Patent Document

This is a continuation of application Ser. No. 07/851,974 filed on Mar. 16, 1992, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention concerns a cloned human neurokinin-3 receptor (hereinafter identified as human NK3R). 
     Neurokinin B (NKB) is a naturally occuring peptide belonging to the neurokinin family of peptides, which also includes substance P (SP) and substance K (SK). NKB binds preferentially to the neurokinin-3 receptor (NK3R), although it also recognizes the other two receptor subtypes (NK1 and NK2) with lower affinity. As is well known in the art, neurokinin B and other tachykinins have been implicated in the pathophysiology of numerous diseases. Neurokinin peptides are reportedly involved in nociception and neurogenic inflammation. The physiological function of NK3R has been implicated in the regulation of enkephalin release, while the NK1 and NK2 receptor subtypes are involved in synaptic transmission (Laneuville et al.,  Life Sci ., 42:1295-1305 (1988)). Since the NKB genomic structure and subcellular distribution are different from those of SP and SK, the physiological function and regulatory mechanism of NKB may be different from SP and SK. 
     More specifically, neurokinin B is a pharmacologically-active neuropeptide that is produced in mammals and possesses a characteristic amino acid sequence that is illustrated below: 
     Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. 
     Several groups have reported the cloning of certain neurokinin receptors. T. M. Fong, et al.,  Mol. Pharmacol ., 41:24-30 (1991) have reported cloned human neurokinin-l and neurokinin-1 short form receptor. J. Yokota, et al.,  J. Biol. Chem ., 264:17649 (1989) have reported cloned rat neurokinin-1 receptor. N. P. Gerard, et al.,  J. Biol. Chem ., 265:20455 (1990), have reported human neurokinin-2 receptor. Cloned rat and bovine neurokinin-2 receptor have likewise been reported. See respectively, Y. Sasi, and S. Nakanishi,  Biochem Biophys. Res. Comm ., 165:695 (1989), and Y. Masu, et al.,  Nature  329:836 (1987). Cloned rat neurokinin-3 receptor has been reported by R. Shigemoto, et al.,  J. Biol. Chem ., 265:623 (1990). The above references, however, neither disclose nor suggest the present invention. 
     The instant invention also concerns an assay protocol which can be used to determine the activity in body fluids of substances that bind human NK3R; these include neurokinin B. The assay can also be used for identifying and evaluating substances that bind NK3R. Thus, the assay can be used to identify neurokinin B antagonists and evaluate their binding affinity. Another method for an assay includes that described by M .A. Cascieri, et al.,  J. Biol. Chem ., 258:5158 (1983). See also, for example, R. M. Snider, et al.,  Science , 251:435 (1991) and S. McLean, et al.,  Science , 251:437 (1991). See also WIPO Patent Publications WO90/05525 and WO90/05729, published May 31, 1990. Methods to date have proven inferior, in part, for failure of the animal receptor (animal NK1R, NK2R or NK3R) activity to accurately reflect that of the human neurokinin-3 receptor. Furthermore, prior to this disclosure, human NK3R has not been available in a highly purified form or in substantial isolation from NK1R and/or NK2R. Use of such neurokinin receptor sources can not accurately depict the affinity of an agonist or an antagonist for a human NK3R. 
     SUMMARY OF THE INVENTION 
     A novel recombinant human neurokinin-3 receptor (hereinafter identified as human NK3R) is disclosed which has been prepared by polymerase chain reaction techniques. Also disclosed is the complete sequence of human NK3R complementary DNA; expression systems, including a CHO (chinese hamster ovarian cell line) stable expression system; and an assay using the CHO expression system. 
     Human NK3R can be used in an assay to identify and evaluate entities that bind neurokinin B receptor or NK3R. The assay can also be used in conjunction with diagnosis and therapy to determine the body fluid concentration of neurokinin-B related substances in patients. In addition, the complete sequence of the human NK3R is useful in the process of developing novel NK3 agonists and antagonists by computer modeling. 
     DETAILED DESCRIPTION OF THE INVENTION 
     One embodiment of the invention concerns human neurokinin-3 receptor, said receptor being free of other human receptor proteins. 
     In one class this first embodiment concerns human neurokinin-3 receptor, said receptor being free of other human proteins. 
     Within this class, this first embodiment concerns human neurokinin-3 receptor from human cells such as glioblastoma, said receptor being free of other human proteins. 
     Also within this class, this first embodiment concerns human neurokinin-3 receptor, the receptor being recombinantly produced from non-human cells. 
     In a second class, this first embodiment concerns a protein corresponding to the amino acid sequence of human neurokinin-3 receptor, the protein comprising 465 amino acids. Within the second class this first embodiment concerns a protein comprising the following 465 amino acid sequence (SEQ ID NO:1:) depicted from the amino to the carboxy terminus: 
     
       
         
               
             
           
               
                 Met Ala Thr Leu Pro Ala Ala Glu Thr Trp Ile Asp 
               
               
                 1               5                   10 
               
               
                   
               
               
                 Gly Gly Gly Gly Val Gly Ala Asp Ala Val Asn Leu 
               
               
                         15                    20 
               
               
                   
               
               
                 Thr Ala Ser Leu Ala Ala Gly Ala Ala Thr Gly Ala 
               
               
                 25                  30                  35 
               
               
                   
               
               
                 Val Glu Thr Gly Trp Leu Gln Leu Leu Asp Gln Ala 
               
               
                             40                  45 
               
               
                   
               
               
                 Gly Asn Leu Ser Ser Ser Pro Ser Ala Leu Gly Leu 
               
               
                     50                  55                  60 
               
               
                   
               
               
                 Pro Val Ala Ser Pro Ala Pro Ser Gln Pro Trp Ala 
               
               
                                 65                  70 
               
               
                   
               
               
                 Asn Leu Thr Asn Gln Phe Val Gln Pro Ser Trp Arg 
               
               
                         75                  80 
               
               
                   
               
               
                 Ile Ala Leu Trp Ser Leu Ala Tyr Gly Val Val Val 
               
               
                 85                  90                  95 
               
               
                   
               
               
                 Ala Val Ala Val Leu Gly Asn Leu Ile Val Ile Trp 
               
               
                             100                 105 
               
               
                   
               
               
                 Ile Ile Leu Ala His Lys Arg Met Arg Thr Val Thr 
               
               
                     110                 115                 120 
               
               
                   
               
               
                 Asn Tyr Phe Leu Val Asn Leu Ala Phe Ser Asp Ala 
               
               
                                 125                 130 
               
               
                   
               
               
                 Ser Met Ala Ala Phe Asn Thr Leu Val Asn Phe Ile 
               
               
                         135                 140 
               
               
                   
               
               
                 Tyr Ala Leu His Ser Glu Trp Tyr Phe Gly Ala Asn 
               
               
                 145                 150                 155 
               
               
                   
               
               
                 Tyr Cys Arg Phe Gln Asn Phe Phe Pro Ile Thr Ala 
               
               
                             160                 165 
               
               
                   
               
               
                 Val Phe Ala Ser Ile Tyr Ser Met Thr Ala Ile Ala 
               
               
                     170                 175                 180 
               
               
                   
               
               
                 Val Asp Arg Tyr Met Ala Ile Ile Asp Pro Leu Lys 
               
               
                                   185                  190 
               
               
                   
               
               
                 Pro Arg Leu Ser Ala Thr Ala Thr Lys Ile Val Ile 
               
               
                         195                 200 
               
               
                   
               
               
                 Gly Ser Ile Trp Ile Leu Ala Phe Leu Leu Ala Phe 
               
               
                 205                 210                 215 
               
               
                   
               
               
                 Pro Gln Cys Leu Tyr Ser Lys Thr Lys Val Met Pro 
               
               
                             220                 225 
               
               
                   
               
               
                 Gly Arg Thr Leu Cys Phe Val Gln Trp Pro Glu Gly 
               
               
                     230                 235                 240 
               
               
                   
               
               
                 Pro Lys Gln His Phe Thr Tyr His Ile Ile Val Ile 
               
               
                                  245                  250 
               
               
                   
               
               
                 Ile Leu Val Tyr Cys Phe Pro Leu Leu Ile Met Gly 
               
               
                         255                 260 
               
               
                   
               
               
                 Ile Thr Tyr Thr Ile Val Gly Ile Thr Leu Trp Gly 
               
               
                 265                 270                 275 
               
               
                   
               
               
                 Gly Glu Ile Pro Gly Asp Thr Cys Asp Lys Tyr His 
               
               
                         280                 285 
               
               
                   
               
               
                 Glu Gln Leu Lys Ala Lys Arg Lys Val Val Lys Met 
               
               
                     290                 295 
               
               
                   
               
               
                 Met Ile Ile Val Val Met Thr Phe Ala Ile Cys Trp 
               
               
                 300                 305                 310 
               
               
                   
               
               
                 Leu Pro Tyr His Ile Tyr Phe Ile Leu Thr Ala Ile 
               
               
                         315                  320 
               
               
                   
               
               
                 Tyr Gln Gln Leu Asn Arg Trp Lys Tyr Ile Gln Gln 
               
               
                 325                 330                 335 
               
               
                   
               
               
                 Val Tyr Leu Ala Ser Phe Trp Leu Ala Met Ser Ser 
               
               
                            10 340                  345 
               
               
                   
               
               
                 Thr Met Tyr Asn Pro Ile Ile Tyr Cys Cys Leu Asn 
               
               
                     350                 355                 360 
               
               
                   
               
               
                 Lys Arg Phe Arg Ala Gly Phe Lys Arg Ala Phe Arg 
               
               
                                 365                  370 
               
               
                   
               
               
                 Trp Cys Pro Phe Ile Lys Val Ser Ser Tyr Asp Glu 
               
               
                         375                  380 
               
               
                   
               
               
                 Leu Glu Leu Lys Thr Thr Arg Phe His Pro Asn Arg 
               
               
                 385                 390                 395 
               
               
                   
               
               
                 Gln Ser Ser Met Tyr Thr Val Thr Arg Met Glu Ser 
               
               
                             400                  405 
               
               
                   
               
               
                 Met Thr Val Val Phe Asp Pro Asn Asp Ala Asp Thr 
               
               
                     410                 415                 420 
               
               
                   
               
               
                 Thr Arg Ser Ser Arg Lys Lys Arg Ala Thr Pro Arg 
               
               
                                 425                  430 
               
               
                   
               
               
                 Asp Pro Ser Phe Asn Gly Cys Ser Arg Arg Asn Ser 
               
               
                         435                  400 
               
               
                   
               
               
                 Lys Ser Ala Ser Ala Thr Ser Ser Phe Ile Ser Ser 
               
               
                 445                 450                 455 
               
               
                   
               
               
                 Pro Tyr Thr Ser Val Asp Glu Tyr Ser 
               
               
                             460                  465. 
               
             
          
         
       
     
     Within the second class this first embodiment also concerns a protein comprising the foregoing amino acid sequence (SEQ ID:NO:1:), the protein being free of other human receptor proteins. 
     A second embodiment concerns a DNA sequence encoding the human neurokinin-3 receptor, the DNA sequence being free of other human DNA sequences. 
     As will be appreciated by those of skill in the art, there is a substantial amount of redundancy in the set of codons which translate specific amino acids. Accordingly, the invention also includes alternative base sequences wherein a codon (or codons) are replaced with another codon, such that the amino acid sequence translated by the DNA sequence remains unchanged. For purposes of this specification, a sequence bearing one or more such replaced codons will be defined as a degenerate variation. Also included are mutations (exchange of individual amino acids) which one of skill in the art would expect to have no effect on functionality, such as valine for leucine, arginine for lysine and asparagine for glutamine. 
     One class of the second embodiment of the invention concerns the following nucleotide sequence (SEQ ID NO:2:) of complementary DNA depicted from the 5′ to the 3′ terminus: 
     
       
         
               
               
               
             
           
               
                 CTATTGCAGT ATCTTTCAGC TTCCAGTCTT ATCTGAAGAC CCCGGCACCA AAGTGACCAG 
                 60 
                   
               
               
                   
               
               
                 GAGGCAGAGA AGAACTTCAG AGGAGTCTCG TCTTGGGCTG CCCGTGGGTG AGTGGGAGGG 
                 120 
               
               
                   
               
               
                 TCCGGGACTG CAGACCGGTG GCGATGGCCA CTCTCCCAGC AGCAGAAACC TGGATAGACG 
                 180 
               
               
                   
               
               
                 GGGGTGGAGG CGTGGGTGCA GACGCCGTGA ACCTGACCGC CTCGCTAGCT GCCGGGGCGG 
                 240 
               
               
                   
               
               
                 CCACGGGGGC AGTTGAGACT GGGTGGCTGC AACTGCTGGA CCAAGCTGGC AACCTCTCCT 
                 300 
               
               
                   
               
               
                 CCTCCCCTTC CGCGCTGGGA CTGCCTGTGG CTTCCCCCGC GCCCTCCCAG CCCTGGGCCA 
                 360 
               
               
                   
               
               
                 ACCTCACCAA CCAGTTCGTG CAGCCGTCCT GGCGCATCGC GCTCTGGTCC CTGGCGTATG 
                 420 
               
               
                   
               
               
                 GTGTGGTGGT GGCAGTGGCA GTTTTGGGAA ATCTCATCGT CATCTGGATC ATCCTGGCCC 
                 480 
               
               
                   
               
               
                 ACAAGCGCAT GAGGACTGTC ACCAACTACT TCCTTGTGAA CCTGGCTTTC TCCGACGCCT 
                 540 
               
               
                   
               
               
                 CCATGGCCGC CTTCAACACG TTGGTCAATT TCATCTACGC GCTTCATAGC GAGTGGTACT 
                 600 
               
               
                   
               
               
                 TTGGCGCCAA CTACTGCCGC TTCCAGAACT TCTTTCCTAT CACAGCTGTG TTCGCCAGCA 
                 660 
               
               
                   
               
               
                 TCTACTGCAT GACGGCCATT GCGGTGGACA GGTATATGGC TATTATTGAT CCCTTGAAAC 
                 720 
               
               
                   
               
               
                 CCAGACTGTC TGCTACAGCA ACCAAGATTG TCATTGGAAG TATTTGGATT CTAGCATTTC 
                 780 
               
               
                   
               
               
                 TACTTGCCTT CCCTCAGTGT CTTTATTCCA AAACGAAAGT CATGCCAGGC CGTACTCTCT 
                 840 
               
               
                   
               
               
                 GCTTTGTGCA ATGGCCAGAA GGTCCCAAAC AACATTTCAC TTACCATATT ATCGTCATTA 
                 900 
               
               
                   
               
               
                 TACTGGTGTA CTGTTTCCCA TTGCTCATCA TGGGTATTAC ATACACCATT 
                 950 
               
               
                   
               
               
                 GTTGGAATTA CTCTCTGGGG AGGAGAAATC CCAGGAGATA CCTGTGACAA GTATCATGAG 
                 1010 
               
               
                   
               
               
                 CAGCTAAAGG CCAAAAGAAA GGTTGTCAAA ATGATGATTA TTGTTGTCAT GACATTTGCT 
                 1070 
               
               
                   
               
               
                 ATCTGCTGGC TGCCCTATCA TATTTACTTC ATTCTCACTG CAATCTATCA ACAACTAAAT 
                 1130 
               
               
                   
               
               
                 AGATGGAAAT ACATCCAGCA GGTCTACCTG GCTAGCTTTT GGCTGGCAAT GAGCTCAACC 
                 1190 
               
               
                   
               
               
                 ATGTACAATC CCATCATCTA CTGCTGTCTG AATAAAAGAT TTCGAGCTGG CTTCAAGAGA 
                 1250 
               
               
                   
               
               
                 GCATTTCGCT GGTGTCCTTT CATCAAAGTT TCGAGCTATG ATGAGCTAGA GCTCAAGACC 
                 1310 
               
               
                   
               
               
                 ACCAGGTTTC ATCCAAACCG GCAAAGCAGT ATGTACACCG TGACCAGAAT GGAGTCCATG 
                 1370 
               
               
                   
               
               
                 ACAGTCGTGT TTGACCCCAA CGATGCAGAC ACCACCAGGT CCAGTCGGAA GAAAAGAGCA 
                 1430 
               
               
                   
               
               
                 ACGCCAAGAG ACCCAAGTTT CAATGGCTGC TCTCGCAGGA ATTCCAAATC TGCCTCCGCC 
                 1490 
               
               
                   
               
               
                 ACTTCAAGTT TCATAAGCTC ACCCTATACC TCTGTGGATG AATATTCTTA ATTCCATTTC 
                 1550 
               
               
                   
               
               
                 CTGAGGTAAA AGATTAGTGT GAGACCATCA TGGTGCCAGT CTAGGACCCC ATTCTCCTAT 
                 1610 
               
               
                   
               
               
                 TTATCAGTCC TGTCCTATAT ACCCTCTAGA AACAGAAAGC AATTTTTAGG CAGCTATGGT 
                 1670 
               
               
                   
               
               
                 CAAATTGAGA AAGGTAGTGT ATAAATGTGA CAAAGACACT AATAACATGT TAGCCTCCAC 
                 1730 
               
               
                   
               
               
                 CCAAAATAAA ATGGGCTTTA AATTT 
                 1755 
               
             
          
         
       
     
     or a degenerate variation thereof. 
     A third embodiment of this invention concerns systems for expressing all or part of the human neurokinin-3 receptor. 
     One class this third embodiment of the invention comprises: 
     A plasmid which comprises: 
     (a) a mammalian expression vector, such as pcDNAI/Neo, and 
     (b) a base sequence encoding human neurokinin-3 receptor protein. 
     Within this first class of the third embodiment the neurokinin-3 receptor comprises the nucleotide sequence (SEQ ID NO:2:) of complementary DNA as shown above. 
     A second class of this third embodiment of the invention concerns a system for the transient expression of human neurokinin-3 receptor in a monkey kidney cell line (COS), the system comprised of a vector which expresses human neurokinin receptor (human NK3R) cDNA. 
     Within this second class of the third embodiment is the sub-class wherein the expression system includes: 
     A plasmid which comprises: 
     (a) a mammalian expression vector, such as pcDNAI/Neo, and 
     (b) a base sequence encoding human neurokinin-3 receptor protein. 
     A third class of this third embodiment of the invention concerns a system for the expression of human neurokinin-3 receptor in a chinese hamster ovarian cell line (CHO), the. system comprising a vector comprising human neurokinin-3 receptor (human NK3R) cDNA. 
     Within this third class of the third embodiment is the sub-class wherein the expression system includes: 
     A plasmid which comprises: 
     (a) a mammalian expression vector, such as pcNDAI/Neo and 
     (b) a base sequence encoding human neurokinin-3 receptor protein. 
     Within this sub-class the neurokinin-3 receptor expression system comprises the nucleotide sequence (SEQ ID NO:2:) of complementary DNA as shown above. 
     It is understood, and is readily apparent to those skilled in the art that a wide variety of commonly used cell lines are suitable for use in the present invention. Suitable cell lines derived from various species include, but are not limited to, cell lines of human, bovine, porcine, monkey, and rodent origin, or from yeast and bacterial strains. 
     A fourth embodiment of the invention concerns a method of using any of the above expression systems for determining the binding affinity of a test sample for human neurokinin-3 receptor. 
     In one class this fourth embodiment concerns a method of using a Chinese hamster ovarian cell line (CHO), the line transplanted with a plasmid, 
     which plasmid comprises: 
     (a) a mammalian expression vector, such as pcDNAI/Neo, and 
     (b) a base sequence encoding human neurokinin-3 receptor protein, the method which comprises: 
     (1) expressing human neurokinin-3 receptor in the CHO cells; 
     (2) adding of a test sample to a solution containing  125 I-eledoisin and the CHO cells; 
     (3) incubating the products of Step (2), the incubation being effective for competitive binding of the  125 I-eledoisin and said test sample to the human neurokinin-3 receptor; 
     (4) separating the  125 I-eledoisin which is bound to the human neurokinin-3 receptor from the  125 I-eledoisin which is not bound; 
     (5) measuring the amount of the  125 I-eledoisin which is bound to the human neurokinin-3 receptor. 
     In a second class this fourth embodiment concerns a method of using a monkey kidney cell line (COS), the line transplanted with a plasmid,. 
     which plasmid comprises: 
     (a) a mammalian expression vector, such as pcDNAI/Neo, and 
     (b) a base sequence encoding human neurokinin-3 receptor protein, the method which comprises: 
     (1) expressing human neurokinin-3 receptor in the COS cells; 
     (2) adding of a test sample to a solution containing  125 I-eledoisin and the COS cells; 
     (3) incubating the products of Step (2), the incubation being effective for competitive binding of the  125 I-eledoisin and said test sample to the human neurokinin-3 receptor; 
     (4) separating the  125 I-eledoisin which is bound to the human neurokinin-3 receptor from the  125 I-eledoisin which is not bound; 
     (5) measuring the amount of the  125 I-eledoisin which is bound to the human neurokinin-3 receptor. 
     In a third class this fourth embodiment concerns a method of using a Chinese hamster ovarian cell line (CHO), the line transplanted with a plasmid, 
     which plasmid comprises: 
     (a) a mammalian expression vector, such as pcDNAI/Neo, and 
     (b) the base sequence encoding human neurokinin-3 receptor protein, the method which comprises: 
     (1) expressing human neurokinin-3 receptor in the CHO cells; 
     (2) equilibrating the product of Step (1) with  3 H-myoinositol; 
     (3) washing the product of Step (2); 
     (4) incubating the product of Step (3) with a test sample and neurokinin-B in the presence of aqueous LiCl, resulting in the production of  3 H-inositol monophosphate; 
     (5) measuring the  3 H-inositol monophosphate. 
     In overview, the present invention describes methods to isolate the human neurokinin-3 receptor (human NK3R) complementary DNA (cDNA) without prior knowledge of its protein sequence or gene sequence. A polymerase chain reaction (PCR) technique was utilized for the isolation of human NK3R cDNA. In the approach, the regions of rat NK3R sequence thought to be similar to human NK3R were identified, oligonucleotide primers corresponding to those region were designed, PCR amplification was carried out to obtain a partial clone of the NK3R cDNA from human cells, and its DNA sequence was determined. The full length cDNA encoding the human NK3R was obtained from human mRNA utilizing the previous sequence information. 
     The complete sequence of the human NK3R CDNA was determined,.and its encoded protein sequence was deduced. Among other things, such sequence information is useful in the process of developing novel neurokinin B antagonists. 
     Three heterologous expression systems were developed to express the cloned human NK3R cDNA. The Xenopus oocyte expression enables one to determine the biological function of human NK3R . The COS (a monkey kidney cell line) expression can be used to measure the ligand binding properties of human NK3R. The CHO (a Chinese hamster ovarian cell line) stable expression is suitable for natural product screen to identify potential therapeutic agents or other substances that bind to neurokinin-3 receptor or human NK3R. The cell line can also be used for determining the concentration of neurokinin B in human samples. 
     Assay protocols were developed to use the heterologously expressed human NK3R for the. determination of the binding affinity and efficacy of neurokinin B agonists/antagonists with therapeutic potential. 
     The following examples are given for the purpose of illustrating the present invention and shall not be construed as being limitations on the scope or spirit of the instant invention. 
    
    
     EXAMPLE 1 
     Isolation of human NK3R cDNA 
     To isolate the human NK3R cDNA in the absence of its sequence information, we developed methods to obtain three separate but overlapping cDNA clones in three steps. (i) We have adopted the homologous cloning strategy (Ohara et al.,  Proc. Nat. Acad. Sci ., 86:5673-5677 (1989)) to isolate cDNA clones encoding the central core region of human NK3R, with the assumption that the human NK3R sequence is similar to the published sequence (Shigemoto et al.,  J. Biol. Chem ., 265:623-628 (1990)) of rat NK3R in certain areas where appropriate PCR primers can be designed. Degenerate primers corresponding to the rat sequence were used in PCR amplification (Mullis and Faloona,  Meth. Enzymol ., 155:335 (1987)) to obtain the cDNA encoding the central transmembrane core region of human NK3R from human mRNA. (ii) After determining the sequence of the core region in human NK3R, new primers corresponding to the human sequence were designed and anchored PCR amplification (Frohman, et al.,  Proc. Nat. Acad. Sci ., 85: 8998-9002 (1988)) was performed using the human primer in the core region. The cDNA encoding the N-terminal region of human NK3R was thus obtained from human mRNA and its sequence was determined. (iii) An anchored PCR strategy was also used to isolate the C-terminal region of human NK3R. To confirm the authenticity of the cDNA encoding human NK3R, an independent PCR amplification was performed to obtain the full length cDNA in a single step using primers from the 5′ and 3′ untranslated regions. 
     A cDNA clone encoding the central region of human NK3 receptor was obtained from human brain mRNA by PCR using degenerate primers based on the rat NK3 receptor sequence. The cDNA synthesis was initiated by the primer “ca” (SEQ ID NO:3:) 
     GGATCCTCRTCRTAGCTGGANAC using reverse transcriptase from BRL (Gaithersburg, Md.). Primary PCR was performed at 50° C. annealing temperature using the cDNA as template and primer “cb” (SEQ ID NO:4:) 
     TTTTGGATCCACTTGGATRAANGGRCA and primer “na” (SEQ ID NO:5:) 
     TTTTGGATCCTTCGTGCAGCCGTCCTGGCG 
     The following basic PCR conditions were used in all PCR experiments: 94° C. denaturation, 72° C. extension and 30 cycles. Secondary PCR was performed using the primary PCR product as template and the primer “cc” (SEQ ID NO:6:) 
     ATATGGATCCGACAGCAGCGAAATGCTCT and primer “nb” (SEQ ID NO:7:) 
     TTTTGAATTCTATGGCTTGGTGGTGGC under the same PCR conditions. A 900 bp cDNA fragment was obtained and sequenced, which was found to encode the central region of human NK3R. 
     A cDNA clone encoding the C-terminal region and 3′ untranslated region of human NK3 receptor was obtained by anchored PCR using sense primers derived from the partial clone described above. The cDNA synthesis was initiated by the oligo-dT primer “notldt” (SEQ ID NO:8:) 
     TTTTGCGGCCGCTTTTTTTTTTTTTTTTT 
     It was followed by a tailing reaction using terminal deoxynucleotide transferase (Promega, Madison, Wis.) to add a poly(A) tail to the 3′ end of the cDNA. Primary PCR was carried out using the cDNA as template and the primers “notldt” and “s1068” (SEQ ID NO:9:) 
     AATTGGATCCTAGATGGAAATACATCCAGC at 55° C. annealing temperature. Secondary PCR was carried out using the primary PCR product as template and the primers “notldt” and “s1106” (SEQ ID NO:10:) 
     AATTGGATCCTTGGCTGGCAATGAGCTCA under the same conditions. Tertiary PCR was carried out using the secondary PCR product as template and the primers “notldt” and “s1137” (SEQ ID NO:11:) 
     AATTGGATCCTCCCATCATCTACTGCTGTC under the same conditions. A 600 bp cDNA fragment was obtained and sequenced, which encodes the C-terminal region of human NK3R and 3′ untranslated region. 
     A cDNA clone encoding the N-terminal region and 5′ untranslated region of human NK3 receptor was obtained by anchored PCR using antisense primers derived from the partial clone encoding the central region of human NK3R. The cDNA synthesis was initiated using the primers “a475”(SEQ ID NO:12:) 
     TGGCGAACACAGCTGTGATA and “a400” (SEQ ID NO:13:) 
     AGCGCGTAGATGAAATTGAC 
     A poly(A) tail was then added to the 3′ end of the cDNA. Primary PCR was performed using the cDNA as template and the primers “not1dt” and “a351” (SEQ ID NO:14:) 
     AATTGCGGCCGCCGGAGAAAGCCAGGTTCACA at 60° C. annealing temperature. Secondary PCR was performed using the primary PCR product as template and the primers “not1dt” and “a332” (SEQ ID NO:15:) 
     AATTGCGGCCGCAGGAAGTAGTTGGTGACAGTC under the same conditions. A 600 bp cDNA fragment was obtained and sequenced, which encodes the N-terminal region of human NK3R and 5′ untranslated region. 
     To confirm the authenticity of the human NK3R cDNA sequence, an independent PCR was carried out to obtain the full length cDNA using primers based on the 5′ and 3′ untranslated regions. The cDNA was initiated using the primers “c1” (SEQ ID NO:16:) 
     AATTGCGGCCGCGACAGGACTGATAAATAGGAG and “c2” (SEQ ID NO:17:) 
     AATTGCGGCCGCCATGATGGTCTCACACTAATC 
     Primary PCR was performed using the cDNA as template and using the primers “c2” and “s50” (SEQ ID NO:18:) 
     AAAGTGACCAGGAGGCAGAGA at 60° C. annealing temperature. Secondary PCR was performed using the primary PCR product as template and the primers “c3” (SEQ ID NO:19:) 
     AATTGCGGCCGCACCTCAGGAAATGGAATTAAG and “s71” (SEQ ID NO:20:) 
     AATTGGATCCAGAACTTCAGAGGAGTCTCG under the same conditions. A 1500 bp cDNA fragment was obtained and its sequence was consistent with the previous partial clones. 
     EXAMPLE 2 
     Expression of the Cloned Human NK3R 
     Three expression systems were developed for the cloned human NK3R. An transient expression in Xenopus oocytes resulted from microinjection of in vitro transcribed mRNA from the cloned cDNA (Xenopus Laevis from XENOPUS ONE, Ann Arbor, Mich.). This system allows the measurement of biological effect of NK3R activation upon ligand binding. Another transient expression in COS (a monkey kidney cell line, ATCC CRL 1651, ATCC Rockville Md.) resulted from the transfection of the cloned cDNA under the control of viral promoter into mammalian cells (e.g., COS). The transfected cells are suitable for determination of the binding affinity of human NK3R for various ligands. Stable expression of human NK3R in mammalian cells (e.g., CHO, a Chinese hamster ovarian cell line, ATCC CRL 9096, ATCC Rockville Md.) was achieved after integration of the transfected cDNA into the chromosomes of the host cells. These stable cell lines will constituently express the cloned human NK3R and can be propagated infinitely. Therefore, a stable expression system is very useful in large scale drug screening, and can be used to determine the concentration of neurokinin-B related substances in biopsy samples of patients. 
     To express the cloned human NK3R, the full length cDNA of human NK3 receptor was subcloned into the expression vector pcDNA-Neo (Invitrogen, San Diego, Calif.). Transient expression in COS cells was achieved by electroporation using the IBI GeneZapper (IBI, New Haven, Conn.). The transfected cells were incubated in 10% fetal calf serum, 100 U/ml penicillin-streptomycin, and 90% DMEM media (Gibco, Grand Island, N.Y.) in a 5% CO 2  incubator at 37° C. for three days before the binding assay. 
     To establish a stable cell line expressing the cloned human NK3R, the cDNA in the expression vector pcDNA-Neo was transfected into CHO cells by electroporation. The transfected cells were incubated in the selection media (10% fetal calf serum, 100 U/ml penicillin-streptomycin, 1/500 hypoxanthine-thymidine, 90% IMDM media (JRH Biosciences, Lenexa, KS), 0.7 mg/ml neomycin) in a 5% CO 2  incubator until colonies were visible. Each colony was separated and propagated to maintain stable cell lines. 
     Both the COS expression and CHO expression allow the determination of binding affinity of various agonists and antagonists at the human NK3R. 
     The cloned human NK3R was expressed in Xenopus oocytes to demonstrate the biological function of human NK3 receptor as an activator of the second messenger inositol trisphosphate. The in vitro MRNA transcript was synthesized from the cDNA in pcDNA-Neo using T7 RNA polymerase (Stratagene, San Diego, Calif.) and injected into Xenopus oocytes. The oocytes were incubated at 19° C. for two days before electrophysiological assay. 
     EXAMPLE 3 
     Assays 
     The binding assay of human NK3R expressed in COS cells or CHO cells is based on the use of  125 I-Bolton Hunter labeled eledoisin (  125 I-BHE, from Du Pont, Boston, Mass.) (or  125 I-NKB) as a radioactively labeled ligand which compete with unlabeled neurokinin peptides or any other ligand for binding to the human NK3R. Monolayer cell culture of COS or CHO was dissociated by the non-enzymatic solution (Specialty Media, Lavallette, N.J.) and resuspended in appropriate volume of the binding buffer (50 mM Tris pH 7.5, 5 mM MnCl 2 , 150 mM NaCl, 0.04 mg/ml bacitracin, 0.004 mg/ml leupeptin, 0.2 mg/ml BSA, 0.01 mM phosphoramidon) such that 0.2 ml of the cell suspension would give rise to about 10,000 cpm of specific  125 I-BHE binding (approximately 50,000 to 200,000 cells). In the binding assay, 0.2 ml of cells were added to a tube containing 0.02 ml of 2.5 nM of  125 I-BHE and 0.02 ml of unlabeled test compound. The tubes were incubated at 4° C. for 1 hour with gentle shaking. The bound radioactivity was separated from unbound radioactivity by GF/C filter (Brandel, Gaithersburg, Md.) which was pre-wetted with 0.1% polyethylenimine. The filter was washed with 3 ml of wash buffer (50 mM Tris pH 7.5, 5 mM MnCl 2 , 150 mM NaCl) three times and its radioactivity was determined by gamma counter. 
     The electrophysiological assay of human NK3R expressed in Xenopus oocytes was based on the fact that NK3R activates the phospholipase C upon agonist binding, and phospholipase C in turn increases the intracellular calcium concentration through inositol trisphosphate (IP 3 ) and IP 3 -gated calcium channel on intracellular membranes. The calcium increase activates calcium-gated chloride channels on plasma membranes which gives rise to a chloride current measurable by two electrode voltage clamp. 
     The oocyte was voltage-clamped at −80 mV by the model 8500 intracellular preamp-clamp (Dagan, Minneapolis, Minn.). The recoding chamber was continuously perfused with recording buffer (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.4). Chloride current was elicited by applying agonist (from 0.1 nM to 1000 nM) to the recording chamber. At least three oocytes were measured for each concentration. The antagonistic activity of any potential NK3 antagonist can be assessed by determining the inhibition of neurokinin B response. Likewise, NK3 agonists can be identified by their ability to stimulate a response in oocytes injected with NK3R mRNA but not in uninjected oocytes. 
     The activation of phospholipase C by the human NK3R may also be measured in CHO cells by determining the accumulation of inositol monophosphate which is a degradation product of IP 3 . CHO cells are seeded in 12-well plate at 250,000 cells per well. After incubating in CHO media for 4 days, cells are loaded with 0.025 mCi/ml of  3 H-myoinositol by overnight incubation. The extracellular radioactivity is removed by washing with phosphate buffered saline. LiCl is added to the well at final concentration of 0.1 mM with or without antagonist, and continued incubation at 37° C. for 15 min. Neurokinin B is added to the well at final concentration of 0.3 nM to activate the human NK3R. After 30 min of incubation at 37° C., the media is removed and 0.1 N HCl is added. Each well is sonicated at 4° C. and extracted with CHCl 3 /methanol (1:1). The aqueous phase is applied to a 1 ml Dowex AG 1×8 ion exchange column. The column is washed with 0.1 N formic acid followed by 0.025 M ammonium formate-0.1 N formic acid. The inositol monophosphate is eluted with 0.2 M ammonium formate-0.1 N formic acid and quantitated by beta counter. 
     In addition to large scale drug screening using the stable CHO cell line expressing the cloned human NK3R, other alternative applications are obvious. For example, the stable cell line can be used in an assay to determine the neurokinin B concentration in human samples. 
     While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the casual variations, adaptations, modifications, deletions, or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents. 
     
       
         
           
             20 
           
           
             
               465 amino acids 
               amino acid 
               single 
               linear 
             
             
               protein 
             
             
               not provided 
             
             1
Met Ala Thr Leu Pro Ala Ala Glu Thr Trp Ile Asp Gly Gly Gly Gly
1               5                   10                  15
Val Gly Ala Asp Ala Val Asn Leu Thr Ala Ser Leu Ala Ala Gly Ala
            20                  25                  30
Ala Thr Gly Ala Val Glu Thr Gly Trp Leu Gln Leu Leu Asp Gln Ala
        35                  40                  45
Gly Asn Leu Ser Ser Ser Pro Ser Ala Leu Gly Leu Pro Val Ala Ser
    50                  55                  60
Pro Ala Pro Ser Gln Pro Trp Ala Asn Leu Thr Asn Gln Phe Val Gln
65                  70                  75                  80
Pro Ser Trp Arg Ile Ala Leu Trp Ser Leu Ala Tyr Gly Val Val Val
                85                  90                  95
Ala Val Ala Val Leu Gly Asn Leu Ile Val Ile Trp Ile Ile Leu Ala
            100                 105                 110
His Lys Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Leu Ala
        115                 120                 125
Phe Ser Asp Ala Ser Met Ala Ala Phe Asn Thr Leu Val Asn Phe Ile
    130                 135                 140
Tyr Ala Leu His Ser Glu Trp Tyr Phe Gly Ala Asn Tyr Cys Arg Phe
145                 150                 155                 160
Gln Asn Phe Phe Pro Ile Thr Ala Val Phe Ala Ser Ile Tyr Ser Met
                165                 170                 175
Thr Ala Ile Ala Val Asp Arg Tyr Met Ala Ile Ile Asp Pro Leu Lys
            180                 185                 190
Pro Arg Leu Ser Ala Thr Ala Thr Lys Ile Val Ile Gly Ser Ile Trp
        195                 200                 205
Ile Leu Ala Phe Leu Leu Ala Phe Pro Gln Cys Leu Tyr Ser Lys Thr
    210                 215                 220
Lys Val Met Pro Gly Arg Thr Leu Cys Phe Val Gln Trp Pro Glu Gly
225                 230                 235                 240
Pro Lys Gln His Phe Thr Tyr His Ile Ile Val Ile Ile Leu Val Tyr
                245                 250                 255
Cys Phe Pro Leu Leu Ile Met Gly Ile Thr Tyr Thr Ile Val Gly Ile
            260                 265                 270
Thr Leu Trp Gly Gly Glu Ile Pro Gly Asp Thr Cys Asp Lys Tyr His
        275                 280                 285
Glu Gln Leu Lys Ala Lys Arg Lys Val Val Lys Met Met Ile Ile Val
    290                 295                 300
Val Met Thr Phe Ala Ile Cys Trp Leu Pro Tyr His Ile Tyr Phe Ile
305                 310                 315                 320
Leu Thr Ala Ile Tyr Gln Gln Leu Asn Arg Trp Lys Tyr Ile Gln Gln
                325                 330                 335
Val Tyr Leu Ala Ser Phe Trp Leu Ala Met Ser Ser Thr Met Tyr Asn
            340                 345                 350
Pro Ile Ile Tyr Cys Cys Leu Asn Lys Arg Phe Arg Ala Gly Phe Lys
        355                 360                 365
Arg Ala Phe Arg Trp Cys Pro Phe Ile Lys Val Ser Ser Tyr Asp Glu
    370                 375                 380
Leu Glu Leu Lys Thr Thr Arg Phe His Pro Asn Arg Gln Ser Ser Met
385                 390                 395                 400
Tyr Thr Val Thr Arg Met Glu Ser Met Thr Val Val Phe Asp Pro Asn
                405                 410                 415
Asp Ala Asp Thr Thr Arg Ser Ser Arg Lys Lys Arg Ala Thr Pro Arg
            420                 425                 430
Asp Pro Ser Phe Asn Gly Cys Ser Arg Arg Asn Ser Lys Ser Ala Ser
        435                 440                 445
Ala Thr Ser Ser Phe Ile Ser Ser Pro Tyr Thr Ser Val Asp Glu Tyr
    450                 455                 460
Ser
465 
           
           
             
               1755 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             2
CTATTGCAGT ATCTTTCAGC TTCCAGTCTT ATCTGAAGAC CCCGGCACCA AAGTGACCAG     60
GAGGCAGAGA AGAACTTCAG AGGAGTCTCG TCTTGGGCTG CCCGTGGGTG AGTGGGAGGG    120
TCCGGGACTG CAGACCGGTG GCGATGGCCA CTCTCCCAGC AGCAGAAACC TGGATAGACG    180
GGGGTGGAGG CGTGGGTGCA GACGCCGTGA ACCTGACCGC CTCGCTAGCT GCCGGGGCGG    240
CCACGGGGGC AGTTGAGACT GGGTGGCTGC AACTGCTGGA CCAAGCTGGC AACCTCTCCT    300
CCTCCCCTTC CGCGCTGGGA CTGCCTGTGG CTTCCCCCGC GCCCTCCCAG CCCTGGGCCA    360
ACCTCACCAA CCAGTTCGTG CAGCCGTCCT GGCGCATCGC GCTCTGGTCC CTGGCGTATG    420
GTGTGGTGGT GGCAGTGGCA GTTTTGGGAA ATCTCATCGT CATCTGGATC ATCCTGGCCC    480
ACAAGCGCAT GAGGACTGTC ACCAACTACT TCCTTGTGAA CCTGGCTTTC TCCGACGCCT    540
CCATGGCCGC CTTCAACACG TTGGTCAATT TCATCTACGC GCTTCATAGC GAGTGGTACT    600
TTGGCGCCAA CTACTGCCGC TTCCAGAACT TCTTTCCTAT CACAGCTGTG TTCGCCAGCA    660
TCTACTCCAT GACGGCCATT GCGGTGGACA GGTATATGGC TATTATTGAT CCCTTGAAAC    720
CCAGACTGTC TGCTACAGCA ACCAAGATTG TCATTGGAAG TATTTGGATT CTAGCATTTC    780
TACTTGCCTT CCCTCAGTGT CTTTATTCCA AAACCAAAGT CATGCCAGGC CGTACTCTCT    840
GCTTTGTGCA ATGGCCAGAA GGTCCCAAAC AACATTTCAC TTACCATATT ATCGTCATTA    900
TACTGGTGTA CTGTTTCCCA TTGCTCATCA TGGGTATTAC ATACACCATT               950
GTTGGAATTA CTCTCTGGGG AGGAGAAATC CCAGGAGATA CCTGTGACAA GTATCATGAG   1010
CAGCTAAAGG CCAAAAGAAA GGTTGTCAAA ATGATGATTA TTGTTGTCAT GACATTTGCT   1070
ATCTGCTGGC TGCCCTATCA TATTTACTTC ATTCTCACTG CAATCTATCA ACAACTAAAT   1130
AGATGGAAAT ACATCCAGCA GGTCTACCTG GCTAGCTTTT GGCTGGCAAT GAGCTCAACC   1190
ATGTACAATC CCATCATCTA CTGCTGTCTG AATAAAAGAT TTCGAGCTGG CTTCAAGAGA   1250
GCATTTCGCT GGTGTCCTTT CATCAAAGTT TCCAGCTATG ATGAGCTAGA GCTCAAGACC   1310
ACCAGGTTTC ATCCAAACCG GCAAAGCAGT ATGTACACCG TGACCAGAAT GGAGTCCATG   1370
ACAGTCGTGT TTGACCCCAA CGATGCAGAC ACCACCAGGT CCAGTCGGAA GAAAAGAGCA   1430
ACGCCAAGAG ACCCAAGTTT CAATGGCTGC TCTCGCAGGA ATTCCAAATC TGCCTCCGCC   1490
ACTTCAAGTT TCATAAGCTC ACCCTATACC TCTGTGGATG AATATTCTTA ATTCCATTTC   1550
CTGAGGTAAA AGATTAGTGT GAGACCATCA TGGTGCCAGT CTAGGACCCC ATTCTCCTAT   1610
TTATCAGTCC TGTCCTATAT ACCCTCTAGA AACAGAAAGC AATTTTTAGG CAGCTATGGT   1670
CAAATTGAGA AAGGTAGTGT ATAAATGTGA CAAAGACACT AATAACATGT TAGCCTCCAC   1730
CCAAAATAAA ATGGGCTTTA AATTT                                         1755 
           
           
             
               23 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             3
GGATCCTCRT CRTAGCTGGA NAC                                             23 
           
           
             
               27 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             4
TTTTGGATCC ACTTGGATRA ANGGRCA                                         27 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             5
TTTTGGATCC TTCGTGCAGC CGTCCTGGCG                                      30 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             6
ATATGGATCC GACAGCAGCG AAATGCTCT                                       29 
           
           
             
               27 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             7
TTTTGAATTC TATGGCTTGG TGGTGGC                                         27 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             8
TTTTGCGGCC GCTTTTTTTT TTTTTTTTT                                       29 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             9
AATTGGATCC TAGATGGAAA TACATCCAGC                                      30 
           
           
             
               29 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             10
AATTGGATCC TTGGCTGGCA ATGAGCTCA                                       29 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             11
AATTGGATCC TCCCATCATC TACTGCTGTC                                      30 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             12
TGGCGAACAC AGCTGTGATA                                                 20 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             13
AGCGCGTAGA TGAAATTGAC                                                 20 
           
           
             
               32 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             14
AATTGCGGCC GCCGGAGAAA GCCAGGTTCA CA                                   32 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             15
AATTGCGGCC GCAGGAAGTA GTTGGTGACA GTC                                  33 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             16
AATTGCGGCC GCGACAGGAC TGATAAATAG GAG                                  33 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             17
AATTGCGGCC GCCATGATGG TCTCACACTA ATC                                  33 
           
           
             
               21 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             18
AAAGTGACCA GGAGGCAGAG A                                               21 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             19
AATTGCGGCC GCACCTCAGG AAATGGAATT AAG                                  33 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             20
AATTGGATCC AGAACTTCAG AGGAGTCTCG                                      30

Technology Category: c