60/153,436, filed September 10,1999, the contents of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable.

REFERENCE TO MICROFICHE APPENDIX Not Applicable.

FIELD OF THE INVENTION The present invention relates to the field of assays for compounds that interact with the receptor for Neuropeptide FF and its related peptides.

BACKGROUND OF THE INVENTION Neuropeptide FF ("NPFF") (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (SEQ ID NO : 1)) and the octadecapeptide neuropeptide AF ("NPAF") (Ala-Gly-Glu- Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (SEQ ID NO : 2)) were originally isolated from bovine brain using an antiserum against the molluscan peptide FMRFamide (Yang et al., 1985, Proc. Natl. Acad. Sci., 82: 7757- 7761). NPFF has also been called mammalian FMRFamide-like peptide, F8Famide, or morphine modulating neuropeptide since NPFF reduces the nociceptive threshold in rats. The human NPFF gene was isolated and shown to encode two related peptides called NPSF and NPAF (Perry et al., FEBS Lett., 409: 426-430). Human NPSF and NPAF appear to be the counterparts of bovine NPFF and NPAF, respectively.

For convenience herein we refer to these related neuropeptides as NPSF and related neuropeptides. A summary of the known NPSF related neuropeptides and their common abbreviations are shown below:

Species Peptide Sequence Abbreviation Bovine FLFQPQRF-NH2 (SEQ ID NO : 1) NPFF AGEGLSSPFWSLAAPQRF-NH2 (SEQ ID NO : 2) NPAF Rat FLFQPQRF-NH2 (SEQ ID NO : 1) NPFF AGEGLSSPFWSLAAPQRF-NH2 (SEQ ID NO : 2) NPAF Human SQAFLFQPQRF-NH2 (SEQ ID NO : 8) NPSF AGEGLNSQFWSLAAPQRF-NH2 (SEQ ID NO : 9) NPAF The potential activities of NPSF and its related peptides in human systems make the peptide a target for studies aimed at identifying compounds that enhance or diminish the biological effects of the peptides. However, while binding sites for NPFF were shown to exist, no binding partner for these peptides was identified. The lack of a known receptor or other binding partner has hampered the design of assays and impeded efforts to discover and study compounds that mimic or alter the biological effects of NPSF or related neuropeptides.

Recent efforts at sequencing mRNAs expressed in humans has lead to the identification of a plethora of receptor polypeptides. Unfortunately, the natural ligands for the vast majority of the identified receptors are unknown. Such"orphan" receptors are of little or no use except as counter-screening agents to assess the specificity of a ligand for a somewhat homologous receptor under study. Because their natural ligands are unknown, orphan receptors are of no use for the study of the true activity of the receptor in an actual biological system.

One such orphan receptor, HG31 was previously identified as weakly homologous to the NPY receptors. The nucleic acid and polypeptide sequences of the human homolog of HG31 was described in U. S. Provisional Application 60/111,432 filed December 08,1998 and EP 0 884 387 A2 published December 16,1998.

Variants of the nucleic acid and polypeptide sequences are also described in those documents. Without wishing to be bound to the teaching of utility of the receptor found in those documents, EP 0 884 387 A2 describes the utility of the sequences of the HG31 receptor (therein referred to as the HLWAR77 receptor) as including the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2 ; pain; cancers; diabeties ; obesity;

anorexia; bulimia ; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others and diagnositc assays for such conditions. That document also purports to describe methods to identify agonists and antagonists using the human HG31 sequences, and methods of treating conditions associated with human HG31 imbalance with the identified compounds.

The HG31 sequences described previously were stated to be similar to the receptors for NPY. Therefore, HG31 sequences reported in the art would be expected to be useful as"minus targets"in counter-screens in connection with screening projects designed to identify compounds that specifically interact with NPY family receptors. (see Hodgson, 1992, Bio/Technology 10: 973-980, at 980).

However, while the homology of HG31 to other receptors was reported, no actual ligand for the HG31 receptor was identified. Therefore, no assay employing competition with an actual ligand for HG31, or employing a comparison to the interaction of the HG31 receptor with an actual ligand, was or could have been described. The present invention solves this problem in the art and provides assays that employ the interaction of HG31 and NPSF and its related neuropeptides to determine whether a candidate compound is a ligand, agonist or antagonist of HG31.

SUMMARY OF THE INVENTION The present invention provides assays for the study of the interaction of NPSF or a related neuropeptide with the HG31 receptor. The assays are useful to identify whether a candidate compound can bind to the HG31 receptor under conditions in which NPSF or a related neuropeptide can bind the receptor. The assays are also useful to determine whether a candidate compound is an agonist or antagonist of HG31 in the presence of NPSF or a related neuropeptide. The above assays can be performed in a variety of formats including competitive, non-competitive and comparative assays in which the interaction of NPSF or a related neuropeptide with HG31 is assessed as a positive or negative control or compared to the result obtained with the candidate compound.

As used herein a"compound"is an organic or inorganic assembly of atoms of any size, and includes small molecules (less than about 2500 Daltons) or

large macromolecules, e. g., peptides, polypeptides, whole proteins, and polynucleotides.

As used herein, a"candidate"is a compound that may be a ligand of a HG31 receptor polypeptide. A candidate compound may also be an agonist or antagonist of the HG31 receptor. Whether or not the candidate is an actual agonist. antagonist or ligand of a HG31 receptor polypeptide is determined in an assay.

As used herein an"agonist"is a compound that interacts with and activates a polypeptide of the HG31 receptor. An activated HG31 receptor polypeptide induces a change in a biochemical pathway linked to the receptor, e. g., can stimulate the cleavage of GTP by a G protein, activate the adenylate cyclase pathway or activate the phospholipase C-P pathway.

As used herein an"antagonist"is a compound that interacts with and inhibits or prevents the activation of a polypeptide of the HG31 receptor.

As used herein"recombinant"refers to the use recombinant genetic techniques to manipulate and/or produce polynucleotides, polypeptides, expression vectors, host cells and the like."recombinant"is also used as an adjective to describe the products of such techniques.

Throughout this specification and the claims appended hereto, the terms HG31 receptor and NPSF receptor are used interchangeably.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1C depict the nucleotide (SEQ ID NO : 3) and amino acid (SEQ ID NO : 4) sequences of short form of HG31.

FIGS. 2A-2D depict the nucleotide (SEQ ID NO : 5) and amino acid (SEQ ID NO : 6) sequences of the long form of HG31.

FIG. 3. Activation of HG31 by human NPSF and its related peptides in the aequorin bioluminescence assay. HEK293/aeql7/Gal5 cells stably expressing HG31 were assayed against NPAF, NPFF, and NPSF. RLU: Random Luminescence Units. Results shown are the means (SEM) of triplicate determinations. The effective half-maximum concentrations (EC50) of NPAF, NPFF, and NPFF are 44, 39,9.0 nM, respectively.

FIG. 4. Activation of HG31 by NPSF and its related peptides in the melanophore assay. Melanophore cells were transfected with HG31 or pcDNA3 (vector control) and assayed against various peptides one day post-transfection.

Results shown are the means (SEM) of duplicate determinations. The effective half-

maximal concentrations (EC50) are NPSF, 1.8 nM; NPFF 9.6 nM; NPAF, 3.5 nM; LPLRF, 143 nM; PrP-20 (Prolactin-releasing peptide), 164 nM.

FIG. 5. The nucleotide sequence (SEQ ID NO : 7) and amino acid sequence (SEQ ID NO : 13) of the human NPFF gene as reported by Perry et al., FEBS Lett., 409: 426-430.

FIG. 6. Radioligand binding analysis of HG31. HEK293/aeql7/GoclS cells were transfected with HG31 and cellular membranes were prepared three days post-transfection. The symbols are: (H), human NPAF; (A), bovine NPFF; (V), human NPSF. Specific binding was defined as the amount of total binding subtracted by the amount of total binding in the presence of 1,000 nM of Tyrl-NPFF. Results shown are the means (SEM) of quadruplicate determinations (n = 4).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides assays that make use of the interaction of Neuropeptide SF (NPSF) and the related mammalian neuropeptides AF (NPAF), and FF (NPFF) with the HG31 receptor. These assays are made possible because of the discovery described herein that NPSF and its related peptides are high affinity ligands of HG31 that activate the receptor when bound thereto and lead to inhibition of adenylate cyclase. The assays provide methods to identify compounds that are ligands of HG31 or are agonists or antagonists of the HG31 receptor. The invention includes compounds identified as agonists or antagonists of HG31 and their use in treatment of a patient. In particular, the invention includes administering an agonist or antagonist of the HG31 receptor as a therapy to pain and opioid tolerance.

Neuropeptide SF (NPSF), AF (NPAF), and FF (NPFF) are related mammalian neuropeptides that are implicated in pain modulation, cardiovascular regulation, and neuroendocrine function. Their involvement in pain modulation includes inducing a vigorous abstinence syndrome in morphine-tolerant rats, regulating the density of opioid receptors, and modulating self-administration of morphine. Agonists and antagonists of the HG31 receptor can be useful in the treatment of pain, opioid tolerance, and other pain-related disorders. Therefore, assays of this invention are useful to discover compounds that mimic the action of these neuropeptides and to discover compounds that are agonists or antagonists of the HG31 receptor.

HG31 was cloned as an orphan G protein-protein coupled receptor as described as U. S. Provisional Application 60/111,432 filed December, 08,1998, and

EP 0 884 287 A2 (therein referred to as HLWAR77) in December 16,1998. The receptor has limited homology to other neuropeptide receptors such as those for neuropeptide Y (NPY). However, no ligand for HG31/HLWAR77 was identified.

Recently, Cikos et al., published an orphan receptor called NPGPR that is identical to HG31/HLWAR77 except an extra 102 amino acids at the N-terminus (Cikos et al., 1999, Biochem. Biophys. Res. Commun., 256: 352-356). Herein, the original sequence of HG31 in the U. S. Provisional Application 60/111,432 is referred to as the Short Form of HG31 (SEQ ID NO : 4) while the form of NPGPR published by Cikos et al. is referred to as the Long Form of HG31 (SEQ ID NO : 6).

Polynucleotides and polypeptides of HG31 that can be usefully employed in the assays of the present invention include those described in U. S Provisional application 60/111, 432 filed December 08,1998, EP 0 884 387 A2 published December 16,1998, Cikos et al., and as described herein.

Polynucleotides As used herein a"polynucleotide"is a nucleic acid of more than one nucleotide. A polynucleotide can be made up of multiple polynucleotide units that are referred to by description of the unit. For example, a polynucleotide can comprise within its bounds a polynucleotide (s) having a coding sequence (s), a polynucleotide (s) that is a regulatory region (s) and/or other polynucleotide units commonly used in the art.

Polynucleotides of particular usefulness in the present invention are those disclosed in FIG. 1 (SEQ ID NO : 3) & FIG. 2 (SEQ ID NO : 5). As used herein in reference to a polynucleotide or gene sequence we may use the terms HG31 polynucleotide. When referring expressly to a gene for a receptor having the particular polypeptide sequence recited in SEQ ID NO : 4, we may refer to the gene by the proper name, the NPSF-Rla gene.

The present invention also relates to the use of recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.

The DNA sequences used in this invention, in whole or in part, can be linked with other DNA sequences, i. e., DNA sequences to which the HG31 sequence is not naturally linked, to form"recombinant DNA molecules"containing HG31. The DNA sequences can be inserted into vectors in order to direct recombinant expression of HG31. Such vectors may be comprised of DNA or RNA; for most purposes DNA

vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode HG31. One skilled in the art can readily determine an appropriate vector for a particular use.

An"expression vector"is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the regulatory regions can direct the expression of the coding sequence. The use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell.

A"regulatory region"is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence. A regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence.

Polynucleotides useful in this invention contain full length or partial length sequences of the HG31 receptor gene. Polynucleotides of this invention can be single or double stranded. If single stranded, the polynucleotides can be a coding, "sense,"strand or a complementary,"antisense,"strand. Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor.

Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.

The polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both. The polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis. Non-natural or modified nucleotides, including inosine, methyl-cytosine, deaza-guanosine, and others known to those of skill in the art, can be present. Natural phosphodiester internucleotide linkages can be appropriate. However, polynucleotides can have non-natural linkages between the nucleotides. Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages. Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.

"Plastic DNA,"having, for example, N-vinyl, methacryloxytethyl, methacrylamide or

ethyleneimine internucleotide linkages, can be used."Peptide Nucleic Acid" (PNA) is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.

As used herein,"purified"and"isolated"are utilized interchangeably for the proposition that the polynucleotides, proteins and polypeptides, or respective fragments thereof in question has been removed from its is vivo environment so that it can be manipulated by the skilled artisan, such as but not limited to sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in quantities that afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially, substantially or wholly purified form. A polynucleotide is considered purified when it is purified away from environmental contaminants. Thus, a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.

Included in the present invention are assays that employ polynucleotides that hybridize to rat and human HG31 sequences under stringent conditions. By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100, ug/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100, ug/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0. 1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.

Other procedures using conditions of high stringency would include either a hybridization step carried out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e. g., Sambrook, et al., 1989, Molecular Cloning: A

Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press. In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.

Polypeptides The present invention also relates to the use of HG31 receptor polypeptides, also referred to as NPSF receptor polypeptides including fragments and mutant or polymorphic forms of HG31, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy- terminal truncations such that these provide for polypeptides or fragments thereof useful for screening for modulators, agonists and/or antagonists of HG31. When referring expressly to a receptor having the particular polypeptide sequence recited in SEQ ID NO : 4, we may refer to the receptor by the proper name, NPSF-Rla.

One of skill in the art can determine whether such naturally occurring forms are mutant or polymorphic forms of HG31 by sequence comparison. One can further determine whether the encoded protein, or fragments of any HG31 protein, is biologically active by routine testing of the protein of fragment in a in vitro or in vivo assay for the binding of the HG31 receptor to NPSF or a related neuropeptide. For example, one can express N-terminal or C-terminal truncations, or internal additions or deletions, in host cells and test for the ability of NPSF or a related neuropeptide to bind to the polypeptide to stimulate the cleavage of GTP by a G protein, activate the adenylate cyclase pathway or activate the phospholipase C-ß pathway. One can also measure increase of cytoplasmic Ca2+ concentration which occurs when receptor activation leads to activation of phospholipase C-, 8 (PLC ß). Intracellular concentration of Ca2+ can be determined by a variety of methods, such as using the real-time, live cell, fluorometric imaging assaying with the FLIPR (Fluorometric Imaging Plate Reader) machine (MOLECULAR DEVICES, U. S. A.), or using reporter genes under the control of a Ca2+-responsive promoter (e. g., using vectors supplied by AURORA BIOSCIENCES, San Diego, CA, U. S. A.).

It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid. Therefore, the present invention discloses codon redundancy which can result in differing DNA molecules expressing an identical protein.

As with many receptor proteins, it is possible to modify many of the amino acids of HG31, particularly those which are not found in the ligand binding domain, and still retain substantially the same biological activity as the original receptor. Thus this invention includes modified HG31 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity, i. e., binding NPSF or related neuropeptides, as the a native HG31 polypeptide. Also included within the scope of this invention are assays using HG31 polypeptides having changes which do not substantially alter the physical or functional properties of the expressed protein. A"conservative amino acid substitution"refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e. g., arginine for lysine ; glutamic acid for aspartic acid). In particular, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine is not expected to cause a change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.

For the purposes of this invention, naturally occurring, or wild-type NPSF receptors have the amino acid sequences shown in FIGS. 1 (SEQ ID NO : 4) and 2 (SEQ ID NO : 6). As used herein, a"functional equivalent"NPSF receptor polypeptide possesses a biological activity that is substantially the same as the biological activity of a wild type HG31. A HG31 polypeptide has"substantially the same biological activity"as the wild type if that polypeptide has a Kd for NPSF or related neuropeptides that is no more than 5-fold greater than the Kd of a HG31 polypeptide shown in FIGS. 1 (SEQ ID NO : 4) or 2 (SEQ ID NO : 6). The term "functional derivative"is intended to include those"fragments,""mutants," "variants,""degenerate variants,""analogs,""homologues"or"chemical derivatives" of a wild type HG31 protein that exhibit substantially the same biological activity.

The term"fragment"is meant to refer to any polypeptide subset of wild-type HG31.

The term"mutant"is meant to refer to a polypeptide that may be substantially similar

to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered binding of NPSF or related neuropeptides, altered affinity for NPSF or related neuropeptides and altered sensitivity to chemical compounds affecting biological activity of the HG31 or functional derivative. The term"variant"is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof.

As used herein in reference to a HG31 gene or encoded protein, a "polymorphic"HG31 is an HG31 that is naturally found as an allele in the population at large. A polymorphic form of HG31 can have a different nucleotide sequence from the particular HG31 alleles shown in FIGS. 1 (SEQ ID NO : 3) and 2 (SEQ ID NO : 5).

However, because of silent mutations, a polymorphic HG31 gene can encode the same or different amino acid sequence as those depicted herein. Further, some polymorphic forms HG31 will exhibit biological characteristics that distinguish the form from wild-type receptor activity, in which case the polymorphic form is also a mutant. Polymorphic forms encompass allelic variants.

A protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature. A protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature.

A protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.

Expression Vectors and Host Cells An important aspect of many assays is the step of providing a host cell expressing a recombinant NPSF receptor polypeptide on the surface thereof. A variety of expression vectors can be used to express recombinant HG31 receptor polypeptides in host cells. Expression vectors are defined herein as DNA sequences that are arranged for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a

potential for high copy number, and promoters. A promoter is defined as a DNA sequence operably linked to a coding region so that it interacts with cellular proteins to direct RNA polymerase to bind to DNA and initiate mRNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. A promoter can be inducible. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

Commercially available mammalian expression vectors which can be suitable for recombinant NPSF receptor polypeptide expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).

Alternatively, a variety of bacterial expression vectors can be used to express recombinant NPSF receptor polypeptide in bacterial cells. Commercially available bacterial expression vectors which are suitable for recombinant human HG31 expression include, but are not limited to pQE (Qiagen), pET1 la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia).

A variety of fungal cell expression vectors can also be used to express recombinant NPSF receptor polypeptide in fungal cells. Commercially available fungal cell expression vectors which are suitable for recombinant HG31 expression include but are not limited to pYES2 (INVITROGEN) and Pichia expression vector (INVITROGEN).

Moreover, a variety of insect cell expression vectors can be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which are suitable for recombinant expression of HG31 receptor polypeptides include but are not limited to pBlueBacIII and pBlueBacHis2 (INVITROGEN), and pAcG2T (PHARMINGEN).

The expression vectors generally described above and containing DNA encoding the NPSF-R1 receptor polypeptide can be used for expression of NPSF receptor polypeptide in an appropriate host cell. Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited

to cell lines derived from Drosophila and silkworm. Briefly, an expression vector is used to transform or transfect the appropriate cells, or cells can be obtained and cultured from an appropriate transgenic animal.

Cell lines derived from mammalian species which can be suitable and which are commercially available, include but are not limited to, L cells L-M (TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).

The expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce a HG31 receptor polypeptide. Identification of HG31 expressing cells can be done by several means, including but not limited to immunological reactivity with anti-HG31 antibodies, labeled ligand binding and the presence of host cell-associated HG31 activity.

A cloned NPSF receptor cDNA can be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant HG31 receptor polypeptide. Techniques for such manipulations can be found described in Sambrook, et al., 1989, and are well known and easily available to the one of ordinary skill in the art.

Expression of NPSF receptor polypeptides from recombinant polynucleotides can also be performed using in vitro produced synthetic mRNA.

Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.

Expression systems can be designed to produce mutant, polymorphic or allelic variants of the HG31 polypeptides shown in FIGS. 1 (SEQ ID NO : 3) and 2 (SEQ ID NO : 5). Fragments of HG31 receptor polypeptides can also be produced.

To determine the HG31 polynucleotide sequence (s) that yield optimal levels of HG31 receptor polypeptide in any particular host cell, recombinant DNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for HG31 as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5'and/or 3'untranslated region of a HG31 cDNA.

The expression levels and activity of HG31 receptor polypeptides can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the HG31 cDNA cassette yielding optimal expression in transient assays, the HG31 construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.

Assays Assays of the present invention can be designed in many formats generally known in the art of screening compounds for biological activity or for binding to receptors.

Assays employing NPSF receptor polypeptides and NPSF or a related neuropeptide The assays of the present invention advantageously exploit the discovery that NPSF and related nueropeptides are high affinity ligands for HG31 receptor polypeptides that activate the HG31 receptor upon binding thereto.

The present invention includes methods of identifying compounds that specifically bind to NPSF receptor polypeptides. Compounds that bind the receptor can be agonists or antagonists. The specificity of binding of compounds having affinity for HG31 can be shown by measuring the affinity of the compounds for recombinant cells expressing a HG31 receptor polypeptide on the surface thereof or affinity for membranes from such cells. Expression of HG31 receptor polypeptides and screening for compounds that bind to HG31 or that inhibit the binding of labeled NPSF or a related neuropeptide to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for HG31. The NPSF or a related neuropeptide can be radiolabeled but can also be labeled by other means known in the art and thereafter can be used to displace bound compounds or can be used as an activator of the HG31 in an assays.

If one desires to produce a fragment of the HG31 receptor or mutant, polymorphic or allelic variants of the receptor, one can test those products in the assays described below and compare the results to those obtained using a HG31 receptor polypeptide of SEQ ID NO : 4 or SEQ ID NO : 6. In this manner one can easily assess the ability of the fragment, mutant, polymorph or allelic variant to bind compounds, be activated by agonists or be inactivated or inhibited by antagonists of the native HG31 receptor.

Therefore, the present invention includes assays by which compounds that are HG31 agonists and antagonists may be identified. The assay methods of the present invention differ from those described in the art because the present assays incorporate at least one step wherein the interaction of NPSF or a related neuropeptide and the HG31 receptor polypeptide is incorporated into the assay.

General methods for identifying ligands, agonists and antagonists of receptors are well known in the art and can be adapted to identify agonists and antagonists of HG31. The order of steps in any given method can be varied or performed concurrently as will be recognized by those of skill in the art of assays.

The following is a sampling of the variety of formats that can be used to conduct an assay of the present invention.

Accordingly, the present invention includes a method for determining whether a candidate compound is a ligand of HG31 the method of which comprises: (a) transfecting cells with an expression vector encoding a HG31 receptor polypeptide; (b) allowing the transfected cells to grow for a time sufficient to allow HG31 to be expressed on the surface of the cells; (c) exposing the cells to labeled NPSF or a related neuropeptide in the presence and in the absence of the compound; (d) measuring the binding of the labeled NPSF or a related neuropeptide to HG31 ; where if the amount of binding of the labeled NPSF or a related neuropeptide is less in the presence of the compound than in the absence of the compound, then the compound is a ligand of HG31 receptor polypeptide and is a potential agonist or antagonist of HG31.

The conditions under which step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e. g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about

55°C. In this step the NPSF or a related neuropeptide and candidate compound can be applied to the cell sequentially or concurrently. Moreover, the assay can be conducted such that the HG31 is presaturated with NPSF or a related neuropeptide and the ability of the candidate compound to displace the bound NPSF or a related neuropeptide is assessed.

The present invention also includes a method of using the interaction of NPSF or a related neuropeptide and a HG31 receptor polypeptide as a positive control for determining whether a compound is capable of binding to a HG31 receptor polypeptide, i. e., whether the compound is a potential agonist or an antagonist of HG31, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells; (b) exposing a first portion of the test cells to the candidate compound; (c) exposing a second portion of the test cells to NPSF or a related neuropeptide; (d) exposing negative control cells lacking a HG31 receptor polypeptide to the candidate compound; (e) measuring the amount of binding of the compound to the test cells; (f) measuring the amount of binding of NPSF or a related neuropeptide to the test cells; (g) measuring the amount of binding of the compound to the negative control cells; (h) comparing the amount of binding of the compound to the first portion of test cells with the amount of binding of the compound to the negative control cells and to the amount of binding of NPSF or a related neuropeptide to the second portion of the test cells; wherein if NPSF or a related neuropeptide binds the test cells and the amount of binding of the compound is greater in the test cells as compared to the negative control cells, the compound is capable of binding to the HG31 receptor polypeptide. Determining whether the compound is actually an agonist or antagonist can then be accomplished by the use of functional assays such as, e. g., the assay involving the use of promiscuous G-proteins described below. In an alternative

embodiment one can also expose negative control cells to NPSF or a related neuropeptide, measure any binding of NPSF or a related neuropeptide to the cells and compare that measurement to the other measurements assessed in the assay.

The above methods can be modified in that, rather than exposing the test cells to the compound, membranes can be prepared from the test cells and those membranes can be exposed to the compound. Such a modification utilizing membranes rather than cells is well known in the art and is described in, e. g., Hess et al., 1992.

Accordingly, the present invention provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a competitive format for determining whether a candidate compound is capable of binding to a HG31 receptor polypeptide in membranes comprising: (a) providing test cells by transfecting cells with an expression vector that directs the expression of HG31 in the cells; (b) preparing membranes containing HG31 from the test cells; (c) exposing the membranes to NPSF or a related neuropeptide under conditions such that the ligand binds to the receptor polypeptide in the membranes; (d) further exposing the membranes to a candidate compound under similar conditions; (e) measuring the amount of binding of the NPSF or a related neuropeptide to the HG31 in the membranes in the presence and the absence of the compound; (f) comparing the amount of binding of the NPSF or a related neuropeptide to HG31 in the membranes in the presence and the absence of the compound where a decrease in the amount of binding of the NPSF or a related neuropeptide to HG31 in the membranes in the presence of the compound indicates that the compound is capable of binding to HG31.

The present invention provides a method of using the interaction of NPSF or a related neuropeptide and a HG31 receptor polypeptide as a positive control for determining whether a candidate compound is capable of binding to a HG31 receptor polypeptide comprising: (a) providing test cells by transfecting cells with an expression vector that directs the expression of HG31 on the surface of the cells;

(b) providing negative control cells (i. e., cells lacking HG31 on their surface); (c) preparing membranes from the test cells and membranes from negative control cells; (d) exposing a first portion of the membranes from the test cells to a candidate; (e) exposing a second portion of the membranes from the test cells to NPSF or a related neuropeptide; (f) exposing a portion of the negative control cells to the candidate; (g) measuring the amount of binding of the compound and the NPSF or a related neuropeptide to the HG31 membranes and measuring the binding of the compound to the negative control membranes; (h) comparing the amount of binding of the compound to the HG31 membranes with the amount of binding of the compound to membranes from the negative control cells, where if the NPSF or a related neuropeptide binds to the HG31 membranes and the amount of binding of the compound to HG31 membranes is greater than the amount of binding of the compound to the membranes from the negative control cells, then the compound is capable of binding to the HG31 receptor polypeptide As a further modification of the above-described methods, RNA encoding HG31 can be prepared as, e. g., by in vitro transcription using a plasmid containing HG31 under the control of a bacteriophage T7 promoter, and the RNA can be microinjected into Xenopus oocytes in order to cause the expression of HG31 in the oocytes. Compounds are then tested for binding to the HG31 expressed in the oocytes. Alternatively, rather than detecting binding, the effect of the compounds on the electrophysiological properties of the oocytes can be determined. As in all assays of this invention, a step using the interaction of NPSF or a related neuropeptide and HG31 is incorporated into the assay.

The present invention includes assays by which HG31 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG31. HG31 belongs to the class of proteins known as G- protein coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotrimeric

G-protein, causing the Goc subunit of the G-protein complex to exchange GDP for GTP, and then disassociate from the Gß and Gy subunits. The Goc subunit can then go on to activate a variety of second messenger systems. After being bound by NPSF or a related neuropeptide, HG31 particularly couples to Gi/o which, when activated, inhibits the adenylate cyclase, resulting in the inhibition of cyclic adenosine monophosphate (cAMP) production.

Accordingly, the present invention provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an agonist of HG31, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells; (b) Preparing membranes containing HG31 from the test cells; (c) Preparing membranes without HG31 from mock-transfected cells.

(d) exposing a portion of the HG31-containing membranes to a binding solution containing the candidate compound and guanosine-5'-O- (3- [35S] thio) triphophate ([35S]) GTPyS) ; (e) exposing another portion of the HG31-containing membranes to a binding solution containing NPSF or a related neuropeptide and guanosine-5'-O- (3- [35s] thio) triphophate ([35S]) GTPyS) ; (f) exposing a portion of the mock-transfected membranes to the same candidate compound and [35S] GTPyS ; (g) after incubation for a sufficient period of time, filter the membranes from steps (d), (e), and (f) through filters that retain [35S] GTPyS associated with protein and washing away the free [35S] GTPyS ; (h) determine and amount of radioactivity on the filters; (i) comparing the amount of radioactivity ([35S]) GTPyS) bound.

Where if NPSF or a related neuropeptide increases the binding of [35S] GTPyS to the HG31 membranes and the amount of binding of [35S] GTPyS to HG31 membranes in the presence of the compound is greater than the amount of binding of [35S] GTPyS to the membranes from the mock-transfected cells, then the compound is an agonist of the HG31 receptor polypeptide.

The present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an antagonist of HG31, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells; (b) Preparing membranes containing HG31 from the test cells; (c) exposing a portion of the HG31-containing membranes to a binding solution containing the candidate compound, NPSF or a related neuropeptide, and guanosine-5'-- (3- [35s] thio) triphophate ([35S] GTPyS) ; (d) exposing a portion of the HG31-containing membranes to a binding solution containing NPSF or a related neuropeptide only and guanosine-5'-O- (3- thio) triphophate ([35S]) GTPyS); (e) after incubation for a sufficient period of time, filter the membranes from steps (c) and (d) through filters that retain [35S] GTPyS associated with protein and washing away the free [35S] GTPyS; (f) determine and amount of radioactivity on the filters; (g) comparing the amount of radioactivity ([35S]) GTPyS bound) from (c) and (d). If the amount of [35S] GTPyS bound by membranes containing HG31 from step (c) is less than the amount bound by membranes from step (d), then the candidate compound is an antagonist of HG31.

The present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an agonist of HG31 based on the finding that activation of HG31 by NPSF or a related neuropeptide leads to inhibition of adenylate cyclase, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells; (b) exposing a first portion of the test cells to the candidate compound; (c) exposing a second portion of the test cells to NPSF or a related neuropeptide;

(d) exposing a first portion of negative control cells lacking a HG31 receptor polypeptide to the same candidate compound; (e) exposing a second portion of negative control cells lacking a HG31 receptor polypeptide to NPSF or a related neuropeptide; (f) exposing cells from (b), (c), (d), and (e) immediately to an activator of adenylate cyclase (e. g., forskolin at 100 nM); (g) measuring the amount of cAMP produced in cells from (b), (c), (d), and (e) after step (f). The total amount of cAMP can be determined by a variety of methods, e. g. the cAMP enzymeimmunoassay (EIA) system (dual range) from AMERSHAM-PHARMACIA (Uppsala, Sweden).

(h) comparing the amount of cAMP produced in cells from (b), (c), (d), and (e). If the amount of cAMP produced by cells from Step (b) is less than the amount produced by cells from Step (d), then the candidate compound is an agonist of HG31. The amount of cAMP produced by cells from step (c) should be less than the amount produced by cells from step (e), which serves as a positive control.

The present invention also provides a method of using the interaction of NPSF or a related neuropeptide and HG31 in a functional assay for determining whether a candidate compound is an antagonist of HG31 based on the finding that activation of HG31 by NPSF or a related neuropeptide leads to inhibition of adenylate cyclase, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of a HG31 receptor polypeptide on the surface of the cells; (b) exposing a first portion of the test cells to the candidate compound; (c) subsequently to step (b), exposing these test cells to NPSF or a related neuropeptide; (d) exposing another portion of the test cells to NPSF or a related neuropeptide only; (e) exposing cells from (c) and (d) to an activator of adenylate cyclase; (f) measuring the amount of cAMP produced in cells from (c) and (d) after activation of adenylate cyclase. Total amount of cAMP can be determined by a variety of methods, e. g. the cAMP enzymeimmunoassay (EIA) system (dual range) from AMERSHAM-PHARMACIA (Uppsala, Sweden).

(g) comparing the amount of cAMP produced in cells from (c) and (d). If the amount of cAMP produced by cells as treated from (c) is more than the amount produced by cells from (d), then the candidate compound is an antagonist of HG31.

Generally, a particular GPCR is only coupled to a particular type of G- protein. Thus, to observe a functional response from the GPCR, it is necessary to ensure that the proper G-protein is present in the system containing the GPCR. It has been found, however, that there are certain G-proteins that are"promiscuous."These promiscuous G-proteins will couple to, and thus transduce a functional signal from, virtually any GPCR. See Offermanns & Simon, 1995 (Offermanns). Offermanns described a system in which cells are transfected with expression vectors that result in the expression of one of a large number of GPCRs as well as the expression of one of the promiscuous G-proteins Gal5 or Gal6. Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via Gal5 or Got16, to activate the ß isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.

Therefore, by making use of these promiscuous G-proteins as in Offermanns, it is possible to set up functional assays for HG31, even in the absence of knowledge of the G-protein with which HG31 is coupled in vivo. One possibility is to create a fusion or chimeric protein composed of the extracellular and membrane spanning portion of HG31 fused to a promiscuous G-protein. Such a fusion protein would be expected to transduce a signal following binding of ligand to the HG31 portion of the fusion protein.

Accordingly, the present invention provides a method of determining whether a candidate compound is an antagonist of HG31 comprising: (a) providing cells that expresses a chimeric HG31 protein fused at its C-terminus to a promiscuous G-protein; (b) exposing a portion of the cells to the candidate; (c) subsequently or concurrently to step (b), exposing the cells to NPSF or a related neuropeptide; (d) measuring the level of inositol phosphates in a portion of the cells exposed only to the NPSF or a related neuropeptide and a portion of the cells exposed to candidate and NPSF or a related neuropeptide; where a decrease in the level of inositol phosphates in the cells in the presence of both the compound and NPSF or a related neuropeptide as compared to

the level of inositol phosphates in the cells in the absence of the compound indicates that the compound is an antagonist of HG31.

Another format for utilizing promiscuous G-proteins in connection with HG31 includes a method of identifying agonists of HG31 comprising: (a) providing cells that expresses both HG31 and a promiscuous G-protein; (b) exposing a first portion of the cells to a candidate; (c) exposing a second portion of the cells to NPSF or a related neuropeptide; (d) measuring the level of inositol phosphates in each portion of the cells; where if the NPSF or a related neuropeptide causes an increase in the level of inositol phosphate in the cells, an increase in the level of inositol phosphates in the cells exposed to the candidate as compared to the level of inositol phosphates in the cells in the absence of the candidate indicates that the compound is an agonist of HG31.

Levels of inositol phosphates can be measured directly using ion- exchange chromatography or indirectly by monitoring calcium mobilization.

Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.

The above-described assay can be easily modified to form a method to identify antagonists of HG31. Such a method is also part of the present invention and comprises: (a) providing cells that expresses both HG31 and a promiscuous G-protein; (b) exposing the cells to a candidate antagonist of HG31; (c) subsequently or concurrently to step (b), exposing a portion of the cells from step (b) to NPSF or a related neuropeptide, (d) measuring the level of inositol phosphates in the cells; where a decrease in the level of inositol phosphates in the cells in the presence of the candidate as compared to the level of inositol phosphates in the cells in the absence of the candidate indicates that the compound is an antagonist of HG31.

The conditions under which the binding steps of above-described methods are practiced are conditions that are typically used in the art for the study of

protein-ligand interactions: e. g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media ; a temperature of about 4°C to about 55°C.

In particular embodiments of the above-described methods, the cells are transfected with expression vectors that direct the expression of HG31 and the promiscuous G-protein in the cells.

In particular embodiments of the above-described methods, the promiscuous G-protein is selected from the group consisting of Ga15 or Gcc16.

Expression vectors containing Gal 5 or Gal 6 are known in the art. See, e. g., Offermanns ; Buhl et al., 1993; Amatruda et al., 1993.

In particular embodiment of the above-described methods, the cells express a chimeric G-protein, e. g. Gqi or Gqo. Assay conditions and expression vectors using chimeric G-proteins are known in the art. See. e. g., Coward et al., 1999, Analytical Biochem., 270,242-248.

Additional types of functional assays that can be used to identify agonists and antagonists of HG31 include transcription-based assays. Transcription- based assays involve the use of a reporter gene whose transcription is driven by an inducible promoter whose activity is regulated by a particular intracellular event such as, e. g., changes in intracellular calcium levels that are caused by the interaction of a receptor with a ligand. Transciption-based assays are reviewed in Rutter et al., 1998.

The transcription-based assays of the present invention rely on the expression of reporter genes whose transcription is activated or repressed as a result of intracellular events that are caused by the interaction of an agonist with a HG31 receptor polypeptide.

An extremely sensitive transcription based assay is disclosed in Zlokarnik et al., 1998 (Zlokarnik) and also in U. S. Patent No. 5,741,657. The assay disclosed in Zlokarnik and U. S. Patent No. 5,741,657 employs a plasmid encoding (3- lactamase under the control of an inducible promoter. This plasmid is transfected into cells together with a plasmid encoding a receptor for which it is desired to identify agonists. The inducible promoter on the (3-lactamase is chosen so that it responds to at least one intracellular signal that is generated when an agonist binds to the receptor.

Thus, following such binding of agonist to receptor, the level of (3-lactamase in the transfected cells increases. This increase in (3-lactamase is made measurable by treating the cells with a cell-permeable dye that is a substrate for P-lactamase. The dye contains two fluorescent moieties. In the intact dye, the two fluorescent moieties

are close enough to one another that fluorescent resonance energy transfer (FRET) can take place between them. Following cleavage of the dye into two parts by ß- lactamase, the two fluorescent moieties are located on different parts, and thus can drift apart. This increases the distance between the fluorescent moieties, thus abolishing the amount of FRET that can occur between them. It is this decrease in FRET that is measured in the assay.

One skilled in the art can modify the assay described in Zlokarnik and U. S. Patent No. 5,741,657 to form an assay for identifying candidate compounds that are agonists of HG31 receptor polypeptides by using an inducible promoter to drive P-lactamase that is activated by an intracellular signal generated by the interaction of agonists and the HG31 receptor. To produce the receptor, a plasmid encoding HG31 is transfected into the cells. The cells are exposed tocandidate compounds for a few hours to allow the transcription and translation of the reporter enzyme 0-lactamase.

The cells are then exposed to the cell-permeable dye for a certain period to time to allow the dyes to load and to be cleaved. Those candidates that cause a decrease in FRET are likely to be actual agonists of the receptor. A portion of the transfected cells are treated with NPSF or a related neuropeptide and the decrease in FRET measured is compared to any decrease in FRET measured in cells exposed to the candidate compound. Additionally, a portion of the transfected cells are treated with NPSF or a related neuropeptide and the decrease in FRET can be monitored as a positive control.

Accordingly, the present invention includes a method for identifying agonists of the HG31 receptor comprising: (a) transfecting cells with an expression vector that directs the expression of HG31 in the cells and an expression vector that directs the expression of (3-lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of agonists and the HG31 receptor; (b) exposing the cells to a candidate compound; (c) exposing the cells to a substrate of (3-lactamase that is a cell- permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by P-lactamase allows the two fluorescent moieties to drift apart; (d) measuring the amount of fluorescent resonance energy transfer (FRET) in the cells;

wherein if the cells exposed to a candidate compound showed a decrease in the amount of FRET, then the candidate is an agonist of the HG31 receptor.

The assay described above can be modified to an assay for identifying antagonists of the HG31 receptor. Such modification would involve the use of P- lactamase under the control of a promoter that is repressed by at least one intracellular signal generated by interaction of an agonist with the HG31 receptor and include conducting the assay in the presence of NPSF or a related neuropeptide. When the cells are exposed to substances suspected of being antagonists of the receptor, lactamase will be decreased, and FRET will be increased.

Accordingly, the present invention includes a method for identifying antagonists of the HG31 receptor comprising: (a) transfecting cells with an expression vector that directs the expression of HG31 in the cells and an expression vector that directs the expression of P-lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of an agonist and the HG31 receptor; (b) exposing a portion of the cells to a candidate antagonist compound; (c) subsequently or concurrently, exposing the cells to NPSF or a related neuropeptide and incubate for a few hours; (d) exposing another portion of the cells to NPSF or a related neuropeptide alone; (e) exposing the cells from step (c) and (d) to a substrate of lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by P-lactamase allows the two fluorescent moieties to drift apart; (f) measuring the amount of fluorescent resonance energy transfer (FRET) in the cells; wherein if the amount of FRET in the cells measured exposed to the candidate antagonist is more than that the amount of FRET measured in the cells not exposed to the candidate antagonists, then the substance is an antagonist of the HG31 receptor.

In certain embodiments, the inducible promoter that is repressed by at least one intracellular signal generated by interaction of an agonist with the HG31

receptor is a promoter that is repressed by decreases in cAMP levels or changes in potassium currents.

In a particular embodiment, the inducible promoter that is activated by at least one intracellular signal generated by interaction of an agonist with the HG31 receptor is a promoter that is activated by decreases in cAMP levels.

In a particular embodiment, P-lactamase is TEM-1 P-lactamase from Escherichia coli.

In particular embodiments, the substrate of (3-lactamase is CCF2/AM (Zlokarnik et al., 1998).

In particular embodiments, the cells express a promiscuous G-protein, e. g., Got15 or Goc16.

In particular embodiment of the above-described methods, the cells express a chimeric G-protein, e. g. Gqi or Gqo. Assay conditions and expression vectors using chimeric G-proteins are known in the art. See. e. g., Coward et al., 1999, Analytical Biochem., 270,242-248.

In particular embodiments, the inducible promoter is a promoter that is activated or repressed by NF-KB or NFAT.

The assays described above could be modified to identify inverse agonists. In such assays, one would expect a decrease in (3-lactamase activity.

Similarly, inverse agonists can be identified by modifying the functional assays that were described previously where those functional assays monitored decreases in cAMP levels. In the case of assays for inverse agonists, increases in cAMP levels would be observed.

Other transcription-based assays that can be used to identify agonists and antagonists of the HG31 receptor rely on the use of green fluorescent proteins or luciferase as reporter genes. An example of such an assay comprises: (a) transfecting cells with an expression vector that directs the expression of HG31 in the cells and an expression vector that directs the expression of green fluorescent protein (GFP) under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of an agonist and the HG31 receptor; (b) measuring the amount of fluorescence from GFP in a first portion of the cells; (c) exposing a second portion of cells to a candidate agonist compound;

(d) measuring the amount of fluorescence from GFP in the second portion of cells after exposure to the candidate; (e) exposing a third portion of cells to NPSF or a related neuropeptide (f) measuring the amount of fluorescence from GFP in the third portion of cells after exposure to the NPSF or a related neuropeptide; wherein if the amount of fluorescence from GFP in the second and third portions of cells are both more that the amount of fluorescence from GFP measured in the first portion of cells, then the candidate is an agonist of the receptor.

In particular embodiments of the above-described methods, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other particular embodiments, the cells are L cells L-M (TK-) (ATCC CCL 1.3), L cells L- M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).

The assays described above can be carried out with cells that have been transiently or stably transfected with an expression vector that directs the synthesis of a HG31 receptor polypeptide. Transfection is meant to include any method known in the art for introducing HG31 into the test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG31, and electroporation.

Where binding of a compound to HG31 is measured, such binding can be measured by employing a labeled compound or agonist. The compound or agonist can be labeled in any convenient manner known to the art, e. g., radioactively, fluorescently, enzymatically.

In particular embodiments of the above-described methods, the HG31 receptor polypeptide has a full length amino acid sequence as shown in FIGS. 1 (SEQ ID NO : 4) or 2 (SEQ ID NO : 6).

Many other assay systems are known in the art and can be adapted to identify compounds that are agonist or antagonists that modulate the activity of the HG31 receptor. Several are described briefly below.

Melanophore system.

The melanophore screening system is described in WO 92/01810, published February 6,1992. Briefly, melanophores are transfected to express a HG31 receptor polypeptide. In an assay for antagonists, the transformed melanophores are exposed to both an activating ligand, e. g., NPSF or a related neuropeptide, and a candidate compound. Inhibition of the signal generated by the activating ligand indicates that the candidate is a potential antagonist of the receptor. In an assay for an agonist, the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor. Cells are exposed to an activating ligand, e. g., NPSF or a related neuropeptide, and used as a control in the later embodiment.

Yeast expressing mammalian adenylate cyclase.

Screening methods employing yeast that express mammalian adenylate cyclase are described in WO 95/30012, published November 9,1995. These yeast can be engineered to co-express a HG31 receptor polypeptide in the presence of an appropriate G-protein. In an assay for antagonists, the transformed yeast are exposed to both an activating ligand, e. g., NPSF or a related neuropeptide, of HG31 and a candidate compound. Inhibition of the signal generated by the activating ligand indicates that the candidate is a potential antagonist of the receptor. In an assay for an agonist, the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor. Cells are exposed to an activating ligand, e. g., NPSF or a related neuropeptide, and used as a control in the later embodiment.

Yeast pheromone protein surrogate screening.

Yeast cells engineered to produce pheromone system protein surrogates can be used to screen for the ability of the surrogate to substitute for the cognate yeast pheromone receptor as described in U. S. Patent No. 5,789,184, August 4,1998. Generally, the method involves expressing a HG31 receptor polypeptide in Saccharomyces cerevisiae in which the receptor is linked to pheromone pathway. In this system, the yeast Goe subunit is generally deleted and replaced with a mammalian Goc protein so that the mammalian G protein-coupled receptor can be coupled to the yeast pheromone pathway. Members of a plasmid library capable of expressing peptides of random sequences are introduced into an appropriate yeast strain. Clones

encoding agonist ligands for the HG31 receptor can be selected for their stimulation of the pheromone pathway. Clones encoding antagonist ligands for the HG31 receptor can be selected for their inhibition of the pheromone pathway in the presence of an HG31 agonist. Alternatively, libraries of chemicals can be screened for their agonist or antagonist activity by testing the chemicals directly.

Phospholipase second signal screening Another screening technique involves expressing the HG31 receptor wherein the receptor is linked to a phospholipase C or D. Cells including CHO, endothelial, embryonic kidney and other cells can be used. As in other screens, ligand and candidates are screened for agonist or antagonist activities by detecting the activation or inhibition or the receptor's activation of the phospholipase second signal. An example of one such system using yeast cells expressing a heterologous phospholipase is found in WO 96/40939, published December 19,1996. Some assays based on phospholipase pathway signals are discussed above.

These and other assay formats can be adapted for high throughput screening (HTPS) of compounds for binding the HG31 receptor or having activity as an agonist or antagonist of HG31. HTPS assays are useful for a variety of large screening projects including screening preassembled chemical libraries, screening the output of combinatorial chemical synthesis of compounds and screening peptide display libraries (e. g., phage display libraries).

The following examples are illustrative of the invention and provide the basis for other embodiments as will be apparent to those of skill in the art.

EXAMPLE 1 NPSF-related neuropeptides as natural ligands of the orphan receptor HG31 Cloning of the HG31 short form sequence into the vector pcDNA3.1 (-) and into the vector pIRESpuromycin.

The complete coding sequence of the HG31 short form was amplified from a cDNA library using the two primers: sense primer, 5'- CTCTGCCCACCTCTTCTCTTC-3' (SEQ ID NO : 10) and antisense primer, 5'- AGAGAGGGCTTTCAGTAAATGTT-3' (SEQ ID NO : 11). The PCR product was purified and subcIoned into pCR2.1 by TA cloning (INVITROGEN, Carlsbad, CA,

USA). The sequence of HG31 was verified by complete sequencing and then subcloned into pcDNA3.1 (-) (INVITROGEN, Carlsbad, CA, USA), resulting in plasmid HG31/pcDNA3. 1 (-). The HG31 sequence was then digested out from the pcDNA3.1 vector and sub-cloned into the vector pIRESpuromcyin (CLONTECH, PALO ALTO, CA, USA), resulting the plasmid HG31/-IRESpuromycin.

Generation of HG31-expressing cells The HEK293/aeql7 cell line was licensed in from NIH (Button and Brownstein, 1993, Cell Calcium, 14: 663-671). The complete coding sequence of mouse promiscuous G protein Gal 5 was cloned into the vector pIRES/zeocin (CLONTECH, Palo Alto, California, USA). The resulting plasmid was transfected into HEK293/aeql7 cells using Lipofectamine (GIBCO-BRL, Gaithersburg, MD, USA) and selected with zeocin. Individual stable colonies were isolated and tested for coupling of various receptors. One clone, #3, showed promiscuous coupling, was named HEK293/aeql7/Gal5 and used for assays thereafter.

HEK/293/aeql7/Gal5 were grown in Dulbecco's Modified Medium (DMEM, GIBCO-BRL, Gaithersburg, MD, USA) + 10% fetal bovine serum (heat inactivated), 1 mM sodium pyruvate, 500 pg/ml Geneticin, 200, ug/ml zeocin, 100 ßg/ml streptomycin, 100 units/ml of penicillin. The HG31/pIRESpuromycin plasmid DNA was transfected into HEK293/aeql7/Gal5 using LIPOFECTAMINE-2000 (Gaithersburg, MD, USA) following the conditions suggested by GIBCO-BRL. Two days after transfection, the cells were trypsinized and plated out in complete culture medium (see above) plus puromycin (0.5 Fg/ml) at 1: 5 dilution. The cells were incubated at 37°C/5% C02 and replaced with fresh complete culture medium twice per week. Two weeks later, puromycin-resistant cells were trypsinized, pooled, and propagated for assays.

Cells were seeded at-20% confluency two days before the assay. For the assay, cells were washed once with DMEM + 0.1 % fetal bovine serum, and then charged for one hour at 37°C/5% C02 in DMEM containing 5 uM coelenterazine cp (MOLECULAR PROBES, Eugene, OR, USA) and 30 uM glutathione. The cells were then washed once with Versene (GIBCO-BRL, Gaithersburg, MD, USA), detached using Enzyme-free cell dissociation buffer (GIBCO-BRL, Gaithersburg, MD, USA), diluted into ECB (Ham's F12 nutrient mixture (GIBCO-BRL) + 0.3 mM CaCl2, 25 mM HEPES, pH7.3,0.1% fetal bovine serum). The cell suspension was centrifuged at 500x g for 5 min. The supernatant was removed, and the pellet was

then resuspended in 10 mL ECB. The cell density was determined by counting with a hemacytometer and adjusted to 500,000 cells/ml in ECB.

Human neuropeptide NPAF and NPSF were custom-synthesized (PHOENIX PHARMACEUTICALS, INC., Belmont, CA, USA). Bovine neuropeptide FF was purchased from commercial sources (PENINSULA LABORATORIES, BELMONT, CA, USA). The peptides were diluted in ECB into 2X concentrates using 5-fold serial dilutions, and aliquoted into assay plates in triplicates at 0.1 ml/well. The cell suspension was injected at 0.1 ml/well, read and integrated for a total of 20 seconds using a Dynex MLX luminometer (DYNEX TECHNOLOGIES, MIDDLESEX, UK). Data were analyzed using the software GRAPHPAD PRISM Version 3.0 (GRAPHPAD SOFTWARE, INC., San Diego, CA, USA). As shown in Fig. 3., HG31-transfected cells showed a robust, dose-dependent response to human neuropeptide NPSF and NPAF, and bovine NPFF. The effective half-maximal concentrations (EC50) of NPAF, NPFF, and NPSF are 44,39,9 nM, indicating that HG31 is a high affinity receptor for NPSF and its related peptides.

HG31-negative cells did not show any response to the peptides (data not shown).

EXAMPLE 2 Activation of HG31 by NPFF peptides in the melanophore assay.

Growth of Xeiiopus laevis melanophores and fibroblasts was performed as described previously (Daniolos, et al., 1992. Pigniefit Cell Res. 3,38- 43; and Lerner 1994. Trends Neurol. Sci. 17,142-146). Briefly, melanophores were grown in Xenopus fibroblast-conditioned growth medium. The fibroblast-conditioned growth medium was prepared by growing Xenopus fibroblasts in 70% L-15 medium (Sigma), pH 7.3, supplemented with 20 % heat inactivated fetal bovine serum (GIBCO-BRL, Gaithersburg, MD, USA), 100 ug/ml streptomycin, 100 units/ml penicillin and 2 mM glutamine at 27°C. The medium from growing fibroblasts was collected, passed through a 0.2 micron filter (referred to as fibroblast-conditioned growth medium) and used to culture melanophores at 27°C. Plasmid DNA was transiently transfected into melanophores by electroporation using a BTX ECM600 electroporator (Genetronics, Inc. San Diego, CA, USA Melanophores were incubated in the presence of fresh fibroblast- conditioned growth medium for 1 hour prior to harvesting of cells. Melanophore

monolayers were detached by trypsinization (0.25% trypsin, JHR BIOSCIENCES, Lenexa, KS, USA), followed by inactivation of the trypsin with fibroblast- conditioned growth medium.

The cells were collected by centrifugation at 200x g for 5 minutes at 4°C. Cells were washed once in fibroblast conditioned growth medium, centrifuged (200 x g, 5 minutes, 4°C) and resuspended at 5 x 106 cells per ml in ice-cold 70% PBS pH 7.0.400 pl aliquots of cells in PBS were added to pre-chilled 1.5 ml tubes containing 2 p, g of HG31/pcDNA3. 1 (-) plasmid DNA, and 22 Rg of pcDNA3 plasmid vector DNA for a total of 24 u, g DNA in a 40 RI total volume. Samples were incubated on ice for 20 minutes with mixing every 7 minutes.

Cell and DNA mixes were transferred to prechilled 0.2 mm gap electroporation cuvettes (BTX ELECTROPORATOR, SAN DIEGO, CA, USA) and electroporated using the following settings: capacitance of 325 microfarad, voltage of 425 volts and resistance of 720 ohms. Immediately following electroporation, cells were mixed with fibroblast-conditioned growth medium and plated onto flat bottom Primaria 96 well microtiter plates (FALCON, LINCOLN PARK, NJ, USA).

Electroporations from multiple cuvettes were pooled together prior to plating to ensure homogenous transfection efficiency.

On the day following transfection, the media was replaced with fresh fibroblast-conditioned growth media and incubated for an additional day at 27°C prior to assaying for receptor expression. On the day of ligand stimulation, medium was removed by aspiration and cells were washed with 70% L-15 media containing 15 mM HEPES, pH 7. 3.

Assays were divided into two separate parts in order to examine Gs/Gq-coupling which results in pigment dispersion in melanophores, or Gi-coupling which results in pigment aggregation. For Gs/Gq-coupling responses, assays were performed as follows. Cells were incubated in 100 jod of 70% L-15 media containing 15 mM HEPES, pH 7.3, for 1 hour in the dark at room temperature, and then incubated in the presence of melatonin (2 nM final concentration) for 1 hour in the dark at room temperature to induce pigment aggregation. Initial absorbance readings at 590 nm were measured using a vmax Microplate reader (MOLECULAR DEVICES, MENLO PARK, CA, USA) prior to addition of ligand. Peptides (100-500 nM, final concentration) were added to wells and control ligands melatonin and oc- MSH were added at 100 nM final concentration, mixed, and incubated in the dark at room temperature for 1 hour, after which the final absorbance at 590 nm was

measured. Absorbance readings were converted to transmittance values using the following formula: 1/As, where A590 = the absorbance reading at 590 nm.

Subsequently, pigment dispersion was quantitated by the formula 1-Tf/Ti, where Ti = the initial transmittance at 590 nm and Tf = the final transmittance at 590 nm.

For Gi-coupled responses, cell monolayers plated in 96-well microtiter plates were incubated in the presence of 100, ul/well of 70% L-15 media containing 1% fibroblast-conditioned growth medium, 2 mM glutamine, 100 Flg/ml streptomycin, 100 units/ml penicillin and 15 mM HEPES, pH 7.3, for 15 minutes in the dark at room temperature to preset the cells to dispersion. Initial absorbance readings at 590 nm were determined, followed by the addition of ligands. After a 1 hour incubation in the dark at room temperature final absorbances were measured.

Absorbance readings were used to quantitate pigment aggregation by the formula Af/Ai-1, where Ai = the initial absorbance at 590 nm and Af = the final absorbance at 590 nm.

Melanophores transiently transfected with plasmid DNAs expressing "HG31", were plated onto 96 well microtiter plates. Following the above pretreatment conditions, cells were incubated for 1 hour in the presence of a collection of 83 known peptides including NPSF. Final concentrations of the peptides were in the range of 100-500 nM.

Pigment aggregation responses (Gi-coupled responses) were detected only with NPSF, NPAF, NPFF, LPRLF and prolactin releasing peptide-20 (PrP-20) with responses ranging from 71%, 92%, 86%, 56%, 48% respectively relative to a 100 nM melatonin control response. Background aggregation responses ranged from 10-22% of the melatonin control. Comparative background responses were seen in pcDNA3 only transfected melanophores, with no values exceeding background for the above peptides.

Pigment dispersion responses (Gs/Gq-coupled responses) were not detected with NPSF, NPAF, NPFF, LPRLF and prolactin releasing peptide.

As shown in FIG. 4, HG31-transfected cells showed strong, dose- dependent pigment aggregation in response to NPSF, NPAF, NPFF, LPLRF (chicken brain peptide), and prolactin-releasing peptide (PrP-20), indicating that the activation of HG31 by NPSF and its related peptides is coupled to the Gi/o pathway. The effective half-maximum response concentrations (ECso) of these peptides are: NPSF, 1.8 nM; NPAF, 3.5 nM; NPFF, 9.6 nM; LPLRF, 142 nM; PrP-20,162 nM, indicating

that HG31 is a high affinity receptor for NPSF, NPAF, and NPFF. All of the peptides were tested in the range of 6X10-1 l-lx10-6 M.

EXAMPLE 3 Transgenic animals In reference to the transgenic animals of this invention, we refer to transgenes and genes. As used herein, a"transgene"is a genetic construct including a gene. The transgene is typically integrated into one or more chromosomes in the cells in an animal or its ancestor by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal.

A gene is a nucleotide sequence that encodes a protein. The gene and/or transgene can also include genetic regulatory elements and/or structural elements known in the art.

The term"animal"is used herein to include all mammals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Preferably the animal is a rodent, and most preferably mouse or rat. A"transgenic animal"is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA. Unless otherwise noted or understood from the context of the description of an animal, the term "transgenic animal"as used herein refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals. The genetic information is typically provided in the form of a transgene carried by the transgenic animal.

The genetic information received by the non-human animal can be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the information can be altered or it can be expressed differently than the native gene. Alternatively, the altered or introduced gene can cause the native gene to become non-functional to produce a "knockout"animal.

As used herein, a"targeted gene"or"Knockout" (KO) transgene is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles of the non-human animal.

An altered HG31 receptor gene should not fully encode the same receptor endogenous to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether. In cases where it is useful to express a non-native HG31 receptor in a transgenic animal in the absence of a endogenous HG31 receptor we prefer that the altered HG31 gene induce a null,"knockout," phenotype in the animal. However a more modestly modified HG31 gene can also be useful and is within the scope of the present invention.

A type of target cell for transgene introduction is the embryonic stem cell (ES). ES cells can be obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al., Nature 292: 154-156 (1981) ; Bradley et al., Nature 309: 255-258 (1984); Gossler et al. Proc. Natl. Acad. Sci. USA 83 : 9065- 9069 (1986); and Robertson et al., Nature 322: 445-448 (1986)). Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)). Animals are screened for those resulting in germline transformants. These are crossed to produce animals homozygous for the transgene.

Methods for evaluating the targeted recombination events as well as the resulting knockout mice are readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.

The creation and study of a transgenic animal may have a therapeutic aim. (Gene therapy is discussed infra.) The presence of a mutant allele or variant sequence within cells of an organism, particularly when in place of a homologous endogenous sequence, may allow the organism to be used as a model in testing and/or studying the role of the HG31 gene or substances which modulate activity of the

encoded polypeptide and/or promoter in vitro or are otherwise indicated to be of therapeutic potential.

Transgenic animals expressing HG31 as a transgene are provided as follows. A polynucleotide having an HG31 nucleotide sequence, e. g., the nucleotide sequence of a cDNA or genomic DNA encoding a full length HG31 receptor, or a polynucleotide encoding a partial sequence of the receptor, sequences flanking the coding sequence, or both, can be combined into a vector for the integration of the polynucleotide into the genome of an animal. The HG31 sequence can be from a human HG31 or from the animal's HG31.

In this example, the target cell for transgene introduction is a murine embryonic stem cell (ES). ES cells can be obtained from pre-implantation embryos of a variety of non-human animals cultured in vitro and fused with embryos (M. J.

Evans et al., (1981); Bradley et al., (1984); Gossler et al. (1986); and Robertson et al., (1986)).

The transgene is introduced into the murine ES cells by microinjection, however, a variety of standard techniques such as DNA transfection, or retrovirus- mediated transduction can be used. The injected ES cells are then combined with blastocysts from a non-human animal. The introduced ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)). The chimeric mice are screened for individuals in which germline transformation has occurred. These are crossed to produce animals homozygous for the transgene.

The targeted recombination events as well as the resulting mice are evaluated by techniques well known in the art, including but not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.

Three basic types of transgenic animals are created depending on the construction of the transgene vector. If the vector is designed to include a nucleotide sequence that encodes a full length human HG31 receptor and to integrate at a site other than the animal's endogenous HG31 gene, the resultant transgenic animal will express both a native and human HG31 receptors. If the vector is designed without a cognate HG31 gene and to integrate at the site of the animal's endogenous HG31 gene such that after integration the endogenous gene is altered to such an extent that the animal lacks a functional HG31 receptor, then a knockout animal is produced.

Finally, if the vector is designed to replace the endogenous HG31 gene with a human gene, or is designed to change the sequence of the endogenous gene to encode the amino acid sequence of the human gene, i. e., is humanized, then the resultant animal lacks a native HG31 receptor and expresses a human HG31 receptor. Animals having a human gene and lacking an endogenous gene can also be created by crossing the first type of animal with a knockout animal to obtain animals homozygous for the knockout and homozygous for the added human HG31 gene. This can be facilitated if the human gene integrates in a chromosome different from the chromosome carrying the endogenous HG31 gene.

Transgenic animals are a source of cells and tissues for use in assays of HG31 modulation, activation or inhibition. Cells can be removed from the animals, established as cell lines and maintained in culture as convenient.

Transgenic HG31 knockout animals can exhibit a variety of phenotypes including lower, little or no tolerance to opiates. In addition to being a valuable resource for the study of HG31 influenced aspects of neuropharmacology, knockout animals can be used to provide negative control cells lacking functional HG31 receptor polypeptides for use in assays.

EXAMPLE 4 Radioligand Binding Assay Transfection and membrane preparation of HEK293/aeql7/Gal5 cells: HEK293/aeql7/Gal5 cells were seeded in T-175 tissue culture flasks at-18 x 106 cells/flask for a total of 13 flasks, and were transfected using LIPOFECTAMINE-2000 (GIBCO-BRL, Gaithersburg, MD, USA) following the manufacturer's protocol. Ten flasks were transfected with HG31/pcDNA3.1 (-) while the remaining three flasks were transfected with vector control (pcDNA3. 1 (+)).

Three days after transfection, cells were washed once with PBS, scraped off into PBS, and spun down at 1, 000g for 5 min at 4°C. All procedures were conducted on ice from here on. The cell pellets were resuspended in 40 ml of harvest buffer [50 mM HEPES, pH 7.4,1 mM EDTA, 4 complete protease inhibitor pellets (BOEHRINGER MANNHEIM, Indianapolis, IN, USA)] and homogenized for 4 bursts at 4°C using a 5 mm probe on a polytron homogenizer (POWERGEN 125, FISHER SCIENTIFIC, Pittsburgh, PA, USA). The suspension was then centrifuged at 48,000g for 20 min at

4°C. The pellet was then resuspended in harvest buffer, dispensed into microcentrifuge tubes at-10 mg of protein per tube, and centrifuged at 12,000g for at 10 min. at 4°C. The pellets were then resuspended in harvest buffer + 250 mM sucrose at a concentration of 8 mg/ml and stored at-70°C.

Binding of bovine 125I-Tyrl-NpFF to cell membranes expressing human HG31: Tyrl-NPFF (Tyr-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) (SEQ ID NO : 12) was custom-synthesized by PHOENIX PHARMACEUTICALS (Belmont, CA, USA) and labeled at the N-terminal Tyr residue with 1251 to a specific activity of - 2,000 Ci/mmol by WOODS ASSAY (Portland, Oregon, USA). The binding solution (0.2 ml in a 96-well plate) contained 80 ptg (total protein) of HG31- expressing cellular membrane, 0.1 nM 125I-Tyrl-NPFF, and varying concentrations of competitor in 25 mM Tris-HCI, pH 7.5,5 mM MgCl2. After incubation for 1 hour at room temperature, binding reactions were harvested onto a GF/B filter (UNIFILTER-96, PACKARD, Downers Grove, IL, USA) and washed with 25 mM Tris-HCI, pH 7.5 (4 cycles, one wash for 1.0 second and another wash of 2.0 second per cycle) using a harvester (HARVESTER-9600, TOMTEC, Hamden, CT, USA).

The filter was then dried for 30 minutes at 50°C and counted by gamma counting (TOPCOUNT NXT, PACKARD, Downers Grove, IL, USA). Data were analyzed and plotted using the software GRAPHPAD PRISM Version 3.0 (GRAPHPAD SOFTWARE, San Diego, CA, USA).

As shown in the FIG. 6, HG31-expressing membranes displayed specific binding to 1251-Tyrl-NPFF which was competed off by bovine NPFF, human NPSF, and human NPAF. The concentration causing 50% inhibition of specific binding (IC50) for bovine NPFF, human NPSF, and human NPAF are 7.5, 1. 9, and 9.7 nM, respectively, consistent with the data from the functional assays.

The results indicate that HG31 is a high affinity receptor for NPSF.

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Many variations of the conduct of assays of the present invention will be apparent to those of skill in the art. Such variations are intended to be within the scope of the claims.