Promiscuous G-protein compositions and their use

Abstract

Disclosed are compositions and methods for their use, such as in identifying G-protein coupled receptors and ligands and compounds that modulate signal transduction. The compositions and methods employ promicuous G-proteins. Activation of the promiscous G-protein can be detected in a variety of assays, including assays in which activation is indicated by a change in fluorescence emission of a sample that contains the composition.

1 . A stable, isolated cell comprising a construct comprising promoter operably linked to a polynucleotide encoding a polypeptide having a biological activity of a promiscuous Gα protein.  
     
     
         2 . The cell of  claim 1 , wherein said promoter is an inducible promoter.  
     
     
         3 . The cell of  claim 2 , wherein said construct permits expression in a mammalian cell.  
     
     
         4 . The cell of  claim 1 , wherein said polynucleotide has a nucleotide sequence with at least 70% sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence of Gα 16  (SEQ ID NO: 1), and the nucleotide sequence of Gα 15  (SEQ ID NO: 2).  
     
     
         5 .- 7 . (canceled)  
     
     
         8 . The cell of  claim 1 , further comprising a second construct comprising a reporter gene operably linked to a second promoter, wherein said second promoter is modulated by a promiscuous Gα protein.  
     
     
         9 . The cell of  claim 8 , wherein the reporter gene encodes a reporter selected from the group consisting of luciferase, green fluorescent protein, chloramphenicol acetyl transferase, β-galactosidase, alkaline phosphatase, β-lactamase, and human growth hormone.  
     
     
         10 .- 11 . (canceled)  
     
     
         12 . The cell of  claim 8 , wherein the reporter gene encodes β-lactamase.  
     
     
         13 . The cell of  claim 8 , wherein said second promoter comprises a protein kinase C-responsive promoter.  
     
     
         14 .- 26 . (canceled)  
     
     
         27 . A method of identifying a GPCR for a given ligand, the method comprising: 
 (i) expressing a putative GPCR in a cell of  claim 1;     (ii) contacting said cell with a ligand; and    (iii) detecting reporter gene expression.    
     
     
         28 . The method of  claim 27 , wherein said detecting comprises fluorescence detection.  
     
     
         29 . The method of  claim 28 , further comprising contacting said cell with a reporter gene substrate.  
     
     
         30 . The method of  claim 27 , further comprising contacting said cell with a compound that increases calcium levels inside said cell.  
     
     
         31 . The method of  claim 30 , wherein said compound is selected from the group consisting of ionomycin and thapsigargin.  
     
     
         32 . The method of  claim 30 , further comprising contacting said cell with phorbol myristate acetate or an analog thereof.  
     
     
         33 . A method of a identifying of a ligand for a given GPCR, the method comprising: 
 a) expressing a GPCR in a cell of  claim 1;     b) contacting said cell with a test chemical; and    c) detecting a signal with a signal transduction detection system.    
     
     
         34 .- 36 . (canceled)  
     
     
         37 . The method of  claim 33 , wherein said detecting comprises reporter gene detection.  
     
     
         38 . A method for identifying a modulator of signal transduction in a cell, the method comprising: 
 a) contacting a cell of  claim 1  with a test chemical;    b) contacting said cell with a ligand that, in the absence of the test chemical, activates signal transduction in said cell, and    c) detecting a signal with a signal transduction detection system.    
     
     
         39 .- 42 . (canceled)  
     
     
         43 . A kit comprising assay reagents and a container containing a cell of  claim 1 .  
     
     
         44 .- 62 . (canceled)