Recombinant C140 receptor, its agonists and antagonists, and nucleic acids encoding the receptor

Abstract

Nucleic acid molecules encoding the C140 cell surface receptor have been cloned and sequenced. The availability of C140 receptor DNA permits the recombinant production of the C140 receptor which can be produced on the surface of a cell, including an oocyte. The nucleic acid molecules are useful in an assay for detecting a substance which affects C140 receptor activity, either receptor agonists or antagonists. Further, the elucidation of the structure of the C140 receptor permits the design of agonist and antagonist compounds which are useful in such assays. The availability of the C140 receptor also permits production of antibodies specifically immunoreactive with one or more antigenic epitopes of the C140 receptor.

1 . A DNA molecule comprising an expression system capable, when transformed into a recombinant host, of producing the C140 receptor at the cell surface of the host, which expression system comprises a nucleotide sequence encoding the C140 receptor operably linked to a control sequence heterologous to said encoding nucleotide and operable in said host cell.  
     
     
         2 . A cell modified to contain the expression system of  claim 1 .  
     
     
         3 . A method to produce cells that contain C140 receptor deployed at their surface, which method comprises culturing the cells of  claim 2  under conditions which effect the expression of the nucleotide sequence encoding the C140 receptor to obtain said cells that contain C140 receptor deployed at their surface.  
     
     
         4 . A cRNA molecule that encodes the C140 receptor.  
     
     
         5 . Cells which are oocytes modified to contain the cRNA of  claim 4 .  
     
     
         6 . A method to produce cells which are oocytes that contain C140 receptor deployed at their surface, which method comprises culturing the oocytes of  claim 5  under conditions which effect the expression of the cRNA encoding the C140 receptor to obtain said cells that contain C140 receptor deployed at their surface.  
     
     
         7 . A method to determine the C140 agonist activity of a candidate substance, which method comprises: 
 incubating the cells of  claim 3  or  6  in the presence and absence of the substance, and    detecting the presence, absence or amount of activation of the C140 receptor in the presence as compared to the absence of said substance whereby an increase in said activation in the presence as compared to the absence of said substance indicates agonist activity of the substance.    
     
     
         8 . A method to assess the ability of a candidate substance to behave as a C140 antagonist, which method comprises: 
 incubating the cells of  claim 3  or  6  in the presence of a C140 agonist and in the presence and absence of said candidate, and    measuring the activation of the C140 receptor in the presence and absence of said candidate, whereby a decrease in said activation in the presence of the candidate indicates the antagonist activity of the candidate.    
     
     
         9 . A method to assess the ability of a candidate substance to bind to C140 receptor, which method comprises: 
 incubating the cells of  claim 3  or  6  in the presence of a C140 agonist or a known C140 antagonist and in the presence and absence of said candidate, and    measuring the binding of said C140 agonist or C140 antagonist to the surface of said cells in the presence and absence of said candidate, whereby a decrease in said binding in the presence of the candidate indicates the ability of the candidate to bind receptor.    
     
     
         10 . An antibody composition specifically immunoreactive with an extracellular region of the C140 receptor protein or a portion thereof.  
     
     
         11 . The antibody composition of  claim 10  wherein said region is the ligand-binding region, or 
 which is specifically immunoreactive with activated C140 receptor, or  
 recognizes an epitope within the receptor sequence SLIGRL, or  
 is specifically reactive with the cleaved activation peptide of the C140 receptor.  
 
     
     
         12 . A method to localize activated C140 receptors in situ, which method comprises: 
 administering to a subject putatively harboring activated C140 receptor an amount of antibody specific to said activated receptor effective to bind to said activated receptor, and    detecting the location of said antibody.    
     
     
         13 . A method for detecting the presence of activated C140 receptor in a mammalian subject, which method comprises: 
 contacting a sample of the biological fluid of said subject with a detection system which measures the presence, absence or amount of the cleaved activation peptide of the C140 receptor; and    detecting the presence, absence or amount of said cleaved peptide.    
     
     
         14 . An agonist peptide capable of activating C140 receptor, which peptide is of the formula  
       AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -Z   (1)  
       wherein AA 1  is a small amino acid or threonine; 
 AA 2  and AA 3  are each independently neutral/nonpolar/large/nonaromatic amino acids;  
 AA 4  is a small amino acid;  
 AA 5  is a basic amino acid;  
 AA 6  may be present or absent and, if present, is a neutral/nonpolar/large/nonaromatic amino acid;  
 AA 7  is absent if AA 6  is absent and may be present or absent if AA 6  is present, and is an acidic amino acid; and  
 Z is a substituent that does not interfere with agonist activity.  
 
     
     
         15 . The peptide of  claim 14  wherein AA 1  is ser, ala, gly, thr, or 2,3-diamino-propionic (2,3-diaP); and/or 
 wherein each of AA 2  and AA 3  is independently selected from the group consisting of ile, val, leu, and Cha; and/or  
 wherein AA 4  is Gly; and/or  
 wherein AA 5  is Arg, Lys or Har; and/or  
 wherein Z comprises OR′, or NR′R′ wherein each R′ is independently H or is a straight or branched chain alkyl or 1-6C, wherein each R′ may optionally be substituted with one or more substituents selected from the group consisting of —OR′, —NR′R′, and —NR′CNR′NR′R′ wherein each R′ is H or is a straight or branched chain alkyl of 1-6C.  
 
     
     
         16 . The peptide of  claim 15  wherein AA 1 -AA 2 -AA 3  is selected from the group consisting of SLI, SLL, SChaI, SChaL, (2,3-diaP)LI and (2,3-diaP)LL; and/or 
 wherein Z includes additional peptide sequence of 1-5 amino acids.  
 
     
     
         17 . The peptide of  claim 14  which is selected from the group consisting of SLIGRLETQPPIT, SLIGRLETQPPI, SLIGRLETQPP, SLIGRLETQP, SLIGRLETQ, SLIGRLET, SLIGRLE, SLIGRL, SLIGR, SLLGKVDGTSHVT, SLLGKVDGTSHV, SLLGKVDGTSH, SLLGKVDGTS, SLLGKVDGT, SLLGKVDG, SLLGKVD, SLLGKV, SLLGK, S(Cha)IGR, S(Cha)LGK, (2,3-diaP)-LIGR, (2,3-diaP)LLGK, SLLGKR—NH 2 , SLIGRR—NH 2 , S(Cha)LGKK—NH 2 , S(Cha)IGRK—NH 2 , (2,3-diaP)-LIGRK—NH 2 , and (2,3-diaP)-LLGKK—NH 2 .  
     
     
         18 . A peptide capable of inhibiting the function of the C140 receptor which peptide is of the formula  
       X-AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -Z  
       wherein X is an amino acid residue other than ser, ala, thr, cys, 2,3-diaP or gly or is a desamino or acylated amino acid, 
 wherein AA 2  and AA 3  are each independently neutral/nonpolar/large/nonaromatic amino acids;  
 AA 4  is a small amino acid;  
 AA 5  is a basic amino acid;  
 AA 6  may be present or absent and, if present, is a neutral/nonpolar/large/nonaromatic amino acid;  
 AA 7  is absent if AA 6  is absent and may be present or absent if AA 6  is present, and is an acidic amino acid; and  
 Z is a substituent that does not interfere with agonist activity.  
 
     
     
         19 . The peptide of  claim 18  wherein X is Mvl, Mpr, Mba, or SMeMpr; and/or 
 wherein each of AA 2  and AA 3  is independently selected from the group consisting of ile, val, leu, Nle, Nva, Cyclopentylalanine and Cha; and/or  
 wherein AA 4  is Gly; and/or  
 wherein AA 5  is Arg, Lys, Orn or Har; and/or  
 wherein Z comprises OH or an ester or salt thereof, or NR′R′ wherein each R′ is independently H or is a straight or branched chain alkyl of 1-6C, wherein each R′ may optionally be substituted with one or more substituents selected from the group consisting of —OR′, —NR′R′, and —NR′CNR′NR′R′ wherein each R′ is H or is a straight or branched chain alkyl of 1-6C.  
 
     
     
         20 . The peptide of  claim 19  wherein AA 2 -AA 3  is selected from the group consisting of LI, LL, ChaI, and ChaL; and/or 
 wherein Z includes a peptide extension of 1-5 amino acid residues.  
 
     
     
         21 . The peptide of  claim 18  which is selected from the group consisting of Mpr-LLGK, Mpr-LIGR, Mpr-(Cha)LKG, Mpr-(Cha)IGR, Mpr-LLGKK-NH 2 , Mpr-LIGRK-NH 2 , Mpr-LIGRKETQP-NH 2 , Mpr-LLGKKDGTS-NH 2 , (n-pentyl) 2 -N-Leu-Ile-Gly-Arg-Lys-NH 2  and (Me-N-(n-pentyl)-Leu-Ile-Gly-Arg-Lys-NH2, and the amidated or acylated forms thereof.  
     
     
         22 . An isolated nucleic acid molecule which encodes a C140 receptor polypeptide or which is complementary to a DNA or RNA molecule encoding a C140 receptor polypeptide.  
     
     
         23 . The nucleic acid molecule of  claim 22  wherein said C140 receptor is the human C140 receptor.  
     
     
         24 . A method to inhibit expression of C140 receptors in a cell comprising providing to said cell an oligonucleotide molecule which is antisense to, or forms a triple helix with, C140 receptor-encoding DNA or with DNA regulating expression of C140 receptor-encoding DNA, in an amount sufficient to inhibit expression of said C140 receptors, thereby inhibiting said expression.  
     
     
         25 . A method to inhibit expression of C140 receptors in a subject, comprising administering to said subject an oligonucleotide molecule which is antisense to, or forms a triple helix with, C140 receptor-encoding DNA or with DNA regulating expression of C140 receptor-encoding DNA, in an amount sufficient to inhibit expression of said C140 receptors in said subject, thereby inhibiting said expression.  
     
     
         26 . A pharmaceutical composition comprising an oligonucleotide molecule of  claim 25  together with a pharmaceutically acceptable carrier or excipient.