GB2364056A - Human 7-transmembrane receptor HLWAR77 - Google Patents

Abstract

HLWAR77 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Methods for the identification of agonists and antagonists of the polypeptides are disclosed, preferably by competitive binding assays in the presence of peptide ligands such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF). Also disclosed are methods for the utilization of the HLWAR77 polypeptides in the treatment of infections and diseases and in diagnostic assays.

Description

2364056 7 T.M RECEPTOR LLWAR 77

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U S Serial No 09/195,517, filed November 19, 1998, now pending, which is a continuation-in-part application of U S.

Serial No 09/006,140, filed January 13, 1998, now pending, the entire disclosures of which are herein incorporated by reference U S Serial No 09/006,140 in turn claims the benefit of U.S Provisional Application Nos 60/049,332, filed June 11, 1997, and 60/067,253, filed December 2, 1997, the entire disclosures of which are herein incorporated by reference.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production.

More particularly, the polynucleotides and polypeptides of the present invention relate to G-protein coupled receptor family, hereinafter referred to as HILWWAR 77 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e g, c AMP (Lefkowitz, Nature, 1991, 351:353- 354) Herein these proteins are referred to as proteins participating in pathways with G- proteins or PPG proteins.

Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B K, et al, Proc Nail Acad, Sci, USA, 1987 84:46-50; Kobilka, B K, et al, Science, 1987, 238:650-656; Bunzow, J R, et al, Nature, 1988, 336:783-787), G-proteins themselves, effector proteins, e g, phospholipase C, adenyl cyclase, and phosphodicsterase, and actuator proteins, e g, protein kinase A and protein kinase C (Simon, M I, et al, Science, 1991, 252:802-8).

For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell Enzyme activation by hormones is dependent on the prcscnce of the nucleotide, GTP GTP also influences hormone binding.

A G-protein connects the hormone receptor to adenylate cyclase G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor The GTP- carrymg form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.

The membrane protein gene superfamily of G protein coupled receptors has been characterized as having seven putative transmembrane domains The domains are believed to represent transmembrane c-helices connected by extracellular or cytoplasmic loops G- protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.

G-protein coupled receptors (otherwise known as 7 TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops The G-protein family of coupled receptors includes dopamine receptors which bind to neurolepric drugs used for treating psychotic and neurological disorders Other examples of members of this family include, but arc not limited to, calcitonin, adrenergic, endothelin, c AMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation genc-l, rhodopsins, odorant, and cytomegalovirus receptors.

Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure The 7 transmembrane regions are designated as TMI, TM 2, TM 3, TM 4, TM 5, TM 6, and TM 7 TM 3 has been implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors Most G- protcin coupled receptors contain potential phosphorylation sites within the third cyroplasmic loop and/or the carboxy terminus For several G-protein coupled receptors, such as the Padrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.

For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protcin coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors The hydrophilic side of each G-protein coupled receptor transmembranc helix is postulated to face inward and form a polar ligand binding site TM 3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM 3 aspartate residue TM 5 serines, a Ti M 6 asparagine and TM 6 or TM 7 phenylalanines or tyrosines are also implicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al, Endoc Rev, 1959, 10:317-331) Different G-protein c-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors G-protein coupled receptors are found in numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane ( 7 TM) reccptors have been successfully introduced onto the market This indicates that these receptors have an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further receptors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV 1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder, mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome/disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tunor/adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; pituitary growth hormone; adenohypophysis hypofinction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e g, HIV, post-polio syndrome, and post-herpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodegenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism-amyotrophy complex; pallidoponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to HLWAR 77 polypepddes and recombinant materials and methods for their production Another aspect of the invention relates to methods for using such HLWAR 77 polypeptides and polynucleotides Such uses include the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-I or HIV-2 cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder, depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder, anxiety neurosis; dysthymic disorder; behavior disorder, mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome / disease; basophil adcnoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tumor / adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e g, HIV, post-polio syndrome, and post-herperic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodegencrative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism-amyotrophy complex; pallido-pontonigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others.

In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with HLWAR 77 or its ligands imbalance with the identified compounds In particular, the preferred method for identifying agonist or antagonist of a ELWAR 77 receptor of the present invention comprises:

contacting a cell expressing on the surface thereof the receptor, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound to be screened under conditions to permit binding to the receptor; and determining whether the compound binds to and activates or inhibits the receptor by measuring the level of a signal generated from the interaction of the compound with the receptor.

In a further preferred embodiment, the method further comprises conducting the identification of agonist or antagonist in the presence of labeled or unlabeled RF-amide (as defined below).

In another embodiment of the method for identifying agonist or antagonist ofa HLWAR 77 receptor of the present invention comprises:

determining the inhibition of binding of a ligand to cells which have the receptor on the surface thereof, or to cell membranes containing the recetpor, in the presence of a candidate compound under conditions to permit binding to the receptor, and determining the amount of ligand bound to the receptor, such that a compound capable of causing reduction of binding of a ligand is an agonist or antagonist Preferably the ligand is amidated bovine NPAF (SEQ ID N O; 7) or NPFF (SEQ ID NO:8).

Yet more preferably, the amidated bovine NPAF (SEQ ID NO:7) or NPFF (SEQ ID NO:) is labeled.

Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate HLWAR 77 activity or levels.

DESCRIPTION OF THE INVENTION

Definitions The following dcfinitions are provided to facilitate understanding of certain terms used frequently herein.

"HLWAR 77 " or "I-ILWAR 77 rcceptor" refers, among others, to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or an allelic variant thereof.

"Receptor Activity" or "Biological Activity of the Receptor" refers to the metabolic or physiologic function of said HLWAR 77 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and immunogenic activities of said HLWAR 77.

"HLWAR 77 gene" refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:1 or allelic variants thereof and/or their complements.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation single and double-stranded DNA, DNA that is a mixture of single and double- stranded regions, single and double-stranded RNA, and RNA that is mixture of single and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.

The termn polynucleotide also includes DN As or RN As containing one or more modified bases and DN As or RN As with backbones modified for stability or for other reasons "Modified" bases include, for example, tritylated bases and unusual bases such as inosine A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i e, pepride isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere in a polypcptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from posrrranslation natural processes or may be made by synthetic methods Modifications include acctylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GQI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylarion, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed, T E Creighton, W H Freeman and Company, New York, 1993 and Wold, F, Posttranslational Protein Modifications: Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed, Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol ( 1990) 182:626-646 and Rattan et Cl, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci ( 1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide.

Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code A variant of a polynuclcotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by dircct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynuclcotide sequences, as the case may be, as determined by the match between strings of such sequences.

"Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M, ed, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D W, ed, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A M, and Griffin, H.G, eds, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M and Devcrcux, J, eds, M Stockton Press, New York, 1991; and Carillo, H, and Lipman, D, SIAM J Applied Math, 48 1073 ( 1988) Preferred methods to determine identity are designed to give the largest match between the sequences tested Methods to determine identity and similarity are codified in publicly available computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are nor limited to, the GCG program package (Devereux, J, et al., Nucleic Acids Research 12 ( 1): 387 ( 1984)), BLASTP, BLASTN, and FASTA (Arschul, S F et al., J Molec Biol 215: 403-410 ( 1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S, et al, NCBI NLM NIE Bethesda, MD 20894; Altschul, S, et al, J Mol Biol 215:403410 ( 1990) The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following:

1) Algorithm: Necdlenan and Wunsch, J Mol Biol 48: 443453 ( 1970) Comparison matrix: BLOSSUM 62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA.

89:10915-10919 ( 1992) Gap Penalty: 12 Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following:

1) Algorithm: Needlcman and Wunsch, J Mol Biol 48: 443-453 ( 1970) Comparison matrix: matches = + 10, mismatch = O Gap Penalty: 50 Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 1, that is be 100 % identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3 ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequenceor in one or more contiguous groups within the reference sequence The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:1 by the numerical pcrcent of the respective percent identity(divided by 100) and subtracting that product from said total number of nuclcotides in SEQ ID NO: 1, or:

nn < xn (n Y), wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:1, and y is, for instance, 0 70 for 70 %, 0 80 for 80 %, O 85 for 85 %, 0 90 for 90 %, 0 95 for 95 %, etc, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100 % identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than % Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or morc contiguous groups within the reference sequence The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:

na<Xa (a y), wherein na is the number of amino acid alterations, Xa is the total number of amino acids in SEQ ID NO:2, and y is, for instance 0 70 for 70 %, 0 80 for 80 %, 0 85 for 85 % etc, and wherein any non- integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.

"RF-amide" refers to a group of ligands for the HLWAR 77 receptor which include but which is not limited to the following peptides:

Peptide Sequence Species (and common name) SEQID NO:

NPAF AGEGLSSPFWSLAAPQRF Bovine 7 amide Hu NPAF AGEGLNSQFWSLAAPQRF Human 9 amide NPFF FLFQPQRF-amide Bovine 8 Hu NPFF SQAFLFQPQRF-amide Human 10 LPLR Famide LPLRF-amide Chicken 11 FMR Famide FMRF-amide Macrocallista nimbosa (clam) 12 Antho-R Famide p EGRF-amide Anthopleura elegantissima (sea 13 anemone) Antho-R Namide L-beta-Phenyllactyl-LRN Anthopleura elegantissima (sea 14 amide anemone) Antho-R Wamide I p ESLRW-amide Anthopleura elegantissima (sea 15 anemone) Antho-R Wamide II p EGLRW-amide Anthopleura elegantissima (sea 16 anemone) Pol-R Famide Pellggrf-amide Polyorchis penicillatus 17 (hydromedusa) KSAYMR Famide KSAYMRF-amide Panagrellus redivivus (nematode)18 Peptide Fl TNRNFLRF-amide Homarus americanus (lobster) 19 DF 2 DRNFLRF-amide Procambarus clarkii (crayfish) 20 PQR Famide PQRF-amide Synthetic 21 Leu-Enkephalin YGGFLRF-amide Synthetic 22 R Famide Met-Enkephalin YGGFMRF-amide Synthetic 23 R Famide Both the NPAF (SEQ ID NO:7) (also referred to as "A-18-F-NH 2 ") and NPFF (SEQ ID NO:8) (also referred to as "F-8-F-NH 2 ") peptides are described in Yang HY, Fratta W, Majane EA, Costa E Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine Proc Nati Acad Sci U S A 1985 Nov; 52 ( 22):7757-61.

Polypeptides of the Invention In one aspect, the present invention relates to HLWAR 77 polypeptides (or HLWAR 77 proteins) The HLWAR 77 polypeptides include the polypeptides of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO:2; and polypeptides comprising the amino acid sequence which have at least 80 % identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90 % identity, and even still more preferably at least 95 % identity to SEQ ID NO: 2 Furthermore, those with at least 97-99 % are highly preferred Also included within HLWAR 77 polypeptides are polypeptideshaving the amino acid sequence which have at least 80 % identity to the polypeptide having the amino acid sequence of SEQ ID NO: 2 over its entire length, and still more preferably at least 90 % identity, and even still more preferably at least 95 % identity to SEQ ID NO: 2 Furthermore, those with at least 97-99 % are highly preferred Preferably HLWAR 77 polypeptides exhibit at least one biological activity of the receptor.

The ILWAR 77 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments of the HLWAR 77 polypeptides are also included in the invention A fiagment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned HLWAR 77 polypeptides As with ELWAR 77 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region Representative examples of polypeptide fagments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of HLWAR 77 polypeptide In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides having the ammo acid sequence of HLWAR 77 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-shect- forming regions, turn and tun-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic index regions Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity.

Also included are those that are antigenic or irnmunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity Among the most preferred fragment is that having the amino acid sequence of SEQ ID NO: 4 Variants of the defined sequence and fragments also form part of the present invention Preferred variants are those that vary from the referents by conservative amino acid substitutions i e, those that substitute a residue with another of like characteristics Typica such substitutions are among la, Val, Leu and le; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.

Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.

The HLWAR 77 polypeptides of the invention can be prepared in any suitable manner Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods.

Means for preparing such polypeptides are well understood in the art Polynucleotides of the Invention Another aspect of the invention relates to HLWAR 77 polynucleotides HLWAR 77 polynucleotides include isolated polynucleotides which encode the HLWAR 77 polypeptides and fragments, and polynucleotides closely related thereto More specifically, HLWAR 77 polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a HLWAR 77 polypeptide of SEQ ID NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS:1 and 3 HLWAR 77 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80 % identity over its entire length to a nucleotide sequence encoding the HLWAR 77 polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80 % identical to that of SEQ ID NO:1 over its entire length In this regard, polynucleotides at least 90 % identical are particularly preferred, and those with at least % are especially preferred Furthermore, those with at least 97 % are highly preferred and those with at least 98-99 % are most highly preferred, with at least 99 % being the most preferred Also included under HLWAR 77 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker The invention also provides polynucleotides which are complementary to such HLWAR 77 polynucleotides.

HLWAR 77 of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of sequencing the c DNA encoding human ELWAR 77 The c DNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 348 to 1610) encoding a polypeptide of 420 amino acids of SEQ ID NO:2 The amino acid sequence of Table 1 (SEQ ID NO:2) has about 33 % identity (using FASTA) in 300 amino acid residues with Neuropeptide Y receptor Li X, Wu Y, North R Forte M 1992 J Biol Chem L,267, 9-12 The nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 50 % identity (using FASTA) in 730 nucleotide residues with Neuropeptide Y receptor Li X, Wu Y, North R, Forthc M 1992 J Biol Chem 267,9-12 Thus, FLWAR 77 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art Table 13

1 CCATCCTAAT ACGACTCACT ATAGGGCTCG AGCGGCCGCC CGGGCAGGTT 51 CTCGGCTCAC TGCAAGCTCC ACCTCTGGG TTCACGCTAT TCTCCTGCCT 101 CAGCCTCCTG AGTAGCTGGG ACTACAGGCG CCCGCCACCA CGCCTGGCTA 151 ATTTT Tt GT ATTTTTAG Ta GGGACGGCGT TTCACTGTGT TAGCCCAGAT 201 GG Tc TCCGTC TC Cc GACC Tc Gt GATCCACC CA Cc TCGGCT TCCCAAAGTG 251 CTGAGATTAC AGGCGTGAGC CACCGCGCCC GGCCAATTTC CTTTCTTAAT 301 TGCC Tct GCC CACCCTCTTCT c TTCTGCTTC CATATTACAG GTTCATCATG 351 AATGAGAAAT GGGACACAAA CTCTTCAGAA AACTGGCATC CCATCTGGAA 401 TGTCAATGAC ACAAAGCATC ATCTGTACTC AGATATTAAT ATTACCTATG 451 TGAACTACTA Tc TTCACCAG CCTCAAGTGG CAGCAATCTT CATTATTTCC 501 TACTTTCTGA TCTTCTTTTT GTGCATGATG GGAAATACTG TGGTTTGCTT 551 TATTGTAATG AGGAACAAAC ATATGCACAC AGTCACTAAT CTCTTCATCT 601 TAAACCTGGCCATAAGTGAT TTACTAGTTG GCATATTCTG CATGCCTATA 651 ACACTGCTGG ACAATATTAT AGCAGGATGG CCATTTGGAA ACACGATGTG 701 CAAGATCAGT GGATTGGTCC AGGGAATATC TGTCGCAGCT TCAGTCTTTA 751 CGTTAGTTGC AATTGCTGTA GATAGGTTCC AGTGTGTGGT CTACCCTTT 801 AAACCAAAGC TCACTATCAA GACAGCGTTT GTCATTATTA T=ATCATCTG 851 GGTCCTAGCC ATCACCATTA TGTCTCCATC TGC-GTAATG TTACATGTGC 901 AAGAAG-AAA ATATTACCGA GTGAGACTCA ACTCCCAGAA TAAAACCAGT 951 CCAGTCTACT GGTGCCGGGA AGACTGGCCA AATCAGGAAA TSAGGAAGAT 1001 CTACACCACT GTGCTGTTTG CCAACATCTA CCTGGCTCCC CTCTCCCTCA 1051 TTGTCATCAT GTATGGAAGG ATTGGAATTT CACTCTTCAG GGCTGCAGTT 1101 CCTCACACAG GCAGGAAGAA CCAGGAGCAG TGGCACGTGG TGTCCAGGPA 1151 GAAGCAGAAG ATCATTAAGA TGCTCCTGAT TGTGGCCCTG CTTTTATTC 1201 TCTCATGGCT GCCCCTGTGG ACTCTAATGA TGCTCTCAGA CTACGCTGAC 1251 CTTTCTCCAA ATGAACTGCA GATCATCAAC ATCTACATCT ACCCTTTTGC 1301 ACACTGGCTG GCATTCGGCA ACAGCAGTGT CAATCCCATC ATTTATGGTT 1351 TCTTCAACGA GAATTTCCGC CGTGGTTTCC AAGAAGCTTT CCAGCTCCAG 1401 CTCTGCCAAA AAAGAGCAAA GCCTATGGAA GCTTATGCCC TAAAAGCTAA 1451 AAGCCATGTG CTCATAAACA CATCTAATCA GCTTGTCCAG GAATCTACAT 1501 TTCAAAACCC TCATGGGGAA ACCTTGCTTT ATAGGAAAAG TGCTGAAAAA 1551 CCCCAACAGG AATTAGTGAT GGAAGAATTA AAAGAAACTA CTAACAGCAG 1601 TGAGATTTAA AAAGAGCTAG TGTGATAATC CTAACTCTAC TACGCATTAT 1651 ATATTTAAAT CCATTGCTTT TTGTGGCTTT GCACTTCAAA TTTTTCAAAG 1701 AATGTTCTAA ATAAAACATT TACTGAAAGC CCTCTCTGGC AAAAAAATTA 1751 AAAATAAACA AAAATGGTCA TAAGATCATA AACAATCTTA TGTTGTATAA 1801 AAATACGTAG AGTGACTTAG ACATGTTTGC ATGAATAAAT ATATTTCTAG 1 AGAACAGTTA AAAAAAAAAA AAAAA)AAA A AAAAAAAA A nucleotide sequence of a human ILWAR 77 (SEQ ID NO: 1).

Table 2 b

1 MNEKWDTNSS ENWEPIWNVIN DTIHLYSDI NITYVNYYLH QPQVAAIFII 51 SYFLIFFLCM MGNTWCFIV MRNKHMITVT NLFILNLAIS DLLV Gr FCMP 101 ITLLDNIIAG WPPGNTMCKI SGLVQGISVA ASVFTLVAIA V Dt PQCVVYP 151 FKPKLTIKTA FVIIMITWVL AITIMSPSAV MLE VQEEKYY RVILNSQNKT 201 SPVYWCREDW PNQEMRKIYT TVLFANIYLA PLSLIVIMYG RGISLFRAA.

251 VPITGRKIWQE QWHWVSRSMQ K II Mn 4 LILIVA LLFILSWLPL WT l MLSDYA 301 DLSPNELQII NI Yl YPFPAIW LAFGNSSVNP Il YGFFNENF RRGFQEAFQL 351 QLCQKRAKPM EAYALKAUSH VLINTSNQLV QESTFQNPRG ETLL YRKSAE 401 KPQQELVMEE LKETTNSSEI b An amino acid sequence of a human HLWAR 77 (SEQ ID NO: 2).

One polynucleotide of the present invention encoding HLWAR 77 may be obtained using standard cloning and screening, from a c DNA library derived from i RNA in cells of Human placenta using the expressed sequence tag (EST) analysis (Adams, M D, et al Science ( 1991) 252:1651-1656; Adams, M D et al, Nature, ( 1992) 355:632-634; Adams, M D, eta, Nature ( 1995) 377 Supp:3- 174) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries Or can be synthesized using well known and commercially available techniqucs.

The nucleotide sequence encoding HLWAR 77 polypcptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 348 to 1610 of SEQ ID NO:1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the generic code, also encodes the polypeptidc of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinant production of HLWAR 77 polypeptide, the polynucleotide may include the coding sequence for the mature polypepride or a fragment thereof, by itself; the coding sequence for the mature polypeptide Or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro or prepro protein sequence, or other fusion peptide portions For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the p QE vector (Qiagen, Inc) and described in Gentz et al, Proc Natl Acad Sci U 54 ( 1989) 86:821-824, or is an HA tag The polynucleotide may also contain non- coding 5 ' and 3 ' sequences, such as transcribed, non-translatcd sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize m RNA.

Further preferred embodiments are polynucleorides encoding ELWAR 77 variants comprising the amino acid sequence of HLWAR 77 polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination Among the preferred polynucleorides of the present invention is contained in Table 3 (SEQ ID NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).

Table 3 '

1 AGTGAACCGT CAGATCGCCT GGAGACGCCA TCCACGCTGT TTTGACCTC 51 ATAGAAGACA CCGGGACCGA TCCAGCCTCC GGACTCTAGC CTAGGCCGC 101 GGACGGATAA CAATTTCACA CAGGAAACAG CTATGACCAC TAGGCTTTT 151 CAAAAGCTA TTTAGGTGAC ACTATAGAAG GTACGCCTGC AGGTACCGG 201 CCGGAATTCC CGGGTCGACC CACGCGTCCG CATAAGTGAT TTACTAGTT 251 GCATATTCTG CATGCCTATA ACACTGCTC-G ACAATATTAT AGCAGCATG 301 CCATTTGGAA ACACGATGTG CAAGATCAGT GGATTGGTCC AGGGAATAT 351 TGTCGCAGCT TCAGTCTTTA CGTTAGTTGC AATTGCTGTA GATAGGTTC 401 AGTGTGTGGT CTACCCTTTT AAACCAAAGC TCACTATCAA GACAGCGTT 451 GTCATTATTA TGATCATCTG GGTCCTAGCC ATCACCATTA TGTCTCCAT 501 TGCAGTAATG TTACATGTGC AAGAAC-AAA ATATTACCGA GTGAGACTC 551 ACTCCCAGAA TAAAACCAGT CCAGTCTACT GGTGCCGGGA AGACTGGCC 601 AATCAGGAAA TGAGGAAGAT CTACACCACT GTGCTGTTTG CCAACATCT 651 CCTGGCTCCC CTCTCCCTCA TTGTCATCAT GTATGGAAGG ATTGGAATT 701 CACTCTTCAG GGCTGCAGTT CCTCAC-CAG GCAGGAAGAA CCAGGAGCA 751 TGGCACGTGG TGTCCAGGAA GAAGCAGAAG ATCATTAAGA TGCTCCTGA 801 TGTGGCCCTG CTTTTTATTC TCTCATGGCT GCCCCTGG ACTCTAATG 851 TGCTCTCAGA CTACGCTGAC CTTTCTCCAA ATGAACTGCA GATCATCAA 901 ATCTACATCT ACCCTTTTGC ACACTGGCTG GCATTCGGCA ACAGCAGTG 951 CAATCCCATC ATTTATGGTT TCTTCAACGA GAATTTCCGC CGTGGTTTC 1001 AAGAAGCTTT CCAGCTCCAG CTCTGCCAAA AAAGAGCAAA GCCTATGGA 1051 GCTTATGCCC TAAAAGCTAA AAGCCATGTG CTCATAAACA CATCTAATC 1101 GCTTGTCCAG GAATCTACAT TTCAAAACCC TCATGGGGAA ACCTTGCTT 1151 ATAGGAAAAG TGCTGAAAAA CCCCAACAGG AATTAGTGAT GGAAGAATT 1201 AAAGAAACTA CTAACAGCAG TGAGATTTAA AAAGAGCTAG TGTGATAAT 1251 CTAACTCTAC TACGCATTAT ATATTTAAAT CCATTGCTTT TTGTGGCTT 1301 GCACTTCAAA TTTTTCAAAG AATGTTCTAA ATAAAACATT TACTGAAAG 1351 CCTCTCTGGC AAAAAAATTA AAAATAAACA AAAATGGTCA TAAGATCAT 1401 AACAATCTTA TGTTGTATAA AAATACGTAG AGTGACTTAG ACATGTTTG 1451 ATGAATAAAT ATATTTCTAG AGAACAGTTA AAAAPAAAA AAAAAAAA 1501 AAAAAA A c A partial nucleotide sequence of a hunman HLWAR 77 (SEQ ID NO: 3).

Table 4 d

1 PRVRISDLLV GIFCMPITLL DNIIAGWPFG NTMCKISGLV QGISVAASVF 51 TLVAIAVDRF QCVVYPFKPK LTIKTAFVII MIIWVLAITI MSPSAVMXKV 101 QEEKYYRVRL NSQNKTSPVY WCREDWP 5 QE MRKIYTTVLF ANIYLAPLSL 151 IVIMYGRIGI SLFRAAVPHT GRKNQEQWHV VSRKKQKIIK MLLIVALLFI 201 LSWLPLWTLM MLSDYADLSP NELQIINIYI Y Fr AHWLAFG NSSVNPIIYG 251 FFNENPRRGF QEAFQLQLCQ KRAKPMEAYA LKAKSHVLIN TSNQLVQEST 301 FQNPHGETLL Y RSAEKPQQ ELVMEELKET TNSSEI d A partial amino acid sequence of a human HLWAR 77 (SEQ ID NO: 4).

The present invention further relates to polynucleotides that hybridize to the herein above- described sequences In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 80 %, and preferably at least 90 %, and more preferably at least 95 %, yet even more preferably 97-99 % identity between the sequences.

Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for c DNA and genomic DNA, to isolate full-length c DN As and genomic clones encoding RLWAR 77 and to isolate c DNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the HLWAR 77 gene Such hybridization techniques are known to those of sklll in the art Typically these nucleotide sequences are % identical, preferably 90 % identical, more preferably 95 % identical to that of the referent The probes generally will comprise at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotidcs Particularly preferred probes will range between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding HLWAR 77 polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof (including that of SEQ ID NO: 3), and isolating full- length c DNA and genomic clones containing said polynucleotide sequence Such hybridization techniques are well known to those of skill in the art Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 C in a solution comprising: 50 % formamide, 5 x SSC ( 150 m M Nia C 1, 15 rm M trisodium citrate), 50 m M sodium phosphate (p H 7 6), 5 x Denhardt's solution, 10 % dexrran sulfate, and microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O lx SSC at about 650 C.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.

Vectors, Host Cells, Expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques Cell- free translation systems can also be employed to produce such proteins using RN As derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, BASICMETHODSINMOLECULAR BIOLOGY( 1986) and Sambrook et al., MOLECULAR CLONING: A L 4 BORATORYMANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y ( 1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, nucroinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introducdon or infection.

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila $ 2 and Spodoptera Sf 9 cells; animal cells such as CHO, COS, He La, C 127, 3 T 3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used Such systems include, among others, chromosomal, episomal and virus-derived systems, e g, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV 40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elcmcnts, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the HLWAR 77 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptidc be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay If HLWAR 77 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered- ILWAR 77 polypeptides can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.

Most preferably, high performance liquid chromatography is employed for purification Well known techniques for refolding proteins may be employed to regenerate active conormation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays This invention also relates to the use of HLWAR 77 polynucleotides for use as diagnostic reagents Detection of a mutated form of HLWAR 77 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of HLWAR 77 Individuals carrying mutations in the HLWAR 77 gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as fror blood, urine, saliva, tissue biopsy or autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other-amplification techniques prior to analysis RNA or c DNA may also be used in similar fashion Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype Point mutations can be identified by hybridizing amplified DNA to labeled ELWAR 77 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by R Nase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing.

See, e g, Myers et at, Science ( 1985) 230:1242 Sequence changes at specific locations may also be revealed by nuclease protection assays, such as R Nase and 51 protection or the chemical cleavage method See Cotton et al, Proc Natl Acad Sci USA ( 1985) 85:4397-4401 In another embodiment, an array of oligonucleotides probes comprising ILWAR 77 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g, genetic mutations Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.

(See for example: M Chee et al, Science, Vol 274, pp 610-613 ( 1996)).

The diagnostic assays offer a process for diagnosing or determining a susceptibility to infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease: impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder, depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder; mood disorder, shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder, disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tumor / adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic- adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency: dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e g, HTV, post-polio syndrome, and post-herpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain:

post-operative pain; neuralgia: and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodegenerative disorders, which includes nosological entities such as disinhibition-dementia- ._ -20 parkinsonism-amyotrophy complex; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, through detection of mutation in the HLWAR 77 gene by the methods described-I addition, bacterial, fungal, protozoan and viral infections, particularly infections caused by HI-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder; mood disorder; shizophrcnia; psychosexual dysfunction; sex disorder, sexual disorder, disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cu hing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolacftnemia; hypopituitarism; hypophysis tumor / adenona; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadisnm Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism.

hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e.g, HIV, post-polio syndrome, and post-herpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other ncurodegenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism-amyorophy complex; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of HLWAR 77 polypeptide or HLWAR 77 m RNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitationof polynucleotides, such as, for example, PCR, RT-PCR, R Nasc protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protein, such as an HLWAR 77, in a sample derived from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Westnm Blot analysis and EUSA assays.

Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, partictlarly infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder, depressive neurosis/disorder; anxiety neurosis; dysthyrnic disorder; behavior disorder; mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachcxia, and obesity; Cushing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tumor / adcnoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadisn; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idioparhic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e.g, HIV, post-polio syndrome, and post-herpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodegencrative disorders, which includes nosological entities such as disinhlbition-dementia-parkinsonism-amyotrophy complex; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrom, which comprises:

(a) a HLWAR 77 polynucleotidc, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof; - 22 (b) a nucleotide sequence complementary to that of (a); (c) a HLWAR 77 polypeptide, preferably the polypeptide of SEQ ID NO: 2, or a fragment thereof; or (d) an antibody to a HLWAR 77 polypeptide, preferably to the polypeptide of SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Chromosome Assays The nucleotide sequences of the present invention are also valuable for chromosome identification The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, in V McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

The differences in the c DNA or genomic sequence between affected anglunaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease- Antibodies The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the HLWAR 77 polypeptides The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against the HLWAR 77 polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, prefcrably a nonhuman, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybridoma technique (Kohler, G and Milsrein, C, Nature ( 1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor er al, Immunology Today ( 1983) 4:72) and the EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc, 1985).

Techniques for the production of single chain antibodies (U S Patent No 4, 946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention Also, ransgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.

The above-descrbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against ILWAR 77 polypeptides may also be employed to treat infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder, depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder, mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tumor / adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idioparhic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e g, HIV, post-polio syndrome, and postherpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodegenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism- amyotrophy complex; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others.

Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with ELWAR 77 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by IV-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease: impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder, depressive neurosis/disorder, anxiety neurosis; dysthymic disorder; behavior disorder; mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituitarism; hypophysis tumor / adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hyperprolactinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; ncuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e g, HTV, post-polio syndrome, and postherpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; ncuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other neurodcgenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism- amyotrophy complcx; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others Yet another aspect of the invention relates to a method of inducing immunological response in a matnral which comprises, delivering HLWAR 77 polypeptide via a vector directing expression of HLWAR 77 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a -LWAR 77 polypepride wherein the composition comprises a HLWAR 77 polypeptide or HLWAR 77 gene The vaccine formulation may further comprise a suitable carrier.

Since HLWAR 77 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection).

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oddants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art.

The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays The HLWAR 77 polypeptide of the present invention may be employed in a process for screening for compounds which bind to and activate the HLWAR 77 polypeptides of the present invention (called agonists), or inhibit the interaction of the HLWAR 77 polypeptides with receptor ligands (called antagonists).

Thus, polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See Coligan et al, Current Protocols in Immunology 1 ( 2):Chapter 5 ( 1991).

HLWAR 77 proteins are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate HLWAR 77 on the one hand and which can inhibit the function of HLWAR 77 on the other hand In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pecroris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder; mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome / disease; basophil adenoma; prolactinoma; hyperprolactinemia; hypopituirarism; hypophysis tumor / adenoma; hypothalamic diseases; Froehlich's syndrome; adenohypophysis disease; hypophysis disease; hypophysis tumor / adenoma; pituitary growth hormone; adenohypophysis hypofunction; adrenohpophysis hyperfunction; hypothalamic hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or psychogenic amenorrhea; hypopituitarism; hypothalamic hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic hypcrprolacinemia; hypothalamic disorders of growth hormone deficiency; idiopathic growth hormone deficiency; dwarfism; gigantism; acromegaly; disturbed biological and circadian rhythms; and sleep disturbances associated with such diseases as neurological disorders, heart and lung diseases, mental illness, and addictions; migraine; hyperalgesia; enhanced or exaggerated sensitivity to pain, such as hyperalgesia, causalgia and allodynia; acute pain; burn pain; atypical facial pain; neuropathic pain; back pain; complex regional pain syndromes I and II; arthritic pain; sports injury pain; pain related to infection, e.g, HIV, post-polio syndrome, and post-herpetic neuralgia; phantom limb pain; labour pain; cancer pain; post-chemotherapy pain; post-stroke pain; post-operative pain; neuralgia; and tolerance to narcotics or withdrawal from narcotics; sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and other ncurodegenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism-amyotrophy complex; pallido-ponto-nigral degeneration; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome.

In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof Such cells include cells from mammals, yeast, Drosophila or E coli In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the HLWAR 77 polypeptide The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which are transfected to express the HLWAR 77 polypeptide of the present invention Such a screening technique is described in PCT WO 92/01810, published February 6, 1992 Such an assay may be employed to screen for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand, such as A- 18-F-NH 2 (SEQ ID NO:7) or F-8-F-NH 2 (SEQ ID NO:8), and a compound to be screened.

Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i e, inhibits activation of the receptor.

The technique may also be employed for screening of compounds which activate the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i e, activates the receptor.

Other screening techniques include the use of cells which express the HLWAR 77 polypeptide (for example, transfected CHO cells) in a system which measures extracellular p H changes caused by receptor activation In this technique, compounds may be contacted with cells expressing the receptor polypeptide of the present invention A second messenger response, e g, signal transduction or p H changes, is then measured to determine whether the potential compound activates or inhibits the receptor.

Another screening technique involves expressing the ELWAR 77 polypeptide in which the receptor is linked to phospholipase C or D Representative examples of such cells include, but are not limited to, endothelial cells, smooth muscle cells, and embryonic kidney cells The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.

Another method involves screening for compounds which are antagonists, and thus inhibit activation of the receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand, an XF-amide, such as A-18-F-NH 2 (SEQ ID NO:7) or F-S-F- NI 2 (SEQ ID NO:8), to cells which have the receptor on the surface thereof, or cell membranes containing the receptor Such a method involves transfecting a eukaryotic cell with DNA encoding the HLWAR 77 polypeptidc such that the cell expresses the receptor on its surface The cell is then contacted with a potential antagonist in the presence of a labeled form of a ligand, an RF-amide, such as A-l 8-F-NH,2 (SEQ ID NO:7) or F-S-F-NII 2 (SEQ ID NO:8) The ligand can be labeled, e g, by radioactivity The amount of labeled ligand bound to the receptors is measured, e g, by measuring radioactivity associated with transfected cells or membrane from these cells If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled ligand which binds to the receptors This method is called binding assay Naturally this same technique can be used to look for an agonist.

Another screening procedure involves the use of mammalian cells (CHO, HEK 293, Xenopus Oocytes, RBL-2 H 3, etc) which are transfected to express the receptor of interest The cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, an RF-amide, such as A S-F-N Hi (SEQ ID NO:7) or F-S-F-NH 2 (SEQ ID NO:8) Any change in fluorescent signal is measured over a defined period of time using, for example, a fluorescence spectrophotometer or a fluorescence imaging plate reader A change in the fluorescence signal pattern generated by the ligand indicates that a compound is a potential antagonist or agonist for the receptor.

Another screening procedure involves use of mammalian cells (CHO, HEK 293, Xenopus Oocytes, RBL-2 H 3, etc) which are transfected to express the receptor of interest, and which are also transfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase or beta-galactosidase behind an appropriate promoter) The cells are contacted with a test substance and the receptor agonist (ligand), an RF-amide, such as A-18-F- NH 2 (SEQ ID NO:7) or F- 8-F-NH 2 (SEQ ID NO:8), and the signal produced by the reporter gene is measured after a defined period of time The signal can be measured using a luminometer, spectrophotometer, fluorimeter, or other such instrument appropriate for the specific reporter construct used Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor.

Another screening technique for antagonists or agonits involves introducing RNA encoding the EILWAR 77 polypeptide into Xenopus oocytes (or CHO, HEK 293, RBL-2 H 3, etc) to transiently or stably express the receptor The receptor oocytes are then contacted with the receptor ligand, an RF-amide, such as A-18-F-NHI 2 (SEQ ID NO:7) or F-8-F-NH 2 (SEQ ID NO:8), and a compound to be screened Inhibition or activation of the receptor is then determined by detection of a signal, such as, c AMP, calcium, proton, or other ions.

Another method involves screening for ILWAR 77 polypeptide inhibitors by determining inhibition or stimulation of HLWAR 77 polypeptide-mediated c AMP and/or adenylate cyclase accumulation or dimunition Such a method involves transiently or stably transfecting a eukaryotic cell with HLWAR 77 polypeptide receptor to express the receptor on the cell surface The cell is then exposed to potential antagonists in the presence of HLWAR 77 polypeptide ligand, an RF-amide, such as A-1 8-F-NH 2 or F-8-F-NI 2 The changes in levels of c AMP are then measured over a defined period of time, for example, by radio-immuno or protein binding assays (for example using Flashplates or a scintillation proximity assay) Changes in c AMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase, in broken cell preparations If the potential antagonist binds the receptor, and thus inhibits HLWAR 77 polypeptide-ligand binding, the levels of HLWAR 77 polypeptide-mediated c AMP, or adenylare cyclase activity, will be reduced or increased.

Another screening method for agonists and antagonists relies on the endogenous pheromone response pathway in the yeast, Saccharomyces cerevisiae Heterothallic strains of yeast can exist in two mitotically stable haploid mating types, MA Ta and MA Ta Each cell type secretes a small peptide hormone that binds to a G-protein coupled receptor on opposite mating-type cells which triggers a MAP kinase cascade leading to G 1 arrest as a prelude to cell fusion Genetic alteration of certain genes in the pheromone response pathway can alter the normal response to pheromone, and hcterologous expression and coupling of human G-protein coupled receptors and humanized G- protein subunits in yeast cells devoid of endogenous pheromone receptors can be linked to downstream signaling pathways and reporter genes (e g, U S Patents 5,063, 154; 5,482,835; 5,691,188) Such genetic alterations include, but are not limited to, (i) deletion of the STE 2 or STE 3 gene encoding the endogenous G-protein coupled pheromone receptors; (ii) deletion of the FAR 1 gene encoding a protein that normally associates with cyclin-dependent kinases leading to cell cycle arrest; and (iii) construction of reporter genes fused to the FUS 1 gene promoter (where FUS 1 encodes a membrane-anchored glycoprotein required for cell fusion) Downstream reporter genes can permit either a positive growth selection (e g, histidine prototrophy using the FU 51-HI 53 reporter), or a colorimetric, fluorimetric or spectrophotometric readout, depending on the specific reporter construct used (e g, b-galactosidase induction using a FUS l-Lac Z reporter).

The yeast cells can be further engineered to express and secrete small peptides from random peptide libraries, some of which can permit autocrine activation of heterologously expressed human (or mammalian) G-protein coupled receptors (Broach, J R and Thorner, J Nature 384: 14-16, 1996; Manfredi et al, Mol Cell Biol 16: 4700-4709, 1996) This provides a rapid direct growth selection (e.g, using the FU 51-HI 53 reporter) for surrogate peptide agonists that activate characterized or orphan receptors Alternatively, yeast cells that functionally express human (or mammalian) G- protein coupled receptors linked to a reporter gene readout (e g, FUS Lac Z) can be used as a platform for high-throughput screening of known ligands, fractions of biological extracts and libraries of chemical compounds for either natural or surrogate ligands Functional agonists of sufficient potency (whether natural or surrogate) can be used as screening tools in yeast cell-based assays for identifying G-protein coupled receptor antagonists For example, agonists will promote growth of a cell with FUS-HI 53 reporter or give positive readout for a cell with FUSI-Lac Z.

However, a candidate compound which inhibits growth or negates the positive readout induced by an agonist is an antagonist For this purpose, the yeast system offers advantages over mammalian expression systems due to its ease of utility and null receptor background (lack of endogenous Gprotein coupled receptors) which often interferes with the ability to identify agonists or antagonists.

The present invention also provides a method for identifying new ligands not known to be capable of binding to an HLWAR 77 polypeptides The screening assays described above for identifying agonists may be used to identify new ligands.

The present invention also contemplates agonists and antagonists obtainable from the above described screening methods.

Examples of potential HLWAR 77 polypeptide receptor antagonists include peptidomimetics, synthetic organic molecules, natural products, antibodies, etc which bind to the receptor but do not elicit a second messenger response such that the activity of the receptor is prevented.

Potential antagonists also include proteins which are closely related to the ligand of the HLWAR 77 polypeptide receptor, i e a fragment of the ligand, which have lost biological function, and when they bind to the LWAR 77 polypeptide receptor, elicit no response.

Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, and ligands for HLWAR 77 polypeptides, which comprises:

(a) a HLWAR 77 polypeptide, preferably that of SEQ ID NO:2; and further preferably comprises labeled or unlabeled RF-amide; (b) a recombinant cell expressing a HLWAR 77 polypeptide, preferably that of SEQ ID NO:2; andfurther preferably comprises labeled or unlabeled RF-amide; or (c) a cell membrane expressing HLWAR 77 polypeptide; preferably that of SEQ ID NO: 2; and further preferably comprises labeled or unlabled RF-amide It will be appreciated that in any such kit, (a), (b), or (c) may comprise a substantial component.

As noted above, a potential antagonist is a small molecule which binds to the HLWAR 77 polypeptide receptor, making it inaccessible to ligands such that normal biological activity is prevented Examples of small molecules include, but are not limited to, small pepides or peptide- like molecules.

Potential antagonists also include soluble forms of HLWAR 77 polypeptide receptor, e g, fragments of the receptor, which bind to the ligand and prevent the ligand from interacting with membrane bound HLWAR 77 polypeptide receptors.

Prophylactic and Therapeutic Methods This invention provides methods of treating an abnormal conditions related to both an excess of and insufficient amounts of HLWAR 77 activity.

If the activity of HL-WAR 77 is in excess, several approaches are available One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the IHLWAR 77, or by inhibiting a second signal, and thereby alleviating the abnormal condition.

In another approach, soluble forms of ILWAR 77 polypeptides still capable of binding the ligand in competition with endogenous HLWAR 77 may be administered Typical embodiments of such competitors comprise fragments of the HLWAR 77 polypeptide.

In still another approach, expression of the gene encoding endogenous HLWAR 77 can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem ( 1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988) Alternatively, oligonucleotides which form triple helices with the gene can be supplied See, for example, Lee et al, Nucleic Acids Res ( 1979) 6:3073; Cooney er al Science ( 1988) 241:456; Dervan et al, Science ( 1991) 251:1360 These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of HLWAR 77 and its activity, several approaches are also available One approach comprises administering to a subject a therapeutically effective amount of a compound which activates HILWAR 77, i e, an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of HLWAR 77 by the relevant cells in the subject For example, a polynucleotidc of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T

Strachan and A P Read, BIOS Scientific Publishers Ltd ( 1996).

Formulation and Administration Peptides, such as the soluble form of IHLWAR 77 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.

Such formulations comprise a therapeutically effective amount of the polypepride or compound, and a pharmaceutically acceptable carrier or excipient Such cariers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used Alternative means for systemic administration include transmucosal and transdernal administration using penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.

The dosage range required depends on the choice ofpeptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1-100 pg/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypcptidc ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject.

Example 1: Mammalian Cell Expression The receptors of the present invention are expressed in either human embryonic kidney 293 (HEK 293) cells or adherent dhfr CHO cells To maximize receptor expression, typically all 5 ' and 3 ' untranslated regions (UT Rs) are removed from the receptor c DNA prior to insertion into a p CDN or p CDNA 3 vector The cells are transfected with individual receptor c DN As by lipofectin and selected in the presence of 400 mg/ml G 418 After 3 weeks of selection, individual clones are picked and expanded for further analysis HEK 293 or CHO cells transfected with the vector alone serve as negative controls To isolate cell lines stably expressing the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis Receptor m RN As are generally detectable in about 50 % of the G 418-resistant clones analyzed.

Example 2 Ligand bank for binding and functional assays.

A bank of over 200 putative receptor ligands has been assembled for screening The bank comprises: transmitters, hormones and chemokines known to act via a human seven transmembrare ( 7 TM) receptor; naturally occurring compounds which may be putative agonists for a human 7 TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found in nature, but which activate 7 TM receptors with unknown natural ligands This bank is used to initially screen the receptor for known ligands, using both functional (i e calcium, c AMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.

Example 3: Ligand Binding Assays Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format The purified ligand for a receptor is radiolabeled to high specific activity ( 50-2000 Ci/mmol) for binding studies A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its receptor Assay conditions for buffers, ions, p H and other modulators, such as nucleotides, arc optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources For these assays, specific receptor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of mulabeled competing ligand Where possible, more than one competing ligand is used to define residual nonspecific binding.

Example 4: Functional Assay in Xenopus Oocytes Capped RNA transcripts from linearized plasmid templates encoding the receptor c DN As of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures In vitro transcripts are suspended in water at a final concentration of 0 2 mg/ml Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts ( 10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure.

Recordings are made in Ca"+ free Barth's medium at room temperature The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.

Example 5: Microphysiometric Assays Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process The p H changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd, Menlo Park, CA) The CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G-protein coupled receptor of the present invention.

Example 6: Extract/Cell Supernatant Screening A large number of mammalian receptors exist for which there remains, as yet, no cornate activating ligand (agonist) Thus, active lgands for these receptors may not be included within the ligands banks as identified to date Accordingly, the 7 TM receptor of the invention is also functionally screened (using calcium, c AMP, microphysiometer, oocyte electrophysiology, etc, functional screens) against tissue extracts to identify natural ligands Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.

Example 7: Calcium and c AMP Functional Assays 7 TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or c AMIP stimulation or inhibition.

Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were obscrved to be in the normal, 100 n M to 200 n M, range HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day > 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of c AMP production using standard c AMP quantitation assays Agonists presenting a calcium transient or c AMP flucuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.

Example 8: Cloning of full length sequence An EST homologous to the 7 TM superfamily sequences was identified Oligonucleotides from these regions were designed to obtain the 5 ' coding region using the Marathon PCR technology The 5 ' oligos were (AAAGACTGAAGCTGCGACAGATATTC (SEQ ID NO:5) AND CCTGGACCAATCCACTGATCTTGC (SEQ ID NO:6)) Fragments of approximately 1200 bp ( 5 ' fragment) were obtained and subcloned into PCR 2 1 vector (Invitrogene) and sequenced Sequence analysis showed an open reading frame starting with nucleotide 348 (ATG) and ending at nucleotide 1610 (TGA) The coding region is 1263 nucleotdes which encodes a protein of 421 amino acids The protein is predicted to have a molecular weight of approximately 46,310.

Example 9:

It has been discovered that the RF-amides (the polypeptides of SEQ ID NOS: 7-23) are ligands for the HLWAR 77 receptor by the following experiment HEK-293 cells were co-ransfectcd with a mammalian expression plasmid encoding HLWAR 77, along with c DN As encoding Gal 5 (Wilkie T M et al Proc Natl Acad Sci USA 1991 88:10049-10053) and a chimeric G-protein referred to as Gqi 5 (Conklin BR ct al, Nature 1993 363:274-276), and assayed on FLIPR for a calcium mobilization response following addition of as A-18-F-NH 2 (SEQ ID NO:7) or F-8-F-l NHZ (SEQ ID NO:8) A robust, dose dependent (EC 50 S 3-5 n M), calcium mobilization response was detected following addition of either of these ligands to cells transfected with HLWAR 77 and the G-proteins.

The agonist peptides did not stimulate a calcium mobilization response in HEK-293 cells transfected only with HLWAR 77, nor was a response detected to these ligands in HEK- 293 cells transfected only with Gal 5 and Gqi 5 The c DN As for both the receptor and the G- proteins had to be expressed in the HEK-293 in order to detect a functional response to these agonists.

Subsequently it has been determined that HEK-293 cells co-transfected with HLWAR 77, and a different chimeric G-protein, Gqo 5 (Conklin BR et al Nature 1993 363:274-276), give a greater calcium mobilization response than cells co-transfected with HLWAR 77 and either Gal 5, Gqi 5, or a mixture of the 2 c DN As Additional G-protein must be present HEK-293 cells in order to detect calcium signaling mediated through HLWAR 77 Thus, in the case of using HEK-293, as described above, additional G-protein(s) is (are) required to run screens for agonists and antagonists It is possible that HLWAR 77 expressed in another cell, for example RBL-2 H 3, may signal through calcium pathways without requiring additional G-protein, as has been noted for the C 5 a receptor (Martino, l A et al J Biol Chem 1994 269:14446-14450), which in some cells also requires additional G-protein for functional coupling (Amatuda T T et al J Biol Chem 1993 268:10139- 10144).

It has also been found that human NPFF (SEQ ID NO:10) (which is 3 residues longer at the N-terminus than bovine NPFF (SEQ ID NO:S)) has equivalent activity to bovine NPFF (SEQ ID NO:S) at HLWAR 77 Human NPFF (SEQ ID NO:10) is 3 residues longer than bovine NPFF (SEQ ID NO:8) at the N-terminus (Human sequence: SQAFLFQPQRF-amide (SEQ ID NO: 10); Bovine sequence: FLFQPQRF-amide (SEQ ID NO:8)) Moreover, a previous publication (see FEBS Letts 1997 vol 409 p 426-30) suggested that the longer human form was inactive in (rat) tissue functional assays Human and bovine peptides are cquiactive at (human) ELWAR 77 in FLIPR They are also equiactive with human and bovine NPAF.

Numerous peptide ligands functioned as agonists generating robust calcium mobilization responses in HLWAR 77/Gal 6 expressing cells The common feature of the diverse peptide ligands which functioned as ELWAR 77/Ga 16 agonists was the occurrence at the carboxyl-terminus of the sequence, Arg-Phe-amide (shown above) The response to the RF-amide peptides of HEK-293 cells expressing HLWAR 77 was dependent on the co-expression of both the c DNA for HLWAR 77 and Ga 16.

Cells expressing only ILWAR 77, or only Ga 16, did not respond to the RFamide peptides, nor did cells transfected with the expression vector, lacking a c DNA insert or which contained c DN As for other orphan GPC Rs The peptides which showed reproducible concentration-dependcnt calcium mobilization responses in HILWAR 77 expressing HEK-293 cells are shown above (SEQ ID NOS:7-23) By far the most potent ligands of the FMRF-amide like peptide agonists were bovine NPFF (F-8-F-amide) (SEQ ID NO:8) and NPAF (A-l -F-a Tmde) (SEQ ID NO:7), with both peptides having E Csos of 10 n M in HLWAR 77/Ga 16 cells.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

SEQUENCE LISTING < 110 > SMITHKLINE BEECHAM CORPORATION SMITKLINE BEECUAM PLC < 120 > 7 TM RECEPTOR HLWAR 77 < 130 > GH-70074-2 GB < 140 > To Be Assigned < 141 > 2001-03-29 < 150 > 09/531,641 < 151 > 2000-03-29 < 150 > 09/195,517 < 151 > 1998-11-19 < 150 > 09/006,140 < 151 > 1998-01-13 < 150 > 60/049,332 < 151 > 1997-06-11 < 150 > 60/067,253 < 151 > 1997-12-02 < 160 > 23 < 170 > Fast SEQ for Windows Version 3 O < 210 > 1 < 211 > 1888 < 212 > DNA < 213 > HOMO SAPIENS < 400 > 1 ccatcctaat acgacncact atagggctcg agcggccgcc cgggcaggzt ctcggctcac 60 igcaagctcc acctcctggg ttcacgctat tctcctgcct cagcctcczg agtagctggg 120 actacaggcg cccgccacca cgcctggcta attttttgt attelcagta gggacggcgt 150 ttcactgtgt tagcccagat ggtctccgtc tcccgacctc gtgatccacc cacctcggct 240 tcccaaagtg ctgggattac aggcgtgagc caccgcgccc ggccaattct ctttcttaat 300 tgcctctgcc cacctcttct cttctgcttc ca:attacag gttcatcatg aatgagaaat 360 gggacacaaa ctcttcagaa aactggcatc ccatctggaa tgtcaatgac acaaagcatc 420 atctgtactc agatattaat attacctatg tgaactacta tcttcaccag cctcaagtgg 430 cagcaatctt cattatttcc taccttctga tctcetttt gtgcatgatg ggaaazactg 540 tggtttgctt tattgtaatg aggaacaaac atatgcacac agtcactaar ctc=-catct 600 =aaacctggc cataagtgat ttactagttg gcatattctg catgccttaa acacrgctgg 660 acaatactat agcaggatgg ccatttggaa acacgatgtg caagatcagt ggattggtcc 720 agggaatatc tgtcgcagct tcagtcttta cgttagttgc aactgctgza gataggttcc 180 atgtgtgggz ctaccctttt aaaccaaagc ccactatcaa gacagcgtzt gtcattatta 840 tgatcatctg ggtcctagcc atcacca;ta tgtctccatc tgcagtaatg ttacatgtgc 900 aagaagaaaa atattaccga gtgagactca actcccagaa taaaaccagt ccagtctact 960 ggtgccggga agactggcca aatcaggaa tgaggaagat ctacaccact gtgc:gtttg 1020 ccaacatcta cctggctccc ctctccctca ttgtcatcat gtatggaagg attggaattt 1080 cactcttcag ggctgcagtt cczcacacag gcaggaagaa ccaggagcag tggcacgtgg 1140 tgtccaggaa gaagcagaag atcattaaga tgctcctgat tgtggccctg cttttattc 1200 tctcatggct gcccetgtgg actctaatga tgctctcaga ctacgctgac ctttctccaa 1260 atgaactgca gatcatcaac atctacatct accttttgc acactggccg gcattcggca 1320 acagcagtgt caatcccatc atttatggtt tcttcaacga gaatttccgc cgtggtttcc 1380 aagaagcttt ccagctccag ctctgccaaa aaagagcaaa gcctatggaa gcttatgccc 1440 taaaagctaa aagccatgtg ctcataaaca catctaatca gcttgtccag gaatctacat 1500 ttcaaaaccc tcatggggaa accttgcttt ataggaaaag tgctgaaaaa ccccaacagg 1560 aattagtgat ggaagaatta aaagaaacta ctaacagcag tgagatttaa aaagagctag 1620 tgtgataatc ctaactctac tacgcattat atatttaaat ccattgcttt ttgtggcttt 1680 gcacttcaaa tttttcaaag aatgttctaa ataaaacatt tactgaaagc cctctctggc 1740 aaaaaaatta aaaataaaca aaaatggtca taagarcata aacaacctta tgttgtataa 1800 aaatacgtag agtgacttag acatgtttgc atgaataaat atarttctag agaacagtta 1860 aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1888 < 210 > 2 < 211 > 420 < 212 > PRT < 213 > HOMO SAPI 3 NS < 400 > 2 Met Asn Glu Lys Trp Asp Thr Asn Ser Ser Glu Asn Trp His Pro lle 1 5 10 15 Trp Asn Val Asn Asp Thr Lys His His Leu Tyr Ser A Sp Ile Asn lie 25 30 Thr Tyr Val Asn Tyr Tyr Leu His Gin Pro Gin val Ala Ala Ile Phe 40 45 lie lie ser Tyr Phe Leu Ile Phe Phe Leu Cys Met Met Gly Asn Thr 55 60 val Val Cys Phe Ile Val Met Arg Asn Lys His Met His Thr Val Thr 70 75 80 Asn Leu Phe Ile Leu Asn Leu Ala lle Ser Asp L Le Leu Val Gly Ile 90 95 Phe Cys Met Pro lle Thr Leu Leu Asp Asn Ile lie Ala Gly Trp Pro 105 110 Phe Gly Asn Thr Met Cys Lys lie ser Gly Leu Val Gin Gly Ile Ser 120 125 Val Ala Ala Ser Val Phe Thr Leu val Ala Ile Ala val Asp Arg Phe 135 140 Gin Cys Val Val Tyr Pro Phe Lys Pro Lys Leu Thr lie Lys Thr Ala 150 155 160 Phe Val Ile lie Met Ile Ile Trp Val Leu Ala Ile Thr Ile Met Ser 170 175 Pro ser Ala Val Met Leu His Val Gin Glu Glu Lys Tyr Tyr Arg Val 185 190 Arg Leu Asn Ser Gin Asn Lys Thr Ser Pro Val Tyr Trp Cys Arg Glu 200 205 Asp Trp Pro Asn Gin Glu Met Arg Lys Ile Tyr Thr Thr Val Leu Phe 210 215 220 Ala Asn Ile Tyr Leu Ala Pro Leu Ser Leu Ile Val Ile Met Tyr Gly 225 230 235 240 Arg Ile Gly Ile Ser Leu Phe Arg Ala Ala Val Pro His Thr Gly Arg 245 250 255 Lys Asn Gin Glu Gin Trp His val Val Ser Arg Lys Lys Gin Lys Ile 260 265 270 le Lys Met Leu Leu lie Val Ala Leu Leu Phe Ile Leu Ser Trp Leu 275 280 285 Pro Leu Trp Thr Leu Met Met Leu Ser Asp Tyr Ala Asp Leu Ser Pro 290 295 300 Asn Glu Leu Gln le Ile Asn Ile Tyr Ile Tyr Pro Phe Ala His Trp 305 310 315 320 Leu Ala Phe Gly Asn Ser Ser Val Asn Pro lie Ile Tyr Gly Phe Phe 325 330 335 Asn Glu Asn Phe Arg Arg Gly Phe Gin Glu Ala Phe Gin Leu Gin Leu 340 345 350 cys Gin Lys Arg Ala Lys Pro Met Glu Ala Tyr Ala Leu Lys Ala Lys 355 360 365 Ser His Val Leu Ile Asn Thr Ser Asn Gin Leu Val Gin Glu Ser Thr 370 375 380 Phe Gin Asn Pro His Gly Glu Thr Leu Leu Tyr Arg Lys Ser Ala Glu 355 390 395 400 Lys Pro Gin Gin Glu Leu Val Met Glu Glu Leu Lys Glu Thr Thr Asn 405 410 415 Ser Ser Glu Ile 420 < 2 i 0 > 3 < 211 > 1508 < 212 > DNA < 213 > HOMO SAPIENS < 400 > 3 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 60 ccgggaccga tccagcctcc ggactctagc ctaggccgcg ggacggataa caatttcaca 120 caggaaacag ctatgaccac taggcttttg caaaaagcta tttaggtgac aetatagaag 180 gtacgcctgc aggtaccggt ccggaattcc cgggtcgacc cacgcgtccg cataagtga: 240 ttactagttg gcatattctg catgcctata acactgctgg acaaatttat agcaggatgg 300 ccatttggaa acacgatgtg caagatcagt ggattggtcc agggaatatc tgtcgcagct 360 tcagtottta cgttagttgc aattgctgta gataggttcc agtgtgtggt ctacccttt: 420 aaaccaaagc tcactatcaa gacagcgttt gtcattatta tgatcatctg ggtcctagcc 480 atcaccatta tgtctccatc tgcagtaatg ttacatgtgc aagaagaaaa atattaccga 540 gtgagactca actcccagaa taaaaccagt ccagtctact ggtgccggga agactggcca 600 aatcaggaaa tgaggaagat ctacaccact gtgctgtttg ccaacatcta cczggctccc 660 ctctccctca ttgtcatcat gtatggaagg attggaattt cactcttcag ggctgcagtt 720 cctccacacag gcaggaagaa ccaggagcag tggcacgtgg tgtccaggaa gaagcagaag 780 atcattaaga tgctcctgat tgtggccctg ctttttattc tctcatggct gcccctgt 9 g 840 actctaatga cgctctcaga ctacgctgac ctttctccaa atgaactgca gatcatcaac 900 atctacatct acccttttgc acactggctg gcattcggca acagcagtgt caatcccatc 960 atttatggtt tcttcaacga gaatttccgc cgtggtttcc aagaagct Ct ccagctccag 1020 ctctgccaaa aaagagcaaa gcctatggaa gcttacgccc taaaagctaa aagccacgtg 1080 ctcataaaca catctaatca gcttgtccag gaatctacat ttcaaaaccc tcatggggaa 1140 accttgcttt ataggaaaag tgctgaaaaa ccccaacagg aattagtgat ggaagaatta 1200 aaagaaacta ctaacagcag tgagatttaa aaagagctag tgtgataatc ctaactctac 1260 tacgcattat atatttaaat ccattgcttt ttgtggcttt gcacttcaaa tttttcaaag 1320 aatgttctaa aeaaaacatt tactgaaagc cctctctggc aaaaaaatta aaaataaaca 1380 aaaatggtca taagatcata aacaatctta tgttgtataa aaatacgtag agcgacttag 1440 acacgtttgc atgaataaat acatttctag agaacagnca aaaaaaaaaa aaaaaaaaaa 1500 aaaaaaaa 1508 < 210 > 4 < 211 > 336 < 212 > PRT < 213 > HOMO SAPIENS Lo < 400 > 4 Pro Arg Val Arg Ile Ser Asp Leu Leu Val Gly Ile Phe Cys Met Pro 1 5 10 15 Ile Thr Leu Leu Asp Asn Ile Ile Ala Gly Trp Pro Phe Gly Asn Thr 25 30 Met Cys Lys lie Ser Gly Leu Val Gin Gly Ile Ser val Ala Ala Ser 40 45 Val Phe Thr Leu Val Ala Ile Ala Val Asp Arg Phe Gin Cys Val Val 55 60 Tyr Pro Phe Lys Pro Lys Leu Thr lie Lys Thr Ala Phe Val lie Ile 70 75 80 Met Ile Ile Trp Val Leu Ala Ile Thr Ile Met Ser Pro Ser Ala Val 90 95 Met Leu His Val Gin Glu Glu Lys Tyr Tyr Arg Val Arg Leu Asn Ser 105 110 Gin Asn Lys Thr Ser Pro Val Tyr Trp Cys Arg Glu Asp Trp Pro Asn 120 125 Gin Glu Met Arg Lys Ile Tyr Thr Thr Val Leu Phe Ala Asn Ile Tyr 135 140 Leu Ala Pro Leu Ser Leu lie Val Ile Met Tyr Gly Arg Ile Gly Ile 150 155 160 Ser Leu Phe Arg Ala Ala Val Pro His Thr Gly Arg Lys Asn Gin Glu 170 175 Gin Trp His Val Val Ser Arg Lys Lys Gin Lys Ile Ile Lys Met Leu 185 190 Leu Ile val Ala Leu Leu Phe Ile Leu Ser Trp Leu Pro Leu Trp Thr 200 205 Leu Met Met Leu ser Asp Tyr Ala Asp Leu Ser Pro Asn Glu Leu Gin 210 215 220 Ile Ile Asn Ile Tyr Ile Tyr Pro Phe Ala His Trp Leu Ala Phe Gly 225 230 235 240 Asn Ser Ser Val Asn Pro Ile Ile Tyr Gly Phe Phe Asn Glu Ase Phe 245 250 255 Arg Arg Gly Phe Gin Glu Ala Phe Gin Leu Gin Leu Cys Gin Lys Arg 260 265 270 Ala Lys Pro Met Glu Ala Tyr Ala Leu Lys Ala Lys Ser His Val Leu 275 280 285 Ile Asn Thr Ser Asn Gin Leu Val Gin Glu Ser Thr Phe Gin Asn Pro 290 295 300 His Gly Glu Thr Leu Leu Tyr Arg Lys Ser Ala Glu Lys Pro Gin Gin 305 310 315 320 Glu Leu Val Met Glu Glu Leu Lys Glu Thr Thr Asn Ser Ser Glu Ile 325 330 335 c 210 > 5 < 211 > 26 < 212 > D 9 A < 213 > HOMO SAPIENS < 400 > 5 aaagactgaa gctgcgacag atattc 26 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > HOMO SAPIENS < 400 > 6 cctggaccaa tccactgatc ttgc 24 < 210 > 7 < 211 > 18 < 212 > PRT < 213 > BOVINE < 400 > 7 Ala Gly Glu Gly Leu Ser Ser Pro Phe Trp Ser Leu Ala Ala Pro Gin 1 S 10 15 Arg Phe < 210 > 8 < 211 > 8 < 212 > PRT < 213 > BOVINE < 400 > 8 Phe Leu Phe Gin Pro Gln Arg Phe 1 5 < 210 > 9 < 211 > 18 < 212 > PRT < 213 > HOMO SAPI El IS < 400 > 9 Ala Gly Glu Gly Leu Asn Ser Gin Phe Trp Ser Leu Ala Ala Pro Gin 1 5 10 15 Arg Phe < 210 > 10 < 211 > 11 < 212 > PRT c 213 > HOMO SAPIENS < 400 > 10 Ser Gin Ala Phe Leu Phe Gin Pro Gin Arg Phe 1 5 10 < 210 > 11 < 211 > 5 < 212 > PRT < 213 > GALLUS GALLUS < 400 > 11 Leu Pro Leu Arg Phe 1 5 < 210 > 12 < 211 > 4 < 212 > PRT < 213 > MACROCALLISTA NIMBOSA 1 ' c 400 > 12 Phe Met Arg Phe < 210 > 13 < 211 > 4 < 212 > PRT < 213 > ANTHOPLEURA ELEGANTISSIMA < 400 > 13 Glu Gly Arg Phe < 210 > 14 < 211 > 3 < 212 > PRT < 213 > ANTHOPLEURA ELEGANTISSIMA < 400 > 14 Leu Arg Asn < 210 > 15 < 211 > 5 < 212 > PRT < 213 > ANTHOPLEURA ELEGANTISSIMA < 400 > 15 Glu Ser Leu Arg Trp 1 5 < 210 > 16 < 211 > 5 < 212 > PRT < 213 > ANTHOPL Et URA ELEGANTISSIMA < 400 > 16 Glu Gly Leu Arg Trp 1 5 < 210 > 17 < 211 > 7 < 212 > PRT < 213 > POLYORCHIS PENICILLATUS < 400 > 17 Glu Leu Leu Gly Gly Arg Phe 1 5 < 210 > 18 < 211 > 7 < 212 > PRT < 213 > PANAGRELLUS REDIVIVWS < 400 > 18 Lys Ser Ala Tyr Met Arg Phe 1 5 ' r' , y 3 < 210 > 19 < 211 > 8 < 212 > PRT < 213 > ROMARUS AMERICANUS < 400 > 19 Thr Asn Arg Asn Phe Leu Arg Phe 1 S < 210 > 20 < 211 > 7 < 212 > PRT < 213 > PROCAMBARUS CLARKII < 400 > 20 Asp Arg Asn Phe Leu Arg Phe 1 5 < 210 > 21 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 400 > 21 Pro Gin Arg Phe < 210 > 22 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 400 > 22 Tyr G Gly y Phe Leu Arg Phe 1 5 < 210 > 23 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 400 > 23 Tyr Gly Gly Phe Met Arg Phe 1 5 ::,s, 7 -, , \,;:.

Claims (14)

What is claimed is;

1 An isolated polynucleotide comprising a nucleotide sequence that has at least 80 % identity over its entire length to a nucleotide sequence encoding the HLWAR 77 polypeptide of SEQ ID NO:2; or a nucleotide sequence complementary to said isolated polynucleotide.

2 The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ID NO: 1 encoding the HLWAR 77 polypeptide of SEQ ID N 02.

3 The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 80 % identical to that of SEQ ID NO: 1 over its entire length.

4 The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO: 1.

The polynucleotide of claim 1 which is DNA or RNA.

6 A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a HLWAR 77 polypeptide comprising an amino acid sequence, which has at least 80 % identity with the polypeptide of SEQ ID NO:2 when said expression system is present in a compatible host cell.

7 A host cell comprising the expression system of claim 6.

8 A process for producing a I-LWAR 77 polypeptide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

9 A process for producing a cell which produces a I-ILWAR 77 polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a HLLWA Rt 77 polypeptide.

A HLWAR 77 polypeptide comprising an amino acid sequence which is at least 80 % identical to the amino acid sequence of SEQ ID NO:2 over its entire length.

11 The polypeptide of claim 10 which comprises the amino acid sequence of SEQ ID NO:2.

12 A method for identifying an agonist or antagonist of a polypepride of claim 10 which comprises:

contacting a cell expressing on the surface thereof the polypeptide, said polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the prlypeptide.

13 A method of claim 12 which further comprises conducting the identification of agonist or antagonist in the presence of a labeled or unlabeled peptide selected from the group consisting of the peptides of SEQ ID NOS:7-23.

14 A method for identifying an agonist or antagonist of a polypeptide of claim 10 which comprises:

determining the inhibition of binding of a ligand to cells which have the polypeptide on the surface thereof, or to cell membranes containing the polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypepride, and determining the amount of ligand bound to the polypeptide, such that a compound capable of causing reduction of binding of a ligand is an agonist or antagonist.

A method of claim 14 in which a ligand is labeled or unlabeled peptide selected from the group consisting of the peptides of SEQ ID NOS:7-23.