Compositions and methods for modulating the activity of G protein-coupled receptor kinases GRK5 and GRK6

Abstract

G protein-coupled receptor kinases (GRK) play an important role in phosphorylating and regulating the activity of G protein-coupled receptors. Complementary DNAs (cDNAs)that encode two novel members of the G protein-coupled receptor kinase (GRK) family are provided in the present invention. These cDNAs encode GRK5 (590 amino acids) and GRK6 (576 amino acids) which represent two new members of the GRK family that have distinct tissue distribution and substrate specificity. The availability of the cDNAs enables the generation of reagents to modulate the activity of endogenous kinases. These include dominant negative mutations and antisense oligonucleotides or stably transfected antisense constructs to block expression of the kinase to generate a cell with a reduced ability to desensitize to various agents. Expression of GRK5 and GRK6 also permits identification of specific inhibitors and activators of these two kinases. Such inhibitors and activators may be used therapeutically to either directly modulate the activity of a given receptor or by augmenting the ability of a given therapeutic agent to stimulate a given receptor.

What is claimed:  
     
         1 . A method of screening for compounds capable of inhibiting the activity of a selected G protein-coupled receptor kinase comprising: 
 (a)contacting a sample containing a selected G protein-coupled receptor kinase encoded by a member of the G protein-coupled receptor kinase gene family with a selected compound under conditions such that the selected G protein-coupled receptor kinase present in the sample and the selected compound interact; and    (b)determining the ability of the selected compound to inhibit the activity of the selected G protein-coupled receptor kinase,    wherein the selected G protein-coupled receptor kinase is GRK5 or GRK6.    
     
     
         2 . The method of  claim 1 , wherein the selected G protein-coupled receptor kinase is GRK5.  
     
     
         3 . The method of  claim 2 , wherein said GRK5 is encoded by a nucleic acid sequence set forth in SEQ ID NO:1.  
     
     
         4 . The method of  claim 1 , wherein the selected G protein-coupled receptor kinase is GRK6.  
     
     
         5 . The method of  claim 4 , wherein said GRK6 is encoded by a nucleic acid sequence set forth in SEQ ID NO:2.  
     
     
         6 . A method of inhibiting the activity of GRK5 or GRK6 comprising administering an effective amount of an inhibitor identified in  claim 9  so that the activity of said GRK5 or GRK6 is inhibited.  
     
     
         7 . A method of screening for compounds capable of inhibiting the activity of a selected G protein-coupled receptor kinase having an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the method comprising: 
 (a) contacting a sample containing the selected G protein-coupled receptor kinase with a selected compound under conditions such that the selected G protein-coupled receptor kinase present in the sample and the selected compound interact; and    (b) determining the ability of the selected compound to inhibit the activity of the selected G protein-coupled receptor kinase.    
     
     
         8 . A method of screening for compounds capable of enhancing the activity of a selected G protein-coupled receptor kinase comprising: 
 (a) contacting a sample containing a selected G protein-coupled receptor kinase encoded by a member of the G protein-coupled receptor kinase gene family with a selected compound under conditions such that the selected G protein-coupled receptor kinase present in the sample and the selected compound interact; and    (b) determining the ability of the selected compound to enhance the activity of the selected G protein-coupled receptor kinase,    wherein the selected G protein-coupled receptor kinase is GRK5 or GRK6.    
     
     
         9 . The method of  claim 8 , wherein the selected G protein-coupled receptor kinase is GRK5.  
     
     
         10 . The method of  claim 9 , wherein said GRK5 is encoded by a nucleic acid sequence set forth in SEQ ID NO:1.  
     
     
         11 . The method of  claim 8 , wherein the selected G protein-coupled receptor kinase is GRK6.  
     
     
         12 . The method of  claim 11  wherein said GRK6 is encoded by a nucleic acid sequence set forth in SEQ ID NO:2.  
     
     
         13 . A method of enhancing the activity of a selected G protein-coupled receptor kinase comprising administering an effective amount of a compound capable of enhancing activity of the selected G protein-coupled receptor kinase identified in  claim 8  so that the activity of the selected G protein-couple receptor kinase is enhanced.  
     
     
         14 . A method of modulating the expression of an endogenous G protein-coupled receptor kinase by a cell comprising contacting the cell capable of expressing the G protein-coupled receptor kinase selected from the group consisting of GRK5 and GRK6 with a corresponding dominant negative mutant of the G protein-coupled receptor kinase which is capable of binding to its receptor substrate but incapable of phosphorylating said receptor substrate.  
     
     
         15 . The method of  claim 14 , wherein said mutant of the G protein-coupled receptor kinase carries a mutation from a lysine residue to an arginine residue.  
     
     
         16 . A method of modulating the expression of an endogenous G protein-coupled receptor kinase by a cell comprising contacting the cell capable of expressing the G protein-coupled receptor kinase selected from the group consisting of GRK5 and GRK6 with a composition containing a selected polyanion or polycation at levels sufficient to influence the expression of the endogenous G protein-coupled receptor kinase.  
     
     
         17 . The method of  claim 16 , wherein the composition comprises a polyanion selected from the group consisting of heparin, dextran sulfate, polyaspartic acid and polyglutamic acid.  
     
     
         18 . The method of  claim 16 , wherein the composition comprises a polycation selected from the group consisting of polylysine, spermine and spermidine.