacttcattgctaaaaatctgatccagagtgcttaca 1381 atcttcccgtggaagggaatatacatgtcaagaagcagattgaatcccggaagcgacttg

1441 ccaagacagtgctggtgtttgtgggcctgttcgccttctgctggctccccaatcatgtca

1501 tctacctgtaccgctcctaccactactctgaggtggacacctccatgctccactttgtca 1561 ccagcatctgtgcccgcctcctggccttcaccaactcctgcgtgaacccctttgccctct 1621 acctgctgagcaagagtttcaggaaacagttcaacactcagctgctctgttgccagcctg 1681 gcctgatcatccggtctcacagcactggaaggagtacaacctgcatgacctccctcaaga

1741 gtaccaacccctccgtggccacctttagcctcatcaatggaaacatctgtcacgagcggt

1801 atgtctagattgacccttgattttgccccctgagggacggttttgctttatggctagaca 1861 ggaacccttgcatccattgttgtgtctgtgccctccaaagagccttcagaatgctcctga 1921 gtggtgtaggtgggggtggggaggcccaaatgatggatcaccattatattttgaaagaag 1981 ccatcaagtcttaagtttttcatttcaacttgtgaacgtttcttctgatgtgaagcaaac 2041 cttcccttttcagaaaagggaacaagtagaaaattattttttaagcctcaagccctgtta 2101 aatggtcgtggccaattatgtcatagaaactgtatgaacaaccagatttacatagcagag 2161 aaatcatacattgaatgcttactttgtgaaagacttcaccttgtcatttctttaagcaga 2221 cgctagtactttagaaatataacttgactctgttttcaggaatatctgtaatacacaaac 2281 caaggaacaacttttatttacactcctaatatgaaaagtcaatcctgtgagagagctcca 2341 tgtatgagggacactctccaagttgataacaatggaagcgagtttaatataaaacaattc

2401 cctaagcatttattttttttttaaaaagatgttactgaggacctagaagaaatgctcaat

2461 acatactttgaaagcaaaaatacaatcaaacacattgacacgtatataaagatccacgcg 2521 tggctgtgcgtgatatctcacactctgaattcttacttgatggaggttttgtttgctgct

2581 acggttttaatcatccagggtgccattccaccatagaagagcaatccttttaggaaaaaa 2641 aaaatcatgctattaattaatcaaatatctataaatgcata DETAILED DESCRIPTION OF EMBODIMENTS

[0065] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0066] These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

[0067] The invention relates, in part, to compositions and methods for use in biological assays. The invention further relates to methods for identifying compounds which alter the interaction between proteins. One example of such an interaction is that between a receptor, such as a GPCR, and a ligand. Thus, the invention may be used to identify compounds which modulate the interaction between a receptor and a ligand. The invention may also be used to identify binding compatible binding partners (e.g., a receptor and a ligand which binds to that receptor). The invention also comprises compositions used in these methods.

[0068] In some aspects, the invention provides compositions and methods for detecting interactions between ligands and their specific GPCRs. Assays of the invention will often rely upon the interaction of a GPRl receptor or GPRl receptor fragment with a ligand (e.g., a chemerin or chemerin fragment).

[0069] Compositions of the invention may be used in a variety of assays, for example, receptor interaction assays (e.g., GPCR assays).

[0070] A number of GPCR assays are known in the art. In many instance, such assays are based upon the principle of binding of a compound to a GPCR followed by detection of either the binding itself or effects resulting from bind.

[0071] Most GPCRs carry information within cells by way of one of two major signaling pathways: (1) regulation of cAMP levels and (2) increases in intracellular Ca²⁺ triggered by inositol (1,4,5) tri-phosphate (IP3). These signaling pathways are activated by the specific G protein associated with the receptor. Gs and Gi coupled receptors result in variations of cAMP while Gq coupled GPCRs activate phospholipase C (PLC) and trigger the inositol phosphate (IP) cascade. Assays which detect GPCR activity may focus on any one or more of these processes and/or products.

[0072] GPCR assays used in the practice of the invention may be "cell containing" or "cell free" assays. A cell free assay is one in which the assay is performed outside of a cell.

[0073] A number of commercially available GPCR assays, as well as assays for other types of receptors, are available. For example, a considerable number of GPCR assays are available from Invitrogen Corporation (Carlsbad, CA) (see, e.g., products associated with cat. nos. K1241 and Kl 130).

[0074] Using the schematic shown in Figure 4 for purposes of illustration, GPCR assays may be designed such that a ligand(s) binds to a membrane associated GPCR resulting in the production of PLC or cAMP. Beta-lactamase is expressed regardless of the GPCR stimulation pathway. In assays such as these, beta-lactamase expression may be detected by monitoring the amount of cleaved beta-lactamase substrate generated. Of course, the invention includes numerous variations of the assays described in Figure 4. For example, any number of reporters may be employed.

[0075] Figure 5 shows a fluorescence resonance energy transfer (FRET) substrate for beta-lactamase which may be used in the practice of the invention. This type of substrate is available from Invitrogen Corporation (e.g., cat. no. Kl 025) and will often be used with a beta-lactamase gene which encodes an expression product which is retained within the cell in which it is expressed (e.g., lacks a functional signal peptide). The beta-lactamase reporter system is described in U.S. Patent Nos. 5,741,657, 6,291,162, and 6,472,205.

[0076] A number of additional GPCR assays are known in the art. For example, Promega Corporations publication Paguio et ah, Cell Notes 16:22-25 (2006) describes the use of their Dual-Glo Luciferase Assay System and pGL4 Luciferase Reporter Vectors in GPCR assays.

[0077] The invention further includes GPCR assays which employ fluorescence polarization. For example, a number of fluorescence polarization GPCR assays are available from Perkin-Elmer (Shelton, CT) (see, e.g., cat. no. FPAlOl and FPA203002KT). In one of these assays, a fluorescent ligand is incubated with a GPCR and, optionally one or more other compounds (e.g., compounds which may inhibit or enhance ligand binding to the GPCR) and fluorescence polarization is read to determine whether ligand-GPRC binding events have occurred or are altered by the compounds. In other assays, the amount of cAMP is measured using fluorescence polarization.

[0078] The invention thus is directed to, in part, assays for measuring GPCR activity and changes in that activity under different conditions. Such methods include those which involve measuring changes in GPCR activity when the GPCR is contacted with one or more compounds (e.g., drug candidates).

[0079] When a GPCR is contacted with one or more compounds, such as a drug candidate, the GPCR may be contacted with one such compound or more than one compound. In some embodiments, more than one GPCR sample may be contacted with separate compounds. As an example, all of the wells of a 96 microtiter plate may contain a GPCR and a known ligand. Further, 90 of the wells may each additionally contain a different compound which is to be tested for the ability to modulate (e.g., enhance or repress GPCR activation) the activation of the GPCR in the presence of the ligand. In some embodiments, the other six wells may be control wells which either contain no added compound or a compound which is known to modulate the activation of the GPCR in the presence of the ligand. Thus, the invention includes methods for screening compounds for their effects on GPCR activation.

[0080] In one aspect, the invention provides TANGO assays which can be used to measure GPRl activity. A number of additional assays are discussed in Szekeres, Receptors and Channels, 5:297-308 (2002).

[0081] Changes in intracellular calcium levels may be used to measure GPCR activity. A number of reagents are available for monitoring calcium fluxes. For example, Invitrogen Corporation provides Fluo-3, Fluo-4 and Fluo-4 NW (no wash) Calcium Indicators which allow for imaging of intracellular calcium fluxes associated with GPCR activation or inhibition. Fluo-3 imaging has been used to study the spatial dynamics of many elementary processes in Ca²⁺ signaling and has been used for flow cytometry for experiments involving photoactivation of caged chelators, second messengers, neurotransmitters and for cell-based pharmacological screening.

[0082] Fluo-4 AM is an analog of the calcium indicator Fluo-3 AM. Fluo-4 AM loads faster and is brighter at equivalent concentrations, which typically makes it a good indicator for confocal microscopy, flow cytometry and microplate screening applications. The Fluo-4 NW (No Wash) Calcium Assay is the next generation calcium indicator in the Fluo calcium indicator family and was specifically developed for automated screening (HTS) applications. The Fluo-4 NW Assay also has the advantage of not requiring a quencher dye. Thus, the invention includes the use of detection methods by which changes in intracellular calcium are measured.

GPRl Molecules

[0083] In some embodiments, the GPCR used in the practice of the invention will be GPRl, a GPRl fragment, or a protein which has the activity of GPRl. By "a protein which has the activity of GPRl" is meant that the protein can bind to a ligand of GPRl such as a chemerin molecule and generate an intracellular activity or response normally associated with activation of GPRl.

[0084] Non-naturally occurring forms of GPRl proteins may be used in the practice of the invention. For example, some embodiments of the invention may employ a GPRl protein in which 0.5%-10% of the amino acids have been deleted. Using the GPRl protein of SEQ ID NO:1 for purposes of illustration, which contains 355 amino acids, this would include GPRl proteins which contain 320 to 354 amino acids.

[0085] Additional modified forms of GPRl which may be used in the practice include those where anywhere from 0.5% to 10% of the amino acids have been substituted. In some instances, these substitutions will be in the ligand binding domain and may either increase or decrease the affinity of GPRl for one or more ligand.

[0086] GPRl coding sequences may be modified to enhance their binding to their interacting protein, in assays of the invention. For example, it is known that certain GPCRs bind arrestins more stably or with greater affinity upon ligand stimulation and this enhanced interaction is mediated by discrete domains, e.g., clusters of serine and threonine residues in the C-terminal tail (Oakley et al., J. Biol. Chem., 274:32248-32257 (1999) and Oakley et al, J. Biol. Chem., 27(5:19452-19460 (2001). Using this as an example, it is clear that the GPRl encoding sequence itself may be modified, so as to increase the affinity of the membrane bound protein, such as the receptor, with the protein to which it binds. Exemplary of such modifications are modifications of the C-terminal region of the membrane bound protein, e.g., receptor, such as those described herein, which involve replacing a portion of it with a corresponding region of another receptor, which has higher affinity for a binding protein, but does not impact the receptor function.

[0087] In addition, a test protein may be modified to enhance its interaction with GPRl . For example, an assay may incorporate point mutants, truncations or other variants of the second test protein, e.g., arrestin, that are known to bind agonist-occupied GPCRs more stably or in a phosphorylation-independent manner (Kovoor et ah, J. Biol. Chem. 274:6831- 6834 (1999).

[0088] Also see U.S. Patent Application No. 60/686,876 which describes GPRl and related assays/methods.

GPRl Ligands and Modulators

[0089] While any number of ligands may be used in the practice of the invention, one ligand for GPRl is a chemerin or a fragment thereof such as SEQ ID NO:5. Chemerin molecules which may be used in the practice of the invention vary widely. An amino acid sequence of a chemerin is set out in SEQ ID NO:7. Chemerin molecules which may be used in the invention include chemerin molecules having the amino acid sequence set out in SEQ ID NO:7, as well as fragments of such molecules. In many instances, as compared to a naturally occurring chemerin molecule, a chemerin molecule will have the same activation activity, an increased, or a decreased activation activity.

[0090] In many instances, chemerin molecules of the invention, with reference to the amino acid sequence in SEQ ID NO:7, will comprise the amino acid sequences of amino acids 1-5 (e.g., the amino acid sequence MRRLL), 2-10, 3-15, 2-20, 2-25, 2-35, 2-50, 2-60, 2-80, 2-90, 2-100, 2-116, 55-116, 55-110, 55-105, 55-102, 55-100, 55-90, 55-85, 55-80, 55- 75, 30-116, 30-110, 30-105, 30-99, 30-89, 30-78, 35-116, 35-110, 50-115, 50-90, 50-80, 60- 116, 60-110, 60-105, 60-100, 70-116, 70-110, 70-105, 70-100, 75-116, 75-110, 80-116, 85- 116, 88-110, 90-110, 100-120, 110-130, 120-140, 130-150, 140-163 and/or 150-163. In many instances, chemerin molecules of the invention will retain at least some portion of an activity associated with a naturally occurring chemerin molecule.

[0091] Assays of the invention may also be used to identify additional ligands for GPRl . Such methods include contacting a GPRl with a potential ligand and then assays for GPRl activation.

[0092] When compounds are screened for their ability to modulate GPRl activity, the formats of these assays may vary considerably. When GPRl is contacted with a chemerin molecule and another compound which is to be tested for modulation of chemerin or GPRl activity, the ratio of the chemerin molecule to the other compound may vary considerably. For example, in a situation where the chemerin molecule and the other compound have identical affinity for GPRl and bind to GPRl competitively, a 1 :1 ratio of the chemerin molecule to the other compound will lead to 1 :1 competitive binding to GPRl. When the other compound does not activate GPRl, a 50% inhibition of GPRl activation by the chemerin molecule would be expected. When the chemerin molecule and the other compound bind competitively but chemerin has a higher affinity for GPRl than the other compound, more of the other compound will need to be present to achieve 50% inhibition of chemerin induced activation of GPRl . This assumes that binding of each molecule results in the same degree of GPRl activity, which may not be the case. In any event, ratios of chemerin molecules to other compounds used in compositions (e.g., kit components) and methods of the invention include the following: about 1 :1, about 1 :1.2, about 1 :1.4, about 1 :1.5, about 1 :1.8, about 1 :2, about 1 :2.5, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :8, about 1 :10, about 1 :20, about 1 :30, about 1 :40, about 1 :50, about 1 :80, about 0.95:1, about 0.90:1, about 0.85:1, about 0.8:1, about 0.75:1, about 0.7:1, about 0.6:1, about 0.5:1, about 0.4:1, about 0.3:1, about 0.2:1, about 0.15:1, about 0.1 :1, about 0.05:1, about 0.02:1, about 0.01 :1, about 0.005:1, about 0.002:1, etc. (e.g., from about 0.002:1 to about 1 :80, from about 1 :1 to about 1 :80, from about 1 :1 to about 1 :50, from about 1 :1 to about 1 :25, from about 1 :1 to about 1 :15, from about 1 :1 to about 1 :10, from about 1 :1 to about 1 :5, from about 0.5:1 to about 1 :10, from about 0.5:1 to about 1 :5, from about 0.5:1 to about 1 :5, from about 0.5:1 to about 1 :5, from about 0.5:1 to about 1 :5, from about 0.1 :1 to about 1 :80, from about 0.1 :1 to about 1 :50, from about 0.1 :1 to about 1 :30, from about 0.1 :1 to about 1 :25, from about 0.1 :1 to about 1 :10, from about 0.1 :1 to about 1 :5, from about 0.05:1 to about 1 :80, from about 0.05:1 to about 1 :20, from about 0.05:1 to about 1 :10, from about 0.05:1 to about 1 :5, etc.).

[0093] Compounds which may be screened for the ability to modulate GPRl activity include peptides/protein and non-protein molecules (e.g., lipids, carbohydrates, small organic molecules, etc.). Such compounds may bind to GPRl competitively with chemerin or non- competitively. Further, such compounds may bind to chemerin competitively with GPRl or non-competitively. Additional, molecular interactions may result in covalent or non-covalent bonds being formed between the respective molecules. The invention also includes methods for using compounds to identify those compounds which interact with either GPRl or chemerin and modulate (e.g., activate or inhibit) one or more GPRl or chemerin activities.

Labels

[0094] As discussed elsewhere herein, molecules used in the practice of the invention may be labeled. A wide variety of labels may be used depending on the particular application. [0095] As used herein, the term "label" means that a compound comprises at least one element, isotope, or chemical compound to enable the detection of the compound by any technique that would enable detection. Labels may be: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, "mTc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb, and ¹⁸⁶Re; b) immune labels, which may be antibodies or antigens, which may be bound to enzymes (such as horseradish peroxidase) that produce detectable agents; or c) colored, luminescent, phosphorescent, or fluorescent dyes.

[0096] It will be appreciated that the labels may be incorporated into a compound at any position that does not substantially interfere with the biological activity or characteristic of the compound that is being detected. In certain embodiments, hydrogen atoms in the compound are replaced with deuterium atoms (²H) to slow the degradation of compound in vivo. Due to isotope effects, enzymatic degradation of the deuterated compounds may be slowed thereby increasing the half- life of the compound in vivo. In other embodiments such as in the identification of the biological target of a natural product or derivative thereof, the compound is labeled with a radioactive isotope, preferably an isotope which emits detectable particles, such as beta-particles.

[0097] In certain other embodiments of the invention, photoaffinity labeling is utilized for the direct elucidation of intermolecular interactions in biological systems. A variety of known photophores can be employed, many of which rely on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (See, Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam). In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p-azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid.

Expression Constructs and Transformation

[0098] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes {e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis, et ah, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, 1990) and Ausubel, et ah, 1994, Current Protocols In Molecular Biology (John Wiley & Sons, 1996).

[0099] The term "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleotide sequences that serve other functions as well and are described infra.

[00100] In certain embodiments, a plasmid vector is contemplated for use in cloning and gene transfer. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.

[00101] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM ^'11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.

[00102] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.

[00103] Many prokaryotic vectors can also be used to transform eukaryotic host cells. However, it may be desirable to select vectors that have been modified for the specific purpose of expressing proteins in eukaryotic host cells. Expression systems have been designed for regulated and/or high level expression in such cells. For example, the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986 and

4,879,236, and which can be bought, for example, under the name MAXBAC® 2.0 from iNviTROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.

[00104] Other examples of expression systems include STRATAGENE®'S

COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.

Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

Regulatory Signals

[00105] The construct may contain additional 5' and/or 3' elements, such as promoters, poly A sequences, and so forth. The elements may be derived from the host cell, i.e., homologous to the host, or they may be derived from distinct source, i.e., heterologous.

[00106] A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

[00107] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of a promoter, one positions the 5 ' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[00108] The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis- acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[00109] A promoter may be one naturally associated with a nucleic acid molecule, as may be obtained by isolating the non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid molecule, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid molecule in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid molecule in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patents Nos. 4,683,202 and 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[00110] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook, et al, 1989). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[00111] Additionally any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, www.epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[00112] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in- frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements. [00113] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature, 334:320- 325 (1988)). IRES elements from two members of the picornavirus family (polio and encephalomyo carditis) have been described (Pelletier and Sonenberg, supra), as well an IRES from a mammalian message (Macejak and Sarnow, Nature, 353:90-94 (1991)). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819.

Other Vector Sequence Elements

[00114] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli, et al, FEMS Microbiol. Lett, 172(1):75-S2 (1999), Levenson, et al, Hum. Gene Ther. PfS,): 1233-1236 (1998), and Cocea, Biotechniques 23^:814-816 (1997)). "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[00115] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler, et al, 1997).

[00116] The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" comprises a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[00117] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 adenosine residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[00118] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not being limited to, for example, the termination sequences of genes, such as the bovine growth hormone terminator, viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as an untranslatable/untranscribable sequence due to a sequence truncation.

[00119] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both of which are convenient, readily available, and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

[00120] In order to propagate a vector in a host cell, it may contain one or more origins of replication (often termed "ori"), sites, which are specific nucleotide sequences at which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. Transformation Methodology

[00121] Suitable methods for nucleic acid delivery for use with the current invention are believed to include virtually any method by which a nucleic acid molecule (e.g., DNA) can be introduced into a cell as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson, et al, Science, 244:1344-1346 (1989), Nabel et al, Science, 244:1342-1344 (1989), by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859), including microinjection (Harlan and Weintraub, J. Cell Biol., 101 (3) : 1094-1099 (1985); U.S. Patent No. 5,789,215); by electroporation (U.S. Patent No. 5,384,253; Tur-Kaspa, et al, MoI. Cell Biol, 6:716-718 (1986); Potter, et al, Proc. Natl. Acad. Sci. USA, 57:7161-7165 (1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467 (1973); Chen and Okayama, MoI. Cell Biol, 7^:2745-2752 (1987); Rippe, et al, MoI. Cell Biol, 70:689-695 (1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, MoI Cell Biol, 5:1188-190 (1985); by direct sonic loading (Fechheimer, et al, Proc. Natl. Acad. Sci. USA, 89(17):%463-%467 (1987); by liposome mediated transfection (Nicolau and Sene, Biochem. & Biophys. Acta., 727:185-190 (1982); Fraley, et al, Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Nicolau, et al, Meth. Enzym., 149:157-176 (1987); Wong, et al, Gene, 70:879-894 (1980); Kaneda, et al, Science, 245:375-378 (1989); Kato, et al, J. Biol. Chem., 266:3361-3364 (1991) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432 (1987); Wu and Wu, 1988); by PEG-mediated transformation of protoplasts (Omirulleh, et al, Plant MoI Biol, 27^:415-428 (1987); U.S. Patent Nos. 4,684,611 and 4,952,500); by desiccation/inhibition-mediated DNA uptake (Potrykus, et al MoI Gen. Genet., 199(2):\69-\77 (1985), and any combination of such methods.

Components of the Assay System Host Cells

[00122] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In some embodiments, a host cell is engineered to express a screenable or selectable marker which is activated by the transcription factor that is part of a fusion protein, along with GPRl. [00123] In the context of expressing a heterologous nucleic acid sequence,

"host cell" refers to a prokaryotic or eukaryotic cell that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. When host cells are "transfected" or "transformed" with nucleic acid molecules, they are referred to as "engineered" or "recombinant" cells or host cells, e.g., a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally-occurring cells which do not contain a recombinantly introduced nucleic acid.

[00124] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as Escherichia coli (e.g., E. coli strain RRl, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DH5, JM109, and KC8, bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.

[00125] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

[00126] In some embodiments, cell division may be arrested. By division arrest, as used herein, is meant that the cells being used have been treated, by means known in the art, so that either their mitotic or meiotic cycle has been stopped, and cellular division can no longer take place. There are many chemical, radiological, and other methods which can be used to accomplish the arrest of cellular division. Mitomycin C is well known for its ability to arrest cell growth by blocking microtubule mobility, thereby arresting cell division. In order to arrest the division of cells, they may be exposed to 10 μg/ml of mitomycin C, for 2.5 hours. The cells may be frozen (e.g., 2.5 hours after treatment) using standard protocols. As an alternative method of division arrest, cells may be exposed to gamma irradiation at a dose of 2Gy(Gray) to 8Gy. Thus, the invention includes methods described herein, wherein those methods (e.g., assays) are performed using division arrested cells. The invention also includes compositions (e.g., cells, kits, etc.) described herein, wherein those compositions contain division arrested cells.

[00127] Compositions and methods related to the preparation and uses of division arrested cells are described in U.S. Patent No. 7,045,281, the entire disclosure of which is incorporated herein by reference.

Test Protein

[00128] The present invention contemplates the use of GPRl and a test protein.

In some embodiments, a GPRl and test protein, will exist as fusions proteins with GPRl fused to a transcription factor, and the test protein fused to a protease that recognizes a cleavage site in the first fusion protein, cleavage of which releases the transcription factor. In some embodiments, test proteins/fusions are such (a) that the first construct be such that the test protein cannot localize to the nucleus prior to cleavage, and (b) that the protease must remain active following both fusion to the test protein and binding of the GPRl to the second test protein.

[00129] With respect to the first construct, either GPRl as a whole and described herein, or portions of GPRl which function in the same manner as the full length first test protein may be used.

Reporters

[00130] The protein which activates a reporter gene may be any protein having an impact on a gene, expression or lack thereof which leads to a detectable signal. Typical protein reporters include enzymes such as chloramphenicol acetyl transferase (CAT), β- glucuronidase (GUS), β-lactamase or β-galactosidase. Also contemplated are fluorescent and chemiluminescent proteins such as green fluorescent protein, red fluorescent protein, cyan fluorescent protein luciferase, beta lactamase, and alkaline phosphatase. Transcriptions Factors and Repressors

[00131] In some embodiments of the invention, transcription factors are used to activate expression of a reporter gene in an engineered host cell. Transcription factors are typically classified according to the structure of their DNA-binding domain, which are generally (a) zinc fingers, (b) helix-turn-helix, (c) leucine zipper, (d) helix-loop-helix, or (e) high mobility groups. The activator domains of transcription factors interact with the components of the transcriptional apparatus (RNA polymerase) and with other regulatory proteins, thereby affecting the efficiency of DNA binding.

[00132] The Rel/Nuclear Factor kB (NF-kB) and Activating Protein-1 (AP-I) are among the most studied transcription factor families. They have been identified as important components of signal transduction pathways leading to pathological outcomes such as inflammation and tumorogenesis. Other transcription factor families include the heat shock/E2F family, POU family and the ATF family. Particular transcription factors, such as tTA and GAL4, are contemplated for use in accordance with the present invention.

[00133] Though transcription factors are one class of molecules that can be used, the assays may be modified to accept the use of transcriptional repressor molecules, where the measurable signal is downregulation of a signal generator, or even cell death.

Proteases and Cleavage Sites

[00134] Proteases are well characterized enzymes that cleave other proteins at a particular site. One family, the Ser/Thr proteases, cleaves at serine and threonine residues. Other proteases include cysteine or thiol proteases, aspartic proteases, metalloproteinases, aminopeptidases, di & tripeptidases, carboxypeptidases, and peptidyl peptidases. The choice of these is left to the skilled artisan and certainly need not be limited to the molecules described herein. It is well known that enzymes have catalytic domains and these can be used in place of full length proteases. Such are encompassed by the invention as well. A specific embodiment is the tobacco etch virus nuclear inclusion A protease, or an active portion thereof. Other specific cleavage sites for proteases may also be used, as will be clear to the skilled artisan.

Modification of GPRl

[00135] GPRl coding sequences may be modified to enhance their binding to their interacting protein, e.g., for use in some assays or some methods of the invention. For example, it is known that certain GPCRs bind arrestins more stably or with greater affinity upon ligand stimulation and this enhanced interaction is mediated by discrete domains, e.g., clusters of serine and threonine residues in the C-terminal tail (Oakley, et al., J. Biol. Chem., 274:32248-32257 (1999) and Oakley, et al, J. Biol. Chem., 276:19452-19460 (2001)). Using this as an example, a GPRl encoding sequence itself may be modified, so as to increase the affinity of the membrane bound protein, such as the receptor, with the protein to which it binds. Exemplary of such modifications are modifications of the C-terminal region of the membrane bound protein, e.g., receptor, such as those described herein, which involve replacing a portion of it with a corresponding region of another receptor, which has higher affinity for the binding protein, but does not impact the receptor function.

[00136] In addition, the test protein may be modified to enhance its interaction with the GPRl . For example, the assay may incorporate point mutants, truncations or other variants of the second test protein, e.g., arrestin that is known to bind agonist-occupied GPCRs more stably or in a phosphorylation-independent manner (Kovoor, et al., J. Biol. Chem., 274:6831-6834 (1999)).

Assay Formats

[00137] As discussed above, the present invention, in one embodiment, offers a straightforward way to assess the interaction of GPRl and a test protein, thereby determining the activity of a test compound. In some embodiments, a first construct, as described herein, comprises a sequence encoding a GPRl, concatenated to a sequence encoding a cleavage site for a protease or protease portion, which is itself concatenated to a sequence encoding a reporter gene activator. By "concatenated" is meant that the sequences described are fused to produce a single, intact open reading frame, which may be translated into a single polypeptide which contains all the elements. These may, but need not be, separated by additional nucleotide sequences which may or may not encode additional proteins or peptides. In some embodiments, a second construct is inserted into recombinant cells as described herein, e.g., it contains both a sequence encoding a second protein, and the protease or protease portion. Together, these elements constitute a basic assay format when combined with a candidate agent whose effect on target protein interaction is sought. EXAMPLES EXAMPLE 1

[00138] To produce a cell based assay using human GPRl, a fusion construct was created, comprising DNA encoding a human GPRl (SEQ ID NO: 1), fused in frame to a DNA sequence encoding amino acids 3-335 of the tetracycline controlled transactivator tTA, described in (Gossen and Bujard, Proc Natl Acad Sci USA 89:5541-51 (1992)). Inserted between these sequences is a DNA sequence encoding the amino acid sequence GSENLYFQL (SEQ ID NO: 2) which includes the low efficiency variant cleavage site for TEV NIa-Pro, ENLYFQL (SEQ ID NO:6), described previously. A CMV promoter was placed upstream of the GPRl coding region, and a polyadenylation sequence was placed downstream of the tTA region. This construct is designated GPRl-L-tTA.

[00139] A second fusion construct was also produced containing a C-terminal

"tail" sequence designed to enhance the affinity of the interacting arrestin fusion partner and the resulting response of the assay. This construct comprises a DNA sequence encoding the first 323 amino acids of human GPRl fused in frame to a DNA sequence encoding the C- terminal 42 amino acids of the human gastrin-releasing peptide receptor (GRPR) (NM 005314) (SEQ ID NO: 3), followed by the ENLYFQL (SEQ ID NO:6) cleavage site described herein, followed by the tTA transactivator sequence. The junction between the GPRl and GRPR tail sequences further contains an Xbal restriction site TCTAGA encoding the amino acids Ser-Arg. This construct was designated GPRl-GRPRct-L-tTA. To produce a cell-based assay for the human CMKLRl gene, a fusion construct was created, comprising DNA encoding the first 329 amino acids of human CMKLRl, which can be found in Genbank under Accession Number NM 004072 (SEQ ID NO:4), fused in frame to a DNA sequence encoding the C-terminal 42 amino acids of human GRPR, followed by the ENLYFQL (SEQ ID NO: 6) cleavage site described herein, followed by the tTA transactivator sequence. The junction between the CMKLRl and GRPR tail sequences further contains an Xbal restriction site TCTAGA encoding the amino acids Ser-Arg. This construct was designated CMKLRl -GRPRct-L-tTA.

[00140] Each of these receptor constructs was individually transfected into a

HEK293 cell line harboring a tTA-dependent luciferase gene and a stably integrated β- arrestin 2 (ARRB2) - TEV NIa protease fusion gene. These are "HTLA-504" cells, which have been described previously. These cells were transfected in a 10 cm cell culture dish with 0.5 μg receptor construct DNA and 7.5 μg carrier DNA using a lipid-based transfection reagent following the vendor's specified instructions. Transfected cells were cultured for about 24 hours before cryopreservation. To conduct the assay, cryopreserved, transiently transfected cells were thawed and plated in 96 well plates at a density of 10,000 cells per well in serum-free medium (SFM). After 5-6 hours of recovery, test ligands were added at various concentrations and the cells were incubated for a period of 8-16 hours. After the incubation period, cells were lysed and luciferase activity was assayed using a standard, commercially available luminescence assay.

[00141] Figure 1 shows dose-response curves for recombinant chemerin protein

(Figure IA) and for a 13-amino acid C-terminal chemerin peptide (H-PHSFYFPGQFAFS- OH, representing chemerin 145-157) (SEQ ID NO: 5) ("peptide 2") (Figure IB) in assays with the GPRl-GRPRct-L-tTA and CMKLRl -GRPRct-L-tTA receptor constructs. These results indicate that GPRl is activated by chemerin with an EC50 of 24OpM, compared to 3nM for CMKLRl; similarly GPRl responds to peptide 2 with an EC50 of InM, compared to 24 nM for CMKLRl. Figure 2 shows a dose-response curve for recombinant chemerin peptide using the GPRl-L-tTA receptor construct. A similar cell-based assay for the human β2-adrenergic receptor (ADRB2) showed no response to either recombinant chemerin protein or chemerin peptide 2.

[00142] The ability of GPRl to respond specifically to chemerin was confirmed using a calcium mobilization assay with a promiscuous Ga- 15 protein designed to couple receptor activity to phospholipase C activity and calcium release. (Offermanns and Simon, J Biol Chem. 270:15175-80 (1995)) Unmodified GPRl and CMKLRl expression constructs were transfected into HEK293 cells, together with a Ga- 15 expression construct at a ratio of 4:1 receptor:G protein, using a lipid-based transfection reagent following the vendor's specified instructions. Following a 24 incubation period, cells were washed and loaded with a calcium sensitive fluorescent dye following the vendor's specified protocol. Fluorescence imaging was performed with an inverted fluorescence microscope equipped with a digital camera and imaging software. The responses of seven representative cells following ligand addition (indicated with an arrow) were averaged. As shown in Figure 3, cells transfected with GPRl and Ga- 15 (Figure 3B), or with CMKLRl and Ga- 15 (Figure 3A), responded to a biologically active 9-amino acid C-terminal chemerin peptide (i.e., amino acids 5-13 of SEQ ID NO: 5) at a concentration of 1 μM. In contrast, cells transfected with Gα-15 alone, or with either GPRl or CMKLRl in the absence of Gα-15, failed to respond to chemerin peptide with calcium release. [00143] The identification of chemerin as a ligand for GPRl thus

"deorphanizes" the receptor and imputes a function thereto.

EXAMPLE 2: FLUO-4 AM Cell Loading Protocol

[00144] On day one, plate cells at 30k/well into 96 black/clear bottom plate coated with PDL. On day two, make the following stock solutions:

[00145] Solution #1 : 25OmM probenecid: 2.5ml IN NaOH in 355mg probenecid (Sigma Cat# P-8761) + 2.5ml of IxHBSS

[00146] Solution #2: IxHBSS (pH7.4) with 2.5mM probenecid and 2OmM

HEPES: IxHBSS: 10ml, IM HEPES: ImI, dH₂O:38.5ml, 25OmM Probenecid (pH7.4):0.5ml

[00147] Pipette off the growth media from each well and replace it with lOOul/well Fluo-4 loading dye, incubate at 37⁰C for 45min-l hour. During this time, turn on FlexStation and warm up to 37⁰C, and setup the program and make 5x compound plates.

[00148] Fluo-4 loading dye: 22.8μl Fluo-4-DMSO (stock: 22.8μl DMSO+50μg

Fluo-4, final 5μM), 10.5μl Pluronic F-127 (stock 20% in DMSO, final 0.02%), 10ml Solution #2

[00149] Pipette off the loading dye and replace it with lOOμl of fresh solution

#2. Set program: compound addition: 25μl (5x compound plate), read: excitation 485nm, emission 525nm, data collection every 1.5s.

[00150] Other features of the invention will be clear to the skilled artisan and need not be reiterated here.

[00151] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in their entirety into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

1. A method for determining if a test compound interacts with aGPRl, comprising contacting (a) the test compound, and (b) (i) chemerin or (ii) a chemerin derivative which interacts with GPRl, and comparing interaction of (b) with GPRl in the presence of (a), to its interaction with GPRl in the absence of (a), as a determination of whether the test compound interacts with GPRl .

2. The method of claim 1, wherein (b) is labeled with a detectable moiety.

3. The method of claim 1, wherein the GPRl is presented on a cell surface.

4. The method of claim 3, comprising determining a downstream activity of GPRl as a determination of the interaction.

5. The method of claim 3, wherein the cell expresses chemerin endogenous Iy.

6. The method of claim 3, wherein the cell has been transformed or transfected with an isolated nucleic acid molecule which encodes GPRl.

7. The method of claim 3, wherein the cell is a eukaryote.

8. The method of claim 3, wherein the cell is a prokaryote.

9. The method of claim 3, comprising contacting the cell with the test compound and chemerin or GPRl binding fragment of chemerin simultaneously.

10. The method of claim 3, comprising contacting the cell with the test compound and chemerin or GPRl binding fragment of chemerin sequentially.

11. A method for determining if a test compound modulates a GPRl activity comprising contacting a compound to a cell which has been transformed or transfected with

(a) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes the GPRl,

(ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and (iii) a nucleotide sequence which encodes a protein which activates a reporter gene in cell, and (b) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes a test protein whose interaction with the GPRl in the presence of the test compound is to be measured, and

(ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site, and determining activity of the reporter gene as a determination of whether the compound modulates GPRl activity.

12. The method of claim 11, further comprising contacting the cell with a chemerin or a GPRl binding fragment thereof.

13. The method of claim 11, wherein the protease is tobacco etch virus nuclear inclusion A protease.

14. The method of claim 11, wherein the protein which activates the reporter gene is a transcription factor.

15. The method of claim 14, wherein the transcription factor is tTA or GAL4.

16. The method of claim 11 , wherein the test protein is an inhibitory protein.

17. The method of claim 16, wherein the inhibitory protein is an arrestin.

18. The method of claim 11 , wherein the cell is a eukaryote.

19. The method of claim 11, wherein the reporter gene is an exogenous gene.

20. The method of claim 19, wherein the exogenous gene encodes β-galactosidase, β- lactamase or luciferase.

21. The method of claim 11, wherein the nucleotide sequence encoding GPRl is modified to increase interaction with the test protein.

22. The method of claim 11, wherein the modification comprises replacing all or part of the nucleotide sequence of the C-terminal region of GPRl with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for the second test protein than the original sequence.

23. The method of claim 22, wherein the nucleotide sequence of the C-terminal region is replaced by a nucleotide sequence encoding all or a part of the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B, CCR4, or GRPR.

24. The method of claim 11, comprising contacting more than one compound to a plurality of samples of cells, each of the samples being contacted by one or more of the compounds, wherein each of the cell samples have been transformed or transfected with (a) and (b), and determining activity of reporter genes in the plurality of the samples to determine if any of the compounds interacts with GPRl.

25. The method of claim 21, comprising contacting each of the samples with one compound, each of which differs from all others.

26. The method of claim 21, comprising contacting each of the samples with a mixture of the compounds.

27. A method for determining if a test compound is a chemerin inhibitor, comprising contacting the test compound and chemerin to GPRl or a GPRl fragment, determining activity of chemerin in the presence of the test compound to activity of chemerin in its absence.

28. The method of claim 27, wherein the chemerin activity is binding to GPRl or GPRl fragment.

29. The method of claim 28, wherein the chemerin is labeled.

30. The method of claim 27, wherein the GPRl or GPRl fragment is expressed by a cell, and the activity of chemerin is a downstream property following binding of chemerin thereto.

31. A method for identifying an analogue of chemerin, comprising contacting a test compound to GPRl or a fragment of GPRl, and determining if the test compound binds to the GPRl or fragment of GPRl and exhibits a property exhibited by chemerin upon binding to the GPRl or the fragment of GPRl, wherein presence of the property indicates the test compound is a chemerin analogue.

32. The method of claim 31, wherein the test compound exhibits greater activity than chemerin upon the binding.

33. The method of claim 31, comprising contacting the test compound and chemerin, to the GPRl or the GPRl fragment, wherein one of chemerin and the test compound are labeled, and determining label bound to the GPRl or the GPRl fragment, to binding of the label in the absence of unlabelled substance.

34. The method of claim 31, wherein the GPRl or fragment of GPRl is expressed by a cell.

35. The method of claim 34, comprising determining a downstream property following binding of chemerin and the test compound thereto.

36. A method for modulating activity of a GPRl, comprising contacting the GPRl with a substance which binds to GPRl but is not chemerin, in an amount sufficient to bind to GPRl and modulate its activity.

37. The method of claim 36, wherein the substance is a non-orthosteric binding partner for GPRl.

38. The method of claim 37, wherein the substance is an allosteric modifier of GPRl .

39. The method of claim 38, wherein the substance is chemerin or a chemerin derivative.

40. The method of claim 39, wherein the chemerin derivative comprises SEQ ID NO: 5 or amino acids 5-13 of SEQ ID NO:5.

41. A recombinant cell, transformed or transfected with:

(a) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes GPRl,

(ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell, and (b) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes a test protein whose interaction with GPRl in the presence of the test compound is to be measured, and

(ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site.

42. The recombinant cell of claim 41, wherein one or both of the nucleic acid molecules are stably incorporated into the genome of the cell.

43. The recombinant cell of claim 41, wherein the cell has been transformed or transfected with the reporter gene.

44. The recombinant cell of claim 41, wherein the protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

45. The recombinant cell of claim 41, wherein the protein which activates the reporter gene is a transcription factor.

46. The recombinant cell of claim 41 , wherein the transcription factor is tTA or GAL4.

47. The recombinant cell of claim 41 , wherein the second protein is an inhibitory protein.

48. The recombinant cell of claim 41 , wherein the inhibitory protein is an arrestin.

49. The recombinant cell of claim 41 , wherein the cell is a eukaryote.

50. The recombinant cell of claim 41 , wherein the cell is a prokaryote.

51. The recombinant cell of claim 41 , wherein the reporter gene is an exogenous gene.

52. The recombinant cell of claim 41, wherein the exogenous gene encodes β- galactosidase, β-lactamase or luciferase.

53. The recombinant cell of claim 41, wherein the nucleotide sequence encoding GPRl is modified to increase interaction with the test protein.

54. The recombinant cell of claim 53, wherein the modification comprises replacing all or part of the nucleotide sequence of the C-terminal region of GPRl with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for the test protein than the original sequence.

55. The recombinant cell of claim 54, wherein the nucleotide sequence of the C-terminal region is replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B, or CCR4.

56. An isolated nucleic acid molecule which comprises, in 5' to 3' order,

(i) a nucleotide sequence which encodes GPRl or GPRl fragment, (ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease, and

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell.

57. The isolated nucleic acid molecule of claim 56, wherein the protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

58. The isolated nucleic acid molecule of claim 56, wherein the protein which activates the reporter gene is a transcription factor.

59. The isolated nucleic acid molecule of claim 58, wherein the transcription factor is tTA or GAL4.

60. An expression vector comprising the isolated nucleic acid molecule of claim 56, operably linked to a promoter.

61. A fusion protein produced by expression of the isolated nucleic acid molecule of claim 56.

62. A test kit useful for determining if a test compound modulates GPRl activity comprising a separate portion of each of:

(a) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes the GPRl,

(ii) a nucleotide sequence encoding a cleavage site for a protease or a portion of a protease,

(iii) a nucleotide sequence which encodes a protein which activates a reporter gene in the cell, and (b) a nucleic acid molecule which comprises:

(i) a nucleotide sequence which encodes a test protein whose interaction with the GPRl in the presence of the test compound is to be measured,

(ii) a nucleotide sequence which encodes a protease or a portion of a protease which is specific for the cleavage site, and

(c) container means for holding each of (a) and (b) separately from each other.

63. The test kit of claim 62, wherein the protease or portion of a protease is tobacco etch virus nuclear inclusion A protease.

64. The test kit of claim 62, wherein the protein which activates the reporter gene is a transcription factor.

65. The test kit of claim 64, wherein the transcription factor is tTA or GAL4.

66. The test kit of claim 62, wherein the test protein is an inhibitory protein.

67. The test kit of claim 66, wherein the inhibitory protein is an arrestin.

68. The test kit of claim 66, further comprising a separate portion of an isolated nucleic acid molecule which encodes a reporter gene.

69. The test kit of claim 68, wherein the reporter gene encodes β-galactosidase, β- lactamase or luciferase.

70. The test kit of claim 62, wherein the nucleotide sequence encoding the GPRl is modified to increase interaction or the GPRl with the second test protein.

71. The test kit of claim 70, wherein the modification comprises replacing all or part of the nucleotide sequence of the C-terminal region of the GPRl with a nucleotide sequence which encodes an amino acid sequence which has higher affinity for the second test protein than the original sequence.

72. The test kit of claim 71, wherein the nucleotide sequence of the C-terminal region is replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, GRPR, F2RL1, CXCR2/IL-8B or CCR4.

73. The test kit of claim 62, further comprising a separate portion of chemerin, or a chemerin derivative which binds to GPRl .

74. A method for measuring the ability of a compound to alter GPRl activity, the method comprising:

(a) contacting a cell which expresses a receptor which binds chemerin and generates an intracellular signal normally associated with a GPRl with (1) a chemerin molecule or fragment thereof and (2) a compound to be screened for modulation of chemerin activity with respect to signal generation,

(b) measuring a signal generated in (a), and

(c) comparing the signal in (b) with a signal generated in the absence of the compound.

75. The method of claim 74, wherein the chemerin molecule is a fragment of a naturally occurring chemerin molecule which, upon interaction with a GPRl, retains the ability to generate an intracellular signal normally associated with a GPRl .

76. A method for determining if a test compound behaves as an analogue of chemerin, comprising contacting (a) the test compound, and (b) (i) chemerin or (ii) another agonist of GPRl, and comparing interaction of (b) with GPRl in the presence of (a), to its interaction with GPRl in the absence of (a), as a determination of whether the test compound interacts with GPRl and modulates activity of GPRl .

77. The method of claim 76, wherein the test compound exhibits greater activity than chemerin upon the binding.

78. The method of claim 76, comprising contacting the test compound and chemerin, to GPRl or the GPRl fragment, wherein one of chemerin and the test compound are labeled, and determining label bound to GPRl or the GPRl fragment, to binding of the label in the absence of unlabelled substance.

79. The method of claim 76, wherein the GPRl or fragment of GPRl is expressed by a cell.

80. The method of claim 79, comprising determining a downstream property following binding of chemerin and the test compound thereto.

81. A method for modulating activity of a GPRl, comprising contacting the GPRl with a substance which binds to GPRl but is not chemerin, in an amount sufficient to bind to GPRl and modulate its activity.

82. The method of claim 81, wherein the substance is a non-orthosteric binding partner for GPRl.

83. The method of claim 82, wherein the substance is an allosteric modifier of GPRl .

84. The method of claim 83, wherein the substance is chemerin or a chemerin derivative.

85. The method of claim 84, wherein the chemerin derivative comprises SEQ ID NO: 5 or amino acids 5-13 of SEQ ID NO:5.