WO1992016220A1 - Controlling nk-2 receptor-mediated responses and relates diagnostics - Google Patents

Abstract

Genomic DNA and cDNA encoding the human NK-2 receptor is cloned and the recombinant protein expressed. Recombinant receptor and receptor fragments are used as therapeutics to treat disease, such as asthma. The recombinant receptor and receptor fragments are also used in methods of screening candidate compounds for their ability to antagonize interaction between the neurokinin A neurotransmitter and NK-2 receptor. Antibodies specific for NK-2 receptor and their use as a therapeutic and/or a vaccine is also disclosed.

Description

CONTROLLING NK-2 RECEPTOR-MEDIATED RESPONSES AND RELATED DIAGNOSTICS

BACKGROUND OF THE INVENTION This invention relates to receptors, particularly receptors involved in neurotransmission.

Neurokinin A (formerly termed substance K) is one member a family of peptide neurotransmitters known as tachykinins (Maggio, An i. Rev. Neurosci . .11:13, 1988). Molecular characterization has revealed that tachykinins are transcribed from alternatively-spliced mRNA molecules (termed, a, β and η mRNAs) and are translated as precursor molecules, termed preprotachykinins (Nawa et al., Nature 306;32. 1983; Kawaguchi et al. , Biochem . Biophys . Res . Comm n . 139:1040. 1986; Krause et al., Proc. Natl . Acad . Sci . USA 84.:881, 1987). Specifically, the β and η messages encode preprototachykinins which include neurokinin A and another tachykinin, substance P; the β mRNA molecule encodes an amino-terminally extended form of neurokinin A, termed neuropeptide K or NpK; and the α form encodes only substance P. Mature tachykinin molecules are produced from the preprotachykinins by proteolytic processing. Structurally, the tachykinin family shares the COOH-terminal protein sequence, Phe-X-Gly-Leu-Met- NH₂ (SEQ ID No.:l), where X is Phe, Tyr, Val, or lie.

Three classes of tachykinin receptors have been identified by bioassay and radioligand binding (Martling et al., Life Sci . _4():1633, 1987; Buck et al.. Science 226:987. 1984; Burcher et al., J. Pharmacol . Exp . Ther. 236:819. 1986). Analysis of these receptors has revealed a COOH-terminal consensus sequence of the receptors which controls biological activity and divergent amino-terminal sequences which determine receptor affinity. The result of such an arrangement is that each tachykinin recognizes each of the three receptor types, but with varying avidity. In particular, the NK-1 receptor preferentially binds substance P; the NK-2 receptor preferentially binds neurokinin A; and the NK-3 receptor preferentially recognizes neurokinin B (another tachykinin, remote from neurokinin A or substance P) (Tatemoto et al., Biochem. Biophys. Res. Comm n. 128:947. 1985) . Synthetic tachykinin analogs designed to act as competitive inhibitors exhibit relative selectivity for each of the three neurokinin receptors ( or ser et al., EMBO J. 5_:2805, 1986; Cavanikas et al., Bur. J. Pharmacol .

77:205, 1982; Regoli et al., Tachykinin Antagonists, eds. R. Hakanson and F. Sundler, pp. 277-287, 1985, Elsevier Science Publisher B.V., Amsterdam).

Tachykinins, in general, have been found to display a wide tissue distribution (Lee et al., Eur. J. Pharmacol . 130:209. 1986; Lundberg et al., Acta Physiol . Scand. 119:243_f 1983), including an association with the central and peripheral nervous system. Neurokinin A, in particular, has been found to be associated with smooth muscle-containing tissues found in the gastrointestinal, respiratory, genitourinary, and vascular systems (Hua et al., -Regrul. Pept. 13:1, 1985).

Within the respiratory system, tachykinins have a number of important physiologic effects. These include bronchoconstriction of large airways, enhancement of vascular permeability, and stimulation of mucus secretion (Naline et al.. Am. Rev. Respir. Dis. 140:679, 1989; Saria et al., Acta Otolaryngol . Suppl . 457:25. 1989; McCormack et al., Life Sci. 45:2405, 1989; Tamura et al., Tohoku J. Exp. hied. 159:69. 1989). Characterization of these responses using tachykinins and structural antagonist analogs have indicated that the NK-2 receptor, i.e., the receptor selective toward neurokinin A, predominates in animal and human respiratory systems (Naline et al. , supra: Tamura et al., supra) . Masu et al. (Nature 329:836. 1987) and Sasai and Nakanishi (Biochem . Biophys . Res . Commun . 165:695. 1989), respectively, have reported the cDNA and deduced protein sequences for the bovine and rat stomach NK-2 receptors. Because such receptors were found to be homologous to the protein rhodopsin, the NK-2 receptor was included as a member of the rhodopsin superfamily. This multigene family is characterized by the presence of seven hydrophobic sequences believed to represent membrane- spanning regions. Proteins included in this family are generally involved in signal transduction, coupled to the GTP-binding proteins (Dohlman et al., Biochemistry .26:2657, 1987; Gilman, Annu . Rev. Biochem . 5.6:615, 1987).

SUMMARY OF THE INVENTION In one aspect, the invention generally features human recombinant NK-2 receptor or a fragment thereof. Preferably, the receptor includes an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 3 (SEQ ID NO.:2). By a "substantially identical" amino acid sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the biological activity of the receptor. Such equivalent growth factors can be isolated by extraction from the tissues or cells of any animal which naturally produce such a receptor or which can be induced to do so, using the methods described below, or their equivalent; or can be isolated by chemical synthesis; or can be isolated by standard techniques of recombinant DNA technology, e.g., by isolation of cDNA or genomic DNA encoding such a receptor. In a related aspect, the invention features a substantially isolated polypeptide which is a fragment of a human NK-2 receptor and includes an extracellular domain capable of binding neurokinin A neurotransmitter. By a "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation) . A "substantially isolated polypeptide" is one which is substantially free of other proteins, carbohydrates and lipids with which it is naturally associated. By an "extracellular domain" is meant any portion of the protein (in this case, the human NK-2 receptor) which is exposed on the outer surface of a receptor-bearing cell, and which contains significant structural information to participate in or define specific binding.

In preferred embodiments, the polypeptide is selected from the group including:

(a) MGTCDIVTEANISSGPESNTTGITAFSMPSW (SEQ. ID N0.:3; amino acid residues 1 to 31 of Fig. 3; SEQ ID NO.:2); (b) NFVYASHNIWYFGR (SEQ. ID N0.:4; amino acid residues 90 to 103 of Fig. 3; SEQ ID N0.:2);

(c) TVTMDQGATKCWAWPEDSGGKTLLLYH (SEQ. ID NO.:5; amino acid residues 171 to 198 of Fig. 3; SEQ ID NO.:2);

(d) GSFQEDIYCHKFIQQVY (SEQ. ID NO.:6; amino acid residues 273 to 289 of Fig. 3; SEQ ID NO.:2); and (e) fragments or analogues of (a) - (d) which are capable of binding a neurokinin A neurotransmitter. Preferably, such a polypeptide is a recombinant polypeptide.

In other related aspects, the invention features purified DNA which encodes a receptor (or fragment thereof) or a polypeptide described above; vectors which contain such DNA and are capable of directing expression of the protein encoded by the DNA in a vector-containing cell; and cells containing such vectors (preferably eukaryotic cells, e.g. , mammalian cells) . By "purified DNA" is meant a DNA molecule which encodes the human NK- 2 receptor (or an appropriate receptor or analog) , but which is free of the genes that, in the naturally- occurring genome of the organism from which the DNA of the invention is derived, flank the gene encoding the NK- 2 receptor.

The expression vectors or vector-containing cells of the invention can be used in a method of the invention to produce human NK-2 receptor and the polypeptides described above. The method involves providing a cell transformed with DNA encoding the NK-2 receptor or a fragment thereof positioned for expression in the cell; culturing the transformed cell under conditions for expressing the DNA; and isolating the recombinant human NK-2 receptor protein. By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of genetic engineering, a DNA molecule encoding the human NK-2 receptor (or a fragment or analog, thereof) . Such a DNA molecule is "positioned for expression" meaning that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of the NK-2 receptor protein, or fragment or analog, thereof) . In yet another aspect, the invention features purified antibody which binds preferentially to the human NK-2 receptor (or a fragment thereof) or a polypeptide described above. By "purified antibody" is meant one which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated to permit therapeutic administration. Such an antibody "preferentially binds" to a human NK-2 receptor (or fragment or analog, thereof), i.e., does not substantially recognize and bind to other antigenically- unrelated molecules. Preferably, the antibody neutralizes the biological activity in vivo of the protein to which it binds. By "biological activity" is meant the ability of the NK-2 receptor to bind neurokinin A and signal the appropriate cascade of biological events. By "neutralize" is meant to partially or completely block (e.g., the biological activity of the human NK-2 receptor) .

In other aspects, the receptor, polypeptides, or antibodies described above are used as the active ingredient of therapeutic compositions. In such therapeutic compositions, the active ingredient may be formulated with a physiologically-acceptable carrier or anchored within the membrane of a cell. These therapeutic compositions are used in methods of treating asthma and treating ulcerative colitis. The methods involve administering to a mammal the therapeutic composition in a dosage effective to antagonize an interaction between neurokinin A neurotransmitter and an NK-2 receptor. Finally, the invention features a method of screening candidate compounds for their ability to antagonize interaction between neurokinin A neurotransmitter and an NK-2 receptor. The method involves: a) mixing a candidate antagonist compound with a first compound which includes a recombinant NK-2 receptor (or fragment) or a polypeptide or an antibody described above on the one hand and with a second compound which includes neurokinin A neurotransmitter or a fragment thereof capable of binding to the first compound on the other hand; b) determining whether the first and second compounds bind; and c) identifying antagonistic compounds as those which interfere with the binding of the first compound to the second compound. By an "antagonist" is meant a molecule which inhibits a particular activity, in this case, the ability of neurokinin A to interact with the NK-2 receptor and/or to trigger the biological events resulting from such an interaction.

The proteins of the instant invention are associated with smooth muscle tissue of the gastrointestinal, respiratory, genitourinary, and vascular systems. They are therefore useful for developing therapeutics which alleviate disorders associated with abnormal smooth muscle cell contraction. These disorders include respiratory diseases such as asthma and gastrointestinal disorders such as peptic ulcers and ulcerative colitis. Preferred therapeutics include antagonists, e.g., peptide fragments, drugs, or antibodies, which block neurokinin A or NK-2 receptor function. Because the receptor component may now be produced by recombinant techniques and because candidate antagonists may be screened in vitro, the instant invention provides a simple and rapid approach to the identification of useful therapeutics. Once identified, a peptide- or antibody-based therapeutic may also be produced, in large quantity and inexpensively, using recombinant and molecular biological techniques.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DETAILED DESCRIPTION The drawings will first briefly be described. Drawings

FIG. 1 shows a restriction map of the human NK-2 receptor gene;

FIG. 2 shows the complete nucleotide sequence and deduced amino acid sequence of the human NK-2 receptor exons and intron-exon junctions (SEQ ID NO.:7, 8, 9, 10, ii); FIG. 3 shows the deduced protein sequence of the human NK-2 receptor (SEQ ID NO.:2); and

FIG. 4 shows the results of a segregation analysis localizing the human NK-2 receptor gene to human chromosome 10.

CLONING AND CHROMOSOMAL LOCALIZATION OF THE HUMAN NK-2 RECEPTO GENE

The human NK-2 receptor gene was cloned as follows. Human tracheal tissue was obtained at autopsy

(approximately 4 h postmortem) from an individual with cystic fibrosis; the tissue was stored at -80°C. RNA was extracted from 3-4 g segments of trachea by first pulverizing in liquid nitrogen, extracting with guanidinium thiocyanate (by the methods of Ullrich et al., Science 196:1313. 1977; and Chirgwin et al.,

Biochemistry 24.:5294, 1979), and centrifuging through cesium chloride by the method of Gilsin et al.

(Biochemistry 13:2633. 1974). Poly(A⁺) RNA was then isolated by passing the material over oligo(dT)-Sepharose

(Pharmacia, Piscataway, NJ) by the method of Aviv and

Leder (Proc. Natl . Acad. Sci . USA 19:1408, 1972) and transcribed into cDNA by the method of Gubler and Hoffman

(Gene (Amst.) 2.5:263, 1983). Synthetic oligonucleotide primers were designed based on the cDNA sequence reported for the bovine NK-2 receptor (Masu et al. , supra) from regions of the molecule enriched in tryptophan or cysteine residues.

These primers extended from nucleotide 91-108 (i.e., the sense primer) and from nucleotide 538-555 (i.e., the antisense primer) ; each included EcόRI restriction site sequences at their 5' ends. Such primers were, respectively, of sequence:

5'-AATGAATTCTGGCAGCTGGCACTGTGG-3• (SEQ. ID NO.:12) δ'-AATGAATTCCCAGGCCACCACGCACTT-S' (SEQ. ID

NO.:13) . The primers were synthesized by standard cyanoethyl phosphoramidite chemistry using an Applied Biosystems Model 318A DNA Synthesizer (Foster City, CA) .

Approximately 100 ng of human tracheal cDNA was combined with 1 μg of each of the synthetic primers and recombinant Taq DNA polymerase (Perkin-Elmer, Norwalk, CT) . Twenty-five cycles of polymerase chain reaction were carried out. Each cycle included: 1 min at 95°C, 2 min at 37°C, and 2 min at 72°C. This was followed by a final extension period of 7 min at 72°C. The PCR product, a 465 base pair fragment including nucleotides 91 to 555, was purified, following electrophoretic separation, using GeneClean (Bio 101, La Jolla, CA) . The fragment was digested with σoRI, repurified as above following a second round of electrophoresis, and ligated to .EcoRI-digested pBluescript SK+ (Stratagene, La Jolla, CA) . Clones were amplified in Escherichia coli XL-lblue (Stratagene, La Jolla, CA) , purified by centrifugation through cesium chloride (by the method of Birnbolm and Doly, Nucl . Acids Res . 7:1513, 1979), and subjected to double-stranded nucleic acid sequencing using DNA polymerase and [ S]dATP by the method of the supplier (Sequenase, United States Biochemical Corporation, Cleveland, OH) . The fragment exhibited >90% sequence identity with the bovine molecule.

A human placental genomic DNA library, prepared by standard techniques, was screened by Southern blot analysis using the 465-bp NK-2 receptor fragment (isolated above) labelled with ³ P. Bacteriophage DNA was transferred to nitrocellulose filters (in duplicate) and hybridized with the probe for 16 h, at 42°C, in 5X SSC (0.75M NaCl, 0.075M sodium citrate)/50% formamide, containing 20mM Tris-HCl, pH 7.5, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS. Filters were washed three times, for 10 min each, in 2X SSC/0.1% SDS, at 22°C; followed by one wash, for 30 min, in 0.2 SSC/0.1% SDS at 68°C; and exposed to X-ray film (Kodak X-Omat, Eastman Kodak, Rochester, NY) at -70°C. Of approximately 1 x 10⁶ bacteriophage plaques screened, four positive clones were obtained; three yielded the Pstl/EcόRl restriction pattern shown in FIG. 1.

To identify the exon-containing fragments, one of these clones, termed NGNK-2, was analyzed by Southern blot hybridization as follows. NGNK-2 was digested with either Pstl or .EσoRI and, following electrophoretic separation on a 1% agarose TAE gel, fragments were blotted to nylon membranes (Gene- Screen, Du Pont, Wilmington, DE) and probed with ³ P- labelled NK-2 receptor fragment (i.e., nucleotides 91- 555, corresponding to exons 1 and 2) , or with ³²P-labelled synthetic oligonucleotides based on exons 3, 4, or 5 of the bovine cDNA sequence. Specifically, these oligonucleotides included the cysteine-rich sequences of exons 4 or 5 (CCLNHR, bovine residues 308-313 of exon 4; SEQ. ID NO.:14; CCPWVT bovine residues 324-329 of exon 5; SEQ. ID NO.:15); and the M5 hydrophobic membrane-spanning sequence of exon 3 (LIVIAL, bovine residues 199-205; SEQ ID NO.:16). It was reasoned that these sequences might represent structurally important regions e.g., due to their potential for disulfide bond formation or insertion into the membrane, and hence might be more likely to be conserved between species. Partial Pstl or EcόRI digests were also performed, the fragments blotted as described above, and probed with a P-labeled Pstl fragment containing a portion of exon 4. For the partial digestion analysis, the restriction map was determined from the ladder of partial digestion products and confirmed by complete digestion with the same enzymes. All mapping distances were accurate to within lOObp. The analysis revealed that five exons encode the NK-2 receptor; these are indicated by thick lines in FIG. 1. No evidence was found for cross-hybridization with related genes or pseudogenes under conditions of medium stringency, indicating that the NK-2 receptor gene is present in a single copy in the human genome.

Fragments identified as exons by hybridization were subcloned into pBluescript (Stratagene, La Jolla, CA) for double-stranded sequencing (as described above) . All of the exons, as well as the 5'- and 3'-flanking regions were sequenced extensively on both strands; intron-exon junctions were assigned by homology with the bovine cDNA sequence and subsequently confirmed by analysis of the human cDNA sequence (see below) . The genomic DNA sequence obtained as well as the deduced protein sequence are shown in FIG. 2 (SEQ. ID NOS.:7, 8, 9, 10, 11). The coding sequence is interrupted by four introns ranging in size from 1 to 4 kilobases. This is unlike the organization of genes for other members of the G-protein-coupled receptor family. To date, only bovine rhodopsin and D₂ dopamine receptor and an unidentified rat molecule contain introns; the coding sequences of the other genes in this family are generally intronless (Nathans and Hogness, Cell 14.:807, 1983; Bunzow et al., Nature 336:783, 1988; Ross et al., Proc. Natl . Acad. Sci . USA ES7.:3052, 1990). The overall sequence identity to the bovine molecule is approximately 90%. The human coding sequence is 42 nucleotides longer than the bovine and 30 nucleotides longer than the rat sequences (Masu et al. , supra: Sasai et al., supra) . Close inspection of exon 1 revealed a single open reading frame which initiated with a methionyl residue, analogous to that of the bovine and rat sequences (Masu et al., supra: Sasai et al., supra) . Inspection of ~500 bp of sequence upstream from the methionine revealed a putative TATA box (at nucleotide -307) and a GC-like box (at nucleotides -356 to -366) . No other characterized transcriptional control signals were observed within 500 bp of the presumed initiating methionine.

The transcription initiation site was determined by primer extension using a synthetic oligonucleotide corresponding to nucleotides -191 to -159 in the 5'- untranslated region. This region was chosen because of the proximity to the putative TATA and GC boxes. The synthetic primer was labeled with [7-³ P]ATP, and 2 x 10⁵ cpm of probe was allowed to anneal to 2 μg of human stomach poly(A⁺) RNA (prepared as described above for human tracheal RNA) for 16 h, at 65°C, in 30 μl of aqueous hybridization buffer, containing 1M NaCl, 167mM HEPES, pH 7.5, and 0.3mM EDTA. The annealed RNA/primer mixture was ethanol precipitated, redissolved in RT 1 buffer (Boehringer Mannheim, Indianapolis, IN) containing 70 units of RNasin (Promega Biotec, Madison, WI) , and 20mM dNTPs, and incubated with 5 units of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN) for 90 min., at 42°C (36, 37) . Any remaining RNA was digested with DNase-free RNase A (Sigma Chemical Co., St. Louis, MO), extracted with phenol/chloroform, ethanol precipitated, and redissolved in 5mM Tris-HCl, pH 7.5, containing 78% formamide, 10 mM EDTA, 0.025% bromphenol blue, and 0.025% xylene cyanol FF. The reaction products were analyzed by electrophoresis on a polyacrylamide sequencing gel. From this analysis, it was determined that transcription initiated at nucleotide -282. A full-length human NK-2 receptor cDNA was cloned as follows.

Human gastrointestinal poly(A)⁺ RNA and cDNA were prepared from resected human stomach tissue as described above for human tracheal tissue RNA and cDNA. An oligonucleotide primer corresponding to the putative 5* end of the cDNA was designed based on an analysis of the genomic exon 1 sequence and the identification of a putative 5' ATG site. At the 3' end, a primer was designed based on extensive homology with the bovine M7 membrane-spanning sequence. Specifically, these primers corresponded to nucleotides 1-18 (i.e., the sense primer) and nucleotides 1173-1196 (i.e., the antisense primer) and each included a nested _EσoRI restriction site at its 5' end. Primers were synthesized as described above. PCR reactions were carried out (also as described above) using about 200 ng of human stomach cDNA and 1 μg of each primer. The PCR amplification involved 5 cycles, each including l min. at 95°C , 1.5 min. at 45°C, and 3 min. at 72°C; followed by 25 cycles, each including 1 min. at 95^βC, 1.5 min at 55^βC, and 3 min. at 72°C; and a final extension of 7 min. at 72^βC. Ten percent of the reaction mixture was subjected to secondary PCR, using the same primers and the same cycling conditions as were used for the initial reaction. The material obtained was purified by agarose electrophoresis, digested with .EσoRI, repurified by agarose electrophoresis, and ligated to .EcoRI-digested pBluescript SK+ (Stratagene; La Jolla, CA) . The PCR product was determined to be a fragment of approximately 1.2 kb. Using standard techniques of Southern blot analysis, this fragment was found to hybridize specifically with the tracheal cDNA clone.

The cDNA clone was sequenced using the methods described above; its deduced protein sequence is shown in FIG. 3 (SEQ. ID NO.:2). By comparison of the NK-2 receptor sequence with that of other members of the rhodopsin superfamily and by examination of the hydrophobic and hydrophilic regions of the molecule, extracellular and intracellular domains may be deduced. Extracellular domains include; amino acids 1 to 31 (SEQ. ID N0.:3); amino acids 90 to 103 (SEQ. ID N0.:4); amino acids 171 to 198 (SEQ. ID N0.:5); and amino acids 273 to 289 (SEQ. ID NO.:6). Intracellular domains include amino acids 60 to 67; 130 to 149; 221 to 250; and 313 to 398.

Below the human sequence are depicted those amino acids which differ in the rat (line 2) and bovine (line 3) NK-2 receptor proteins. In analyzing the sequences of the three species, it is seen that significant variability occurs in the 5*- and 3*-regions of the coding sequences. This is of interest since the model for this class of receptors suggests that the amino- terminal region is part of the extracellular domain and is potentially part of the ligand-binding site (Dixon et al.. Cold Spring Harbor Symp. Quant. Biol . 53.:487, 1988). A single putative site for N-glycosylation is preserved among the three species, at residue 19 of the predicted proteins. Human and bovine, but not rat, proteins have an additional Asn-X-Ser sequence at residues 11-13. Also by analogy with the proposed model for adrenergic receptors, the COOH-terminal segment is the predicted cytoplasmic tail. Recent mutagenesis studies with the jS_j-adrenergiσ receptor indicate that the proposed third cytoplasmic loop and the proximal portion of the COOH terminus form an interaction site with the G_g protein (Dixon et al., supra; O'Dowd et al., J. Biol . Chem. 203:15985. 1988). The least structural diversity is seen in the seven hydrophobic domains, which presumably form a pocket or pore within the membrane for ligand binding. Preliminary data indicated that .EcoRI and Pstl restriction digests of mouse and human genomic DNA yielded different hybridization patterns with the tracheal NK-2 receptor fragment (i.e., nucleotides 91 to 555) . This fact was exploited to determine the genomic map position of the human NK-2 receptor as follows.

Genomic DNA was prepared from mouse liver and from human leukocytes as previously described (Blin et al., Nucleic Acids Res . 3.:2303, 1976; Baas et al.. Hum . Genet . 12:301, 1984). Aliquots of 7μg each were digested with EcoRI, Pstl, or Hindlll (Boehringer Mannheim, Indianapolis, IN) . Products were separated on a 1% agarose TAE gel and the DNA blotted to a nylon membrane (GeneScreen, Du Pont, Wilmington, DE) . The blots were hybridized with the ³²P-labelled NK-2 receptor cDNA fragment (i.e., nucleotides 91-555) in a hybridization solution of 50% formamide/ 1% SDS/ 1M NaCl/ 10% dextran sulfate/ salmon sperm DNA (lOOμg/ml) , for 16 h, at 42°C. Nonspecifically bound probe was removed by washing twice in 2X SSC, for 5 min, at room temperature; followed by washing twice in 2X SSC/1% SDS, at 65°C, for 30 min. The blots were then exposed to X-ray film (Kodak X-Omat) at - 70^βC. The restriction fragments hybridizing to the NK-2 receptor probe generated by .EcoRI and Pstl were also found to be different for human and mouse DNA. Chromosome localization for the NK-2 receptor gene was accomplished using DNA from 40 cell hybrids involving 18 unrelated human cell lines and 4 mouse cell lines (Shows et al.. Adv. Hum . Genet . 12.:341, 1982; Shows et al.. Somatic Cell Hoi . Genet . 10:315, 1984; Shows et al., Cytogenet . Cell Genet . £1:99, 1978). The hybrids were characterized by karyotypic analysis and by mapped enzyme markers (Shows et al.. Adv. Hum . Genet . 12.:341, 1982; Shows et al., Cytogenet . Cell Genet . 21:99, 1978; Shows et al., Isozymes Curr. Top. Biol . Med. Res . 10:323, 1983). The human NK-2 receptor cDNA fragment (i.e., nucleotides 91-555) was hybridized to Southern blots containing .EcoRI-digested DNA from the human-mouse hybrids as described above. Scoring was determined by the presence or absence of human bands in the hybrids on the blots. Concordant hybrids have either retained or lost the human bands together with a specific human chromosome. Discordant hybrids have either retained the human bands but not a specific chromosome, or the reverse. Percent discordancy indicates the degree of discordant segregation for a marker and a chromosome. A 0% discordancy is the basis for chromosome assignment. This analysis is summarized in FIG. 4. The hybrid XTR- 3BSAGB, exhibiting the 10q-, 10pter→10q23: band and lacking intact chromosome 10, localized the NK-2 receptor to the pter→q23 region of human chromosome 10. POLYPEPTIDES ACCORDING TO THE INVENTION

Polypeptides according to the invention include the entire hτυnan NK-2 receptor as described in FIG. 2

(SEQ. ID N0S.:7, 8, 9, 10, 11) or FIG. 3 (SEQ. ID N0.:2). Alternatively, any analog or fragment of the NK-2 receptor capable of interacting with neurokinin A is useful in the invention. Such an interaction may be readily assayed using any of a number of standard in vitro methods (see e.g., Regoli et al., Tachykinin Antagonists, eds. Hakanson and Sundler, Elsevier Science Publisher, .Amsterdam, 1985) . In one particular assay, neurokinin A is adhered to a microtiter plate (using methods similar to those for adhering antigens for an ELISA assay) and the ability of labelled NK-2 receptor fragment- or receptor analog-expressing cells (e.g., labelled with ³H-thymidine) to bind the immobilized nerokinin A is used to detect an interaction between neurokinin A and the receptor component. Specific receptor analogues of interest include full-length or partial receptor proteins including an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the receptors' ability to interact with neurokinin A. Specific receptor fragments of interest include the portions of the receptor deduced to be extracellular. Such regions may be identified by comparison with related proteins of similar structure (e.g., other members of the rhodopsin superfamily) ; useful regions are those exhibiting homology to the extracellular domains of well- characterized members of the family. Examples of these regions include, but are not limited to, amino acid residues 1 to 31 (SEQ. ID NO.:3), 90 to 103 (SEQ. ID N0.:4), 171 to 198 (SEQ. ID N0.:5), and 273 to 289 (SEQ. ID N0.:6) as shown in FIG. 3 (SEQ. ID NO.:2).

Alternatively, from the primary amino acid sequence, the secondary protein structure and, therefore, the extracellular domain regions may be deduced semi- empirically using a hydrophobicity/hydrophilicity calculation such as the Chou-Fasman method (see, e.g., Chou and Fasman, Ann . Rev. Biochem . 7:251, 1978). Hydrophilic domains, particularly ones surrounded by hydrophobic stretches (e.g., transmembrane domains) present themselves as strong candidates for extracellular domains. Finally, extracellular domains may be identified experimentally using standard enzymatic digest analysis, e.g., tryptic digest analysis.

Candidate fragments would be tested for interaction with neurokinin A by the assays described herein (e.g., the assay described above). POLYPEPTIDE EXPRESSION

Polypeptides according to the invention may be produced by transformation of a suitable host cell with a full or partial NK-2 receptor-encoding cDNA or genomic DNA fragment in a suitable expression vehicle, and expression of the receptor.

Those skilled in the field will understand that any of a wide variety of expression systems may be used to provide the recombinant receptor protein. The precise host cell used is not critical to the invention, however the following host cells are preferred: Chinese Hamster Ovary (CHO) cells, Madin-Darby Canine Kidney (MDCK) cells, COS cells, and fibroblast cells, such as mouse 3T3 cells. Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockville, MD; ATCC Accession Nos. CCL 61, CCL34, CRL 1650, and CCL 163, respectively) . The method of transfection and the choice of expression vehicle will depend on the host system selected. Mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) ; expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987) .

One particularly preferred expression system is the mouse 3T3 fibroblast host cell transfected with a pMAMneo expression vector (Clontech, Palo Alto, CA) . pMAMneo provides: an RSV-LTR enhancer linked to a dexamethasone-inducible MMTV-LTR promoter, an SV40 origin of replication (which allows replication in mammalian systems) , a selectable neomycin gene, and SV40 splicing and polyadenylation sites. DNA encoding the NK-2 receptor or an appopriate receptor fragment or analog (as described above) would be inserted into the pMANneo vector in an orientation designed to allow expression. The recombinant receptor protein would be isolated as described below. Other preferable host cells which may be used in conjunction with the pMAMneo expression vehicle include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively) .

In another preferred expression system, the NK-2 receptor protein (or receptor fragment or analog) is produced by a stably-transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra) ; methods for constructing such cell lines are also publically available, e.g., in Ausubel et al. (supra) . In one example, cDNA encoding the receptor (or receptor fragment or analog) is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plas id and, therefore, the NK-2 receptor-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (as described in Ausubel et al., supra) . This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra) ; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra) . Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR~cells; ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification.

Yeast cells may also be used as a host system. Yeast vectors into which the NK-2 receptor- or receptor fragment- or analog-encoding DNA may be cloned are publically available, and many are described in Pouwels et al. (supra) . Methods of yeast transformation are described in Ausubel et al. (supra) .

Once the recombinant NK-2 receptor protein (or fragment or analog, thereof) is expressed, it is isolated, e.g. , using affinity chromatography. In one example, neurokinin A or an i-NK-2 receptor antibodies (described below) may be attached to a column and used to isolate intact receptor or receptor fragments or analogues. Lysis and fractionation of receptor-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra) . Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography.

Receptors of the invention, particularly short receptor fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed. , 1984, The Pierce Chemical Co., Rockford, IL) .

SCREENING FOR NK-2 RECEPTOR ANTAGONISTS

As discussed above, one aspect of the invention features screening for compounds that antagonize the interaction between neurokinin A and the NK-2 receptor, thereby preventing or reducing the cascade of events that are mediated by that interaction. The elements of the screen are the neurokinin A neurotransmitter (or a suitable receptor-binding fragment or analog thereof) and recombinant NK-2 receptor (or a suitable receptor fragment or analog, as outlined above) configured to permit detection of binding.

Neurokinin A (also known as Substance K) may be obtained from the Sigma Chemical Co. (St. Louis, MO) . Alternatively, it may be produced by standard methods of chemical synthesis or recombinant DNA technology given its known amino acid sequence: His-Lys-Thr-Asp-Ser-Phe- Val-Gly-Leu-Met-NH₂ (SEQ. ID NO.:17).

Preferably, the NK-2 receptor component is produced by a cell that naturally presents substantially no receptor, e.g., by engineering such a cell to contain nucleic acid encoding the receptor component in an appropriate expression system. Suitable cells are, e.g., those discussed above with respect to the production of recombinant receptor, most preferably mouse 3T3 fibroblast cells.

The assay is preferably performed by fixing the cell expressing the NK-2 receptor component to a solid substrate (e.g., a test tube or microtiter well) by means well known to those in the art, and presenting labelled neurokinin A or a fragment or analog thereof to the cell in the presence of the candidate antagonist. Binding is assayed by the detection label in association with the receptor component (and, therefore, in association with the solid substrate) . Molecules which specifically interfere with labelled neurokinin A binding are considered to be useful in the invention.

The assay format may be any of a number of suitable formats for detecting specific binding, such as a radioimmunoassay format. Preferably, cells transiently or stably transfected with an NK-2 receptor expression vector are immobilized on a microtiter plate and reacted with neurokinin A (or an active fragment or analog thereof) which is detectably labelled, e.g., with a radiolabel or an enzyme which can be assayed, e.g., alkaline phosphatase or horseradish peroxidase.

Alternatively, neurokinin A may be adhered to the microtiter plate (using methods similar to those for adhering antigens for an ELISA assay) and the ability of labelled NK-2 receptor expressing cells (e.g., labelled with ³H-thymidine) can be used to detect specific receptor binding to the immobilized neurokinin A.

In one particular example, a vector expressing the NK-2 receptor (or receptor fragment or analog) is transfected into C0S-7 cells (ATCC Accession No. CRL 1651) by the DEAE dextran-chloroquine method (Ausubel et al., supra) . Expression of the receptor protein confers binding of the neurokinin A neurotransmitter to the cells; neurokinin A does not bind to untransfected host cells or cells bearing the parent vector alone. 10 cm. tissue culture dishes are seeded with freshly trypsinized NK-2 receptor-expressing COS-7 cells (750,000 cells, dish) 12-18h post-transfection. Forty-eight hours later, triplicate dishes are incubated with 2nM radioiodinated neurokinin A (-300 cpm/fmol) . This is used as a "control" against which antagonist assays are measured. Such antagonist assays involve incubation of the NK-2 receptor-expressing cells with an equivalent amount of labelled neurokinin A in combination with an appropriate amount of candidate antagonist. An antagonist useful in the invention blocks labelled neurokinin A binding to the immobilized receptor-expressing cells.

Appropriate candidate antagonists include NK-2 receptor fragments, particularly fragments containing an extracellular domain (described above) ; such fragments would preferably including five or more amino acids.

Other candidate antagonists include non-peptide compounds designed or derived from analysis of the receptor, as well as anti-NK-2 receptor antibodies. ANTI-NK-2 RECEPTOR ANTIBODIES NK-2 receptor or receptor fragments or analogues (described above) may be used to raise antibodies by any of the conventional methods well known to those skilled in the art. For example, the cDNA sequence of the NK-2 receptor can be used to select short peptide sequences which can be synthesized (e.g., by chemical synthesis or recombinant DNA techniques) and used to immunize animals, e.g., rabbits, in order to generate antibodies. The antibodies may be polyclonal or monoclonal. Polyclonal antibodies may be enriched in anti-receptor activity, e.g., by column purification (i.e., by using receptor or receptor fragments or analogues immobilized on a column to screen out the desired antibody, see, e.g. , Ausubel et al. , supra) . Desired monoclonal antibody-producing hybridomas may also be selected by stimulating and then screening with receptor or receptor fragments or analogues, using standard immunological techniques (see, e.g., Ausubel et al., supra) . THERAPEUTICS AND VACCINES

Particularly suitable therapeutics are the antagonistic receptor fragments (described above) formulated in an appropriate buffer such as physiological saline. Where it is particularly desirable to mimic the receptor conformation at the membrane interface, the fragment may include transme brane residues adjacent to the extracellular domain of the receptor. In this case, the fragment may be associated with an appropriate lipid fraction (e.g., in lipid vesicles or attached to fragments obtained by disrupting a cell membrane) . Alternatively, anti-NK-2 receptor antibodies produced as described above may be used as a therapeutic. Again, the antibodies would be administered in a pharmaceutically- acceptable buffer (e.g., physiological saline). If appropriate, the antibody preparation may be combined with a suitable adjuvant. The therapeutic preparation is administered in accordance with the condition to be treated. Ordinarily, it will be administered intravenously, at a dosage that provides suitable competition for neurokinin A binding. Alternatively, it may be convenient to administer the therapeutic orally, nasally, or topically, e.g., as a liquid or a spray. Again, the dosage would be adjusted to provide suitable competition for neurokinin A binding. Treatment may be repeated as necessary for alleviation of disease symptoms. Because neurokinin A has been shown to have a spasmogenic effect on the smooth muscle cell-containing tissue of the gastrointestinal, respiratory, genitourinary, and vascular systems, NK-2 receptor antagonists may provide relief from respiratory diseases such as asthma and gastrointestinal diseases such as peptic ulcers and ulcerative colitis.

The antibodies of the invention, in a suitable buffer and, if appropriate, including an adjuvant, may also be used as a protective vaccine. Such a vaccine would be administered in a dosage that provides suitable competition for neurokinin A binding over the long-term. Such a vaccine would be useful, e.g., to individuals suffering from frequent asthma attacks. Other embodiments are within the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (1) APPLICANTS: Gerard, Norma P. Gerard, Craig

(ii) TITLE OF INVENTION: CONTROLLING NK-2 RECEPTOR- MEDIATED RESPONSES AND RELATED DIAGNOSTICS

(Hi) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: IBM P.C. DOS (Version 3.30)

(D) SOFTWARE: WordPerfect (Version 5.0)

(▼1) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/670,066

(B) FILING DATE: 15-Mar-1991

(▼iii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Freeman, John W.

(B) REGISTRATION NUMBER: Reg. No. 29,066

(C) REFERENCE/DOCKET NUMBER: 00108/063WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Phe Tyr Val lie

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 368

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Thr Cys Asp lie Val Thr Glu Ala Asn lie Ser Ser Gly Pro

5 10 15

Glu Ser Asn Thr Thr Gly lie Thr Ala Phe Ser Met Pro Ser Trp Gin 20 25 30

Leu Ala Leu Trp Ala Thr Ala Tyr Leu Ala Leu Val Leu Val Ala Val 35 40 45

Thr Gly Asn Ala lie Val lie Trp lie lie Leu Ala His Arg Arg Met 50 55 60

Arg Thr Val Thr Asn Tyr Phe lie Val Asn Leu Ala Leu Ala Asp Leu 65 70 75 80

Cys Met Ala Ala Phe Asn Ala Ala Phe Asn Phe Val Tyr Ala Ser His 85 90 95

Asn lie Trp Tyr Phe Gly Arg Ala Phe Cys Tyr Phe Gin Asn Leu Phe 100 105 110

Pro lie Thr Ala Met Phe Val Ser lie Tyr Ser Met Thr Ala lie Ala 115 120 125 Ala Asp Arg Tyr Met Ala He Val His Pro Phe Gin Pro Arg Leu Ser 130 135 140

Ala Pro Ser Thr Lye Ala Val He Ala Gly He Trp Leu Val Ala Leu 145 150 155 160

Ala Leu Ala Ser Pro Gin Cys Phe Tyr Ser Thr Val Thr Met Asp Gin 165 170 175

Gly Ala Thr Lys Cys Val Val Ala Trp Pro Glu Asp Ser Gly Gly Lys 180 185 190

Thr Leu Leu Leu Tyr His Leu Val Val He Ala Leu He Tyr Phe Leu 195 200 205

Pro Leu Ala Val Met Phe Val Ala Tyr Ser Val He Gly Leu Thr Leu 210 215 220

Trp Arg Arg Ala Val Pro Gly His Gin Ala His Gly Ala Asn Leu Arg 225 230 235 240

His Leu Gin Ala Lys Lys Lys Phe Val Lys Thr Met Val Leu Val Val 245 250 255

Leu Thr Phe Ala He Cys Trp Leu Pro Tyr His Leu Tyr Phe He Leu 230 235 240

Gly Ser Phe Gin Glu Asp He Tyr Cys His Lys Phe He Gin Gin Val 245 250 255

Tyr Leu Ala Leu Phe Trp Leu Ala Met Ser Ser Thr Met Tyr Asn Pro 260 265 270

He Thr Tyr Cys Cys Leu Asn His Arg Phe Arg Ser Gly Phe Arg Leu 275 280 285 290

Ala Phe Arg Cys Cys Pro Trp Val Thr Pro Thr Lys Glu Asp Lys Leu 295 300 305

Glu Leu Thr Pro Thr Thr Ser Leu Ser Thr Arg Val Asn Arg Cyβ His 310 315 320

Thr Lys Glu Thr Leu Phe Met Ala Gly Asp Thr Ala Pro Ser Glu Ala 325 330 335

Thr Ser Gly Glu Ala Gly Arg Pro Gin Asp Gly Ser Gly Leu Trp Phe 340 345 350

Gly Tyr Gly Leu Ala Ala Pro Thr Lys Thr His Val Glu He 355 360 365

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Gly Thr Cys Asp He Val Thr Glu Ala Asn He Ser Ser Gly Pro

5 10 15

Glu Ser Asn Thr Thr Gly He Thr Ala Phe Ser Met Pro Ser Trp 20 25 30

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Asn Phe Val Tyr Ala Ser His Asn He Trp Tyr Phe Gly Arg

5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Thr Val Thr Met Asp Gin Gly Ala Thr Lys Cys Val Val Ala Trp Pro

5 10 15

Glu Asp Ser Gly Gly Lys Thr Leu Leu Leu Tyr His 20 25

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 6: (i) SEQUENCE CHARACTERISTICS:

(A) .LENGTH: 17

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Gly Ser Phe Gin Glu Asp He Tyr Cys His Lys Phe He Gin Gin Val

5 10 15

Tyr

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 845

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCTCTGATCT GGAAGGTTTC CTGAATTACG TGACGAGAAA CCTGGGTTCG AGTCCTAACT 60

TGTCACCAAC TTCCTGAGTG ACCTGGGCTG GTCCCGTCCC CTCTTGGAAT CTCTGTCTTC 120

CATCTCTTCA GCGAAGGGGT TGATTTATAA GGGTGTTTTC TGCTCTGACA CTGTGATTTG 180

AATTCTGTGT TTCCACATGA TATTCGAGAA GTCTGGCCGG AAGGATGGAA TCTGAAATGA 240

CAATGGTTCT GGACTGGGCT TTGTGCTCAG CCCAGCTCAT CTTTGCCTGA GACCTAGGAG 300

TGGCCCCAGG CTCTCCTGAT GTGCCACCAC GCTTGGCATC TGCTCCTCTC CCTGCCCCCA 360

TATTCCCATG CTCTGAAGGG AGTTCTCTTT CATAGCAAAT CCGAGAGGAG CCGAGGAGCC 420

AGGTCCTTTG TTCCAGACCC AGAAGCAGCC 450

ATG GGG ACC TGT GAC ATT GTG ACT GAA GCC AAT ATC TCA TCT GGC CCT 498

Met Gly Thr Cys Asp He Val Thr Glu Ala Asn He Ser Ser Gly Pro

5 10 15

GAG AGC AAC ACC ACG GGC ATC ACA GCC TTC TCC ATG CCC AGC TGG GAG 546

Glu Ser Asn Thr Thr Gly He Thr Ala Phe Ser Met Pro Ser Trp Gin 20 25 30 CTG GCA CTG TGG GCC ACA GCC TAC CTG GCC CTG GTG CTG GTG GCC GTG 594

Leu Ala Leu Trp Ala Thr Ala Tyr Leu Ala Leu Val Leu Val Ala Val 35 40 45

ACG GGT AAT GCC ATC GTC ATC TGG ATC ATC CTG GCC CAT CGG AGG ATG 642

Thr Gly Asn Ala He Val He Trp He He Leu Ala His Arg Arg Met 50 55 60

CGC ACA GTC ACC AAC TAC TTC ATC GTC AAT CTG GCG CTG GCT GAC CTC 690

Arg Thr Val Thr Asn Tyr Phe He Val Asn Leu Ala Leu Ala Asp Leu 65 70 75 80

TGC ATG GCT GCC TTC .AAT GCC GCC TTC AAC TTT GTC TAT GCC AGC CAC 738

Cys Met Ala Ala Phe Asn Ala Ala Phe Asn Phe Val Tyr Ala Ser His 85 90 95

AAC ATC TGG TAC TTT GGC CGT GCC TTC TGC TAC TTC CAG AAC CTC TTC 786

Asn He Trp Tyr Phe Gly Arg Ala Phe Cys Tyr Phe Gin Asn Leu Phe 100 105 110

CCC ATC ACA GCC ATG TTT GTC AGC ATC TAC TCC ATG ACC GCC ATT GCT 834

Pro He Thr Ala Met Phe Val Ser He Tyr Ser Met Thr Ala He Ala 115 120 125

GCC GAC 836

Ala Asp 130

ACAGAGAGG 845

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 219

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CTGCCCTCTG CTCAC 15

AGG TAC ATG GCC ATC GTC CAC CCC TTC CAG CCT CGG CTT TCA GCT CCC 63 Arg Tyr Met Ala He Val His Pro Phe Gin Pro Arg Leu Ser Ala Pro

5 10 15 AGC ACC AAG GCG GTT ATT GCT GGC ATC TGG CTG GTG GCT CTC GCC CTG 111

Ser Thr Lys Ala Val He Ala Gly He Trp Leu Val Ala Leu Ala Leu 20 25 30

GCC TCC CCT CAG TGC TTC TAC TCC ACC GTC ACC ATG GAC CAG GGT GCC 159

Ala Ser Pro Gin Cys Phe Tyr Ser Thr Val Thr Met Asp Gin Gly Ala 35 40 45

ACC AAG TGC GTG GTG GCC TGG CCC GAA GAC AGC GGG GGC AAG ACG CTC 207

Thr Lys Cys Val Val Ala Trp Pro Glu Asp Ser Gly Gly Lys Thr Leu 50 55 60

CTC CTG 213

Leu Leu

65

TAAGCC 219

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 168

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CTCCCTGCAG 10

TAC CAC CTC GTG GTG ATC GCC CTC ATC TAC TTC CTG CCG CTC GCG GTG 58

Tyr His Leu Val Val He Ala Leu He Tyr Phe Leu Pro Leu Ala Val

5 10 15

ATG TTT GTA GCC TAC AGC GTC ATC GGC CTC ATG CTC TGG AGG CGC GCA 106

Met Phe Val Ala Tyr Ser Val He Gly Leu Thr Leu Trp Arg Arg Ala 20 25 30

GTG CCC GGA CAT CAG GCG CAC GGT GCC AAC CTC CGC CTA CTG CAG GCC 154

His Gly Ala Asn Leu Arg Leu Leu Gin Ala Val Pro Gly His Gin Ala 35 40 45

AAG AAG AAG 163

Lys Lys Lys 50

GTGGG 168 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 213

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CCTGGACCAG 10

TTT GTG AAG ACC ATG GTG CTG GTG GTG CTG ACG TTT GCC ATC TGC TGG 58 Phe Val Lys Thr Met Val Leu Val Val Leu Thr Phe Ala He Cys Trp

5 10 15

CTG CCC TAC CAC CTC TAC TTC ATC CTG GGC AGC TTC CAG GAG GAC ATC 106

Leu Pro Tyr His Leu Tyr Phe Leu Leu Gly Ser Phe Gin Glu Asp He 20 25 30

TAC TGC CAC AAG TTC ATC CAG CAA GTC TAC CTG GCA CTC TTC TGG TTG 154 Tyr Cys His Lys Phe He Gin Gin Val Tyr Leu Ala Leu Phe Trp Leu 35 40 45

GCC ATG AGC TCT ACC ATG TAC AAT CCC ATC ATC TAC TGC TGT CTC AAC 202 Ala Met Ser Ser Thr Met Tyr Asn Pro He He Tyr Cys Cys Leu Asn 50 55 60

CAC AGG 208

His Arg

65

TGAGC 213

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CGCTCCCCCA GG 12

TTT CGC TCT GGA TTC CGG CTT GCC TTC CGC TGC TGC CCA TGG GTC ACA 60

Phe Arg Ser Glu Phe Arg Leu Ala Phe Arg Cys Cys Pro Trp Val Thr

5 10 15

CCC ACC AAG GAA GAT AAG CTC GAG CTG ACT CCC ACG ACC TCC CTC TCC 108

Pro Thr Lys Glu Asp Lys Leu Glu Leu Thr Pro Thr Thr Ser Leu Ser 20 25 30

ACG AGA GTC AAC AGG TGT CAC ACT AAG GAG ACT TTG TTC ATG GCT GGG 156

Thr Arg Val Asn Arg Cys His Thr Lys Glu Thr Leu Phe Met Ala Gly 35 40 45

GAC ACA GCC CCC TCC GAG GCT ACC AGT GGG GAG GCG GGG CGT CCC CAG 204

Asp Thr Ala Pro Ser Glu Ala Thr Ser Gly Glu Ala Gly Arg Pro Gin 50 55 60

GAT GGA TCA GGG CTA TGG TTT GGG TAT GGT TTG CTT GCC CCC ACC AAA 252

Asp Gly Ser Gly Leu Trp Phe Gly Tyr Gly Leu Leu Ala Pro Thr Lys 65 70 75 80

ACT CAT GTT GAA ATT 300

Thr His Val Glu He 85

TGATCCCAAT GTGGCAGTGT TGGGCAGGTA GGGGTTAGTG GGAGGTGTTT GGGCTATTGC 360

AGGGATGCGA TCCCTTATGA ATAGATTAAC TGACCCTTCC AGTTGAAGTG AATCATCACT 420

CTC 423

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION:, SEQ ID NO: 12:

AATGAATTCT GGCAGCTGGC ACTGTGG 27 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AATGAATTCC CAGGCCACCA CGCACTT 27

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Cys Cys Leu Asn His Arg

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Cys Cys Pro Trp Val Thr

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Leu He Val He Ala Leu

5

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

is Lys Thr Asp Ser Phe Val Gly Leu Met

5 10

Claims

Claims 1. Human recombinant NK-2 receptor or a fragment thereof.

2. The receptor of claim 1 comprising an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 3 (SEQ ID NO.:2).

3. A substantially isolated polypeptide which is a fragment of a human NK-2 receptor comprising an extracellular domain capable of binding neurokinin A neurotransmitter.

4. A polypeptide selected from the group comprising: (a) MGTCDIVTEANISSGPESNTTGITAFSMPSW (SEQ. ID NO.:3; amino acid residues 1 to 31 of Fig. 3; SEQ ID NO.:2); (b) NFVYASHNIWYFGR (SEQ. ID NO.:4; amino acid residues 90 to 103 of Fig. 3; SEQ ID N0.:2); (c) TVTMDQGATKCWAWPEDSGGKTLLLYH (SEQ. ID NO.:5; amino acid residues 171 to 198 of Fig. 3; SEQ ID NO.:2); (d) GSFQEDIYCHKFIQQVY (SEQ. ID NO.:6; amino acid residues 273 to 289 of Fig. 3; SEQ ID NO.:2); and7 (e) fragments or analogues of (a) - (d) which are capable of binding a neurokinin A neurotransmitter.

5. The polypeptide of claim 4 further characterized in that said polypeptide is a recombinant polypeptide.

6. Purified DNA which encodes a receptor or fragment of claim 1 or a polypeptide of claims 2, 3, or 4.

7. A vector containing the DNA of claim 6, said vector being capable of directing expression of the protein encoded by said DNA in a vector-containing cell.

8. A cell which contains the vector of claim 7.

9. The cell of claim 8, said cell being a eukaryotic cell.

10. The cell of claim 9, said cell being a mammalian cell.

11. A method of producing recombinant NK-2 receptor protein or a fragment thereof comprising, providing a cell transformed with DNA encoding the NK- 2 receptor of a fragment thereof positioned for expression in said cell; culturing said transformed cell under conditions for expressing said DNA, and isolating said recombinant human NK-2 receptor protein.

12. A purified antibody which binds preferentially to a receptor of claim 1 or a polypeptide of claims 2 or 3.

13. The antibody of claim 12, wherein said antibody neutralizes the biological activity in vivo of a receptor of claim 1 or a polypeptide of claims 2 or 3.

14. A therapeutic composition comprising as an active ingredient recombinant receptor according to claim 1, a polypeptide according to claim 2 or 3, or an antibody according to claim 12 or 13, said active ingredient being formulated in a physiologically-acceptable carrier.

15. The therapeutic composition of claim 14, wherein said active ingredient comprises the recombinant NK-2 receptor of claim 1 or a polypeptide of claims 2 or 3 anchored within the membrane of a cell.

16. A method of treating asthma in a mammal comprising administering the therapeutic composition of claims 14 or 15 to said mammal in a dosage effective to antagonize an interaction between neurokinin A neurotransmitter and an NK-2 receptor.

17. A method of treating ulcerative colitis is a mammal comprising administering the therapeutic composition of claims 14 or 15 to said mammal in a dosage effective to antagonize an interaction between neurokinin A neurotransmitter and an NK-2 receptor.

18. A method of screening candidate compounds for the ability to antagonize interaction between a neurokinin A neurotransmitter and an NK-2 receptor, said method comprising: a) mixing a candidate antagonist compound with a first compound comprising the recombinant NK-2 receptor or fragment of claim 1, or the polypeptide of claims 2 or 3, or an antibody of claims 12 or 13 on the one hand and with a second compound comprising neurokinin A neurotransmitter or a fragment thereof capable of binding said first compound on the other hand; b) determining whether said first and second compounds bind; and c) identifying antagonistic compounds as those which interfere with the binding of the first compound to the second compound.