Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, human orphan G protein-coupled receptors.

1. A method for identifying a compound for regulating insulin concentration in the blood of a mammal comprising the steps of:
 contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO: 8; and  
 measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating insulin concentration in the blood of a mammal.  
 
     
     
       2. The method of  claim 1  wherein said compound for regulating insulin concentration in the blood of a mammal is a therapeutic for treating diabetes. 
     
     
       3. The method of  claim 1  wherein the compound for regulating insulin concentration in the blood of a mammal is selected from agonist, partial agonist, and inverse agonist of the receptor. 
     
     
       4. The method of  claim 1  wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. 
     
     
       5. The method of  claim 1  where said host cell is produced by a method comprising:
 transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8;  
 wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO: 8.  
 
     
     
       6. A method for identifying a compound for regulating glucose concentration in the blood of a mammal comprising the steps of:
 contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO: 8; and  
 measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucose concentration in the blood of a mammal.  
 
     
     
       7. The method of  claim 6  wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. 
     
     
       8. The method of  claim 6  where said host cell is produced by a method comprising:
 transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8;  
 wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO: 8.  
 
     
     
       9. A method for identifying a compound for regulating glucagon concentration in the blood of a mammal comprising the steps of:
 contacting one or more candidate compounds with a host cell that expresses a receptor comprising the amino acid sequence of SEQ ID NO: 8; and  
 measuring the ability of the compound or compounds to inhibit or stimulate said receptor, wherein said inhibition or stimulation of said receptor is indicative of a compound for regulating glucagon concentration in the blood of a mammal.  
 
     
     
       10. The method of  claim 9  wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. 
     
     
       11. The method of  claim 9  where said host cell is produced by a method comprising:
 transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8;  
 wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising said amino acid sequence of SEQ ID NO: 8.  
 
     
     
       12. A method for identifying a compound for inhibiting or stimulating a receptor comprising:
   a )  the amino acid sequence of SEQ ID NO:  8 ;        b )  a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 ;        c )  an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO: 7  at  42 ° C., followed by washing in  0 . 1 ×SSC at  65 ° C.;        d )  an amino sequence encoded by the nucleotide sequence of SEQ ID NO:  7 ;        e )  a G protein - coupled receptor having at least  95   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        f )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  95   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels,        comprising the steps of:      i )  contacting one or more candidate compounds with a host cell or membrane thereof,        
       
         wherein said host cell or membrane expresses a receptor comprising:  
           a )  the amino acid sequence of SEQ ID NO:  8 ;    
           b )  a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 ;    
           c )  an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO: 7  at  42 ° C., followed by washing in  0 . 1 ×SSC at  65 ° C.;    
           d )  an amino sequence encoded by the nucleotide sequence of SEQ ID NO:  7 ;    
           e )  a G protein - coupled receptor having at least  95   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or    
           f )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  95   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; and      ii )  measuring the ability of the compound or compounds to inhibit or stimulate said receptor.     
       
     
     
       13. The method of  claim 12 , wherein the compound is selected from agonist, partial agonist, and inverse agonist of the receptor. 
     
     
       14. The method of  claim 13 , wherein the compound is an agonist of the receptor. 
     
     
       15. The method of  claim 13 , wherein the compound is a partial agonist of the receptor. 
     
     
       16. The method of  claim 13 , wherein the compound is an inverse agonist of the receptor. 
     
     
       17. The method of  claim 12 , wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:
   a )  a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:  8 ;        b )  a nucleotide sequence encoding a polypeptide comprising a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 ;        c )  a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO: 7  at  42 ° C., followed by washing in  0 . 1 ×SSC at  65 ° C.;        d )  the nucleotide sequence of SEQ ID NO:  7 ;        e )  a nucleotide sequence encoding a G protein - coupled receptor having at least  95   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        f )  a nucleotide sequence having at least  95   %  identity to the nucleotide sequence of SEQ ID NO:  7 , wherein said nucleotide sequence encodes a G protein - coupled receptor capable of modulating insulin or glucagon levels.     
     
     
       18. The method of  claim 12 , wherein said host cell is produced by a method comprising:
   transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:      a )  a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:  8 ;        b )  a nucleotide sequence encoding a polypeptide comprising a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 ;        c )  a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO: 7  at  42 ° C., followed by washing in  0 . 1 ×SSC at  65 ° C.;        d )  the nucleotide sequence of SEQ ID NO:  7 ;        e )  a nucleotide sequence encoding a G protein - coupled receptor having at least  95   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        f )  a nucleotide sequence having at least  95   %  identity to the nucleotide sequence of SEQ ID NO:  7 , wherein said nucleotide sequence encodes a G protein - coupled receptor capable of modulating insulin or glucagon levels,          wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising:      a )  the amino acid sequence of SEQ ID NO:  8 ;        b )  a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 ;        c )  an amino acid sequence encoded by a nucleotide sequence that hybridizes to the complete complement of SEQ ID NO: 7  at  42 ° C., followed by washing in  0 . 1 ×SSC at  65 ° C.;        d )  an amino sequence encoded by the nucleotide sequence of SEQ ID NO:  7 ;        e )  a G protein - coupled receptor having at least  95   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        f )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  95   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels.       
     
     
       19. The method of  claim 12 , wherein the receptor comprises the amino acid sequence of SEQ ID NO:  8 . 
     
     
       20. The method of  claim 12 , wherein the receptor is a mutant of SEQ ID NO:  8 , wherein lysine is substituted for leucine at amino acid residue  224 . 
     
     
       21. The method of  claim 12 , wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the activity of a second messenger. 
     
     
       22. The method of  claim 21 , wherein the second messenger is selected from the group consisting of adenyl cyclase and phospholipase C. 
     
     
       23. The method of  claim 12 , wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the level of a second messenger. 
     
     
       24. The method of  claim 23 , wherein the second messenger is selected from the group consisting of cAMP, diacyl glycerol, and inositol  1 , 4 , 5 - triphosphate.   
     
     
       25. The method of  claim 12 , wherein the ability of the compound or compounds to inhibit or stimulate said receptor is measured by measuring the binding of GTPγS to a membrane comprising said G protein- coupled receptor.   
     
     
       26. The method of  claim 12 , wherein the host cell is a mammalian host cell. 
     
     
       27. The method of  claim 12 , wherein the host cell is a yeast host cell. 
     
     
       28. The method of  claim 12 , wherein the host cell comprises a reporter system comprising multiple cAMP responsive elements operably linked to a reporter gene. 
     
     
       29. The method of  claim 12 , wherein said receptor is a constitutively activated receptor. 
     
     
       30. The method according to  claim 12 , wherein said method comprises identifying a compound for inhibiting or stimulating a receptor comprising:
   a )  a G protein - coupled receptor having at least  98   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        b )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  98   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels, comprising the steps of:      contacting one or more candidate compounds with a host cell or membrane thereof,        
       
         wherein said host cell or membrane expresses a receptor comprising:  
           a )  a G protein - coupled receptor having at least  98   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or    
           b )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  98   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels, and measuring the ability of the compound or compounds to inhibit or stimulate said receptor.   
       
     
     
       31. The method of  claim 17 , wherein said host cell comprises an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:
   a )  a nucleotide sequence encoding a G protein - coupled receptor having at least  98   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        b )  a nucleotide sequence having at least  98   %  identity to the nucleotide sequence of SEQ ID NO:  7 , wherein said nucleotide sequence encodes a G protein - coupled receptor capable of modulating insulin or glucagon levels.     
     
     
       32. The method of  claim 18 , wherein said host cell is produced by a method comprising:
   transfecting a cell with an expression vector, said expression vector comprising a polynucleotide, said polynucleotide comprising:      a )  a nucleotide sequence encoding a G protein - coupled receptor having at least  98   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        b )  a nucleotide sequence having at least  98   %  identity to the nucleotide sequence of SEQ ID NO:  7 , wherein said nucleotide sequence encodes a G protein - coupled receptor capable of modulating insulin or glucagon levels,          wherein said host cell, under appropriate culture conditions, produces a polypeptide comprising:      a )  a G protein - coupled receptor having at least  98   %  identity to the amino acid sequence of SEQ ID NO:  8 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels; or        b )  a G protein - coupled receptor encoded by a nucleotide sequence having at least  98   %  identity to the nucleotide sequence of SEQ ID NO: 7 , wherein said G protein - coupled receptor is capable of modulating insulin or glucagon levels.