G protein coupled receptor protein, production, and use thereof

Abstract

DNA primers effective in screening G protein coupled receptor protein-encoding DNA fragments are provided. The primers which are complementary to nucleotide sequences that are in community with (homologous to) the nucleotide sequences encoding amino acid sequences corresponding to or near the first membrane-spanning domain or the sixth membrane-spanning domain each of known various G protein coupled receptor proteins were designed and synthesized. Methods of amplifying G protein coupled receptor protein-encoding DNAs using the above DNA primers, and novel target G protein coupled receptor protein-encoding DNAs are also provided. Screening of DNA libraries can be efficiently carried out. Human pituitary gland or amygdala-derived and mouse pancreas-derived G protein coupled receptor proteins, etc. or salts thereof, partial peptides thereof, DNAs coding for the above G protein coupled receptor proteins, processes for producing the above G protein coupled receptor proteins, methods of determining ligands for the above G protein coupled receptor proteins, methods of screening compounds that inhibit the binding between the ligand and the G protein coupled receptor proteins or screening kits therefor, compounds or salts thereof obtained by the above screening method or the screening kit, pharmaceutical compositions containing the above compounds or salts thereof, and antibodies against the above protein coupled receptor proteins or partial peptides thereof are provided.

1 . A DNA which comprises a nucleotide sequence represented by a SEQ ID NO selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 19.  
     
     
         2 . A method for amplifying a DNA coding for a G protein coupled receptor protein by polymerase chain reaction techniques, which comprises: 
 (i) carrying out a polymerase chain reaction in the presence of a mixture of 
 a DNA coding for a G protein coupled receptor protein, said DNA being capable of acting as a template,  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19; or  
   (ii) carrying out a polymerase chain reaction in the presence of a mixture of 
 a DNA coding for G protein coupled receptor protein, said DNA being capable of acting as a template,  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13.  
   
     
     
         3 . A method for screening a DNA library for a DNA coding for a G protein coupled receptor protein, which comprises: 
 (i) carrying out a polymerase chain reaction in the presence of a mixture of 
 said DNA library,  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 3, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 5, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 6, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 7, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 10, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 14, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 16 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 18, and  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 2, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 4, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 8, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 9, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 11, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 15, DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 17 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 19,  
   under conditions to amplify selectively a template DNA coding for the G protein coupled receptor protein, contained in the DNA library and selecting said DNA; or    (ii) carrying out a polymerase chain reaction in the presence of a mixture of 
 said DNA library  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 1 and DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 12, and  
 at least one DNA primer selected from the group consisting of DNA primers comprising a nucleotide sequence represented by SEQ ID NO: 13,  
 under conditions to amplify selectively a DNA coding for the G protein coupled receptor protein, contained in the DNA library and selecting said DNA.  
   
     
     
         4 . A DNA coding for a G protein coupled receptor protein or a fragment thereof, which is obtained by the method according to  claim 2  to  3 .  
     
     
         5 . A G protein coupled receptor protein encoded by the DNA according to  claim 4 , a peptide segment or fragment thereof or a salt thereof.  
     
     
         6 . A G protein coupled receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence represented by SEQ I D NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 28, an amino acid sequence represented by SEQ ID NO: 34, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 38, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 56, and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 56; a peptide segment (or fragment) thereof, a modified peptide derivative thereof or a salt thereof.  
     
     
         7 . The G protein coupled receptor protein according to  claim 6 , comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 38, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 56 and substantial equivalents to the amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 56.  
     
     
         8 . The G protein coupled receptor protein according to claims  6  or  7 , wherein said receptor is a purinoceptor.  
     
     
         9 . The G protein coupled receptor protein according to any of  claims 6  to  8 , wherein an agonist to said receptor is useful as an immunomodulator or an antitumor agent, in addition it is useful in therapeutically or prophylactically treating hypertension, diabetes or cystic fibrosis, and an antagonist to said receptor is useful as a hypotensive agent, an analgesic, or an agent for therapeutically or prophylactically treating incontinence of urine.  
     
     
         10 . A DNA which comprises a nucleotide sequence coding for a G protein coupled receptor protein of  claim 6 .  
     
     
         11 . The DNA according to  claim 10  comprising a nucleotide sequence coding for the G protein coupled receptor protein according to  claim 7 .  
     
     
         12 . The DNA according to  claim 11  comprising a nucleotide sequence represented by SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 57.  
     
     
         13 . A transformant containing a vector comprising the DNA according to  claim 4  or  10 ; or an expression system comprising an open reading frame (ORF) of DNA derived from a G protein coupled receptor protein DNA according to  claim 4  or  10 , wherein the ORF is operably linked to a control sequence compatible with a desired host cell.  
     
     
         14 . A method for determining a ligand to the G protein coupled receptor protein according to any of  claims 5  to  8 , which comprises contacting 
 (i) at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of  claims 5  to  8 , peptide segments or salts thereof, and mixtures thereof, with  
 (ii) at least one compound to be tested and determining whether said compound to be tested bound to the component of (i).  
 
     
     
         15 . A screening method for a compound capable of inhibiting the binding of a G protein coupled receptor protein according to any of  claims 5  to  8  with a ligand, which comprises carrying out a comparison between: 
 (i) at least one case where said ligand is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of  claims 5  to  8 , peptide segments or salts thereof, and mixtures thereof, and  
 (ii) at least one case where said ligand together with a compound to be tested is contacted with at least one component selected from the group consisting of G protein coupled receptor proteins or salts thereof according to any of  claims 5  to  8 , peptide segments or salts thereof, and mixtures thereof.  
 
     
     
         16 . A compound which is determined through the method according to  claim 15  or a salt thereof.  
     
     
         17 . The compound according to  claim 16 , which is an agonist or antagonist to a G protein coupled receptor protein according to any of  claims 5  to  8 .  
     
     
         18 . A ligand to a G protein coupled receptor protein according to any of  claims 5  to  8 , which is determined through the method according to  claim 14.