Methods for improving the function of heterlogous G protein-coupled receptors

Abstract

This invention relates to mutant G protein-coupled receptors with improved G-protein coupling and receptor response, yeast cells expressing such receptors, vectors useful for making such cells, and methods of making and using same.

1 - 38 . (canceled)  
   
   
       39 . A yeast cell comprising a nucleic acid sequence encoding a modified, heterologous G protein-coupled receptor (GPCR), wherein the modification comprises a mutation in an intracellular domain of the G protein-coupled receptor and results in an improved functional response and a cell-based assay as compared to a wild-type form of the heterologous G protein-coupled receptor, and wherein the modified G protein-coupled receptor is selected from the group consisting of a muscarinic acetylcholine receptor, a cholecystokinin CCKB receptor, a somatostatin receptor, an alpha 2A adrenergic receptor, and a serotonin receptor.  
   
   
       40 . The yeast cell according to  claim 39 , wherein the modification promotes agonist stimulated growth, and wherein the agonist is a G protein-coupled receptor agonist.  
   
   
       41 . The yeast cell according to  claim 40 , wherein the modification results in improved coupling between the receptor and a heterotrimeric G protein or failure of the receptor to interact with cell desensitization or sequestration-internalization machinery or proper plasma membrane localization.  
   
   
       42 . The yeast cell according to  claim 39 , wherein the muscarinic acetylcholine receptor is a rat M3 muscarinic acetylcholine receptor or a  D. melanogaster  muscarinic acetylcholine receptor.  
   
   
       43 . The yeast cell according to claim  1 , wherein the muscarinic acetylcholine receptor is a rat M3 muscarinic acetylcholine receptor or a  D. melanogaster  muscarinic acetylcholine receptor.  
   
   
       44 . The yeast cell according to claim  1 , wherein the cholecystokinin CCKB receptor is a rat cholecystokinin CCKB receptor.  
   
   
       45 . The yeast cell according to claim  1 , wherein the somatostatin receptor is a rat somatostatin receptor subtype  3 .  
   
   
       46 . A yeast cell comprising a nucleic acid sequence encoding a chimeric G protein-coupled receptor, wherein the chimeric G protein-coupled receptor comprises a first heterologous G protein-coupled receptor in which an intracellular domain has been replaced with a modified intracellular domain of a second heterologous G protein-coupled receptor, and wherein the modified intracellular domain of the second heterologous G protein-coupled receptor confers an improved functional response to the chimeric G protein coupled-receptor in a cell-based assay as compared to a wild-type form of the first heterologous G protein-coupled receptor.  
   
   
       47 . The yeast cell according to  claim 46 , wherein the modified intracellular domain is the third intracellular loop.  
   
   
       48 . The yeast cell according to  claim 39  or  46 , further comprising a plasmid comprising an inducible reporter gene.  
   
   
       49 . The yeast cell according to  claim 48 , wherein the reporter gene is a green fluorescent protein.  
   
   
       50 . The cell according to  claim 49 , wherein the green fluorescent protein is operably linked to a FUS2 promoter.  
   
   
       51 . A method for screening compounds capable of binding to G protein-coupled receptors comprising: 
 (a) subjecting the yeast cell according to  claim 39  or  47  to a test compound; and    (b) measuring the effect of the test compounds on cell growth.