Expression of G protein-coupled receptors with altered ligand binding and/or coupling properties

Abstract

Modified forms of G protein-coupled receptors which display altered ligand binding and/or coupling properties are provided as well as cells expressing such receptors and assays utilizing these cells for screening and identifying pharmaceutically effective compounds that specifically modulate the activity of a these modified forms of G protein coupled receptors. Yeast or mammalian cells can be used to express such receptors. The subject assays enable rapid screening of large numbers of compounds (e.g., compounds in a library) to identify those which are receptor agonists or antagonists. Compositions of matter, such as these novel receptors, novel recombinant yeast cells and novel gene constructs, are also provided. The instant assays provide a convenient format for discovering compounds which can be useful in modulating cellular function, as well as in understanding the pharmacology of compounds that specifically interact with these modified forms G protein coupled receptors.

1 - 14 . (canceled)  
     
     
         15 . A recombinant yeast cell having a mutant form of a mammalian G protein-coupled receptor expressed in a membrane of said yeast cell, said mutant receptor having a sequence that varies from a wild type G protein-coupled receptor having a wild type amino acid sequence comprising an amino acid motif [X 1 X 2 X 3 X 4 ] lying near the carboxy terminal end of said domain, wherein: 
 X 1  denotes an amino acid residue at position 1 of said motif and is selected from the group consisting of Phe, Leu, Val, and Tyr;    X 2  denotes an amino acid residue at position 2 of said motif and is selected from the group consisting of Phe, Lys and Gln;    X 3  denotes an amino acid residue at position 3 of said motif and is selected from the group consisting of Leu, Arg, Glu, Asn, Gln, Ser, Ala, Leu; and    X 4  denotes an amino acid residue at position 4 of said motif and is selected from the group consisting of Ala, Cys, Asp, Glu, Gly, Ser, Thr and Tyr; and wherein said mutant receptor comprises:    a seventh transmembrane domain with a carboxy terminal end; and    at least one point mutation at a position on said amino acid motif; wherein said mutant receptor acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone response pathway of the yeast cell, such that upon interaction with a ligand to modulate signal transduction in said pheromone response pathway, a signal generated by said mutant receptor is greater than a signal generated upon interaction of said ligand with a wild type G protein-coupled receptor.    
     
     
         16 . The yeast cell of  claim 15 , comprising a human receptor.  
     
     
         17 . The yeast cell of  claim 16 , wherein said receptor is selected from the group consisting of human galanin-1 receptor, somatastatin receptor type I, somatastatin receptor type II, somatastatin receptor type III, and human nociceptin receptor.  
     
     
         18 . The yeast cell of  claim 15 , comprising an IL8A receptor.  
     
     
         19 . The yeast cell of  claim 16 , which expresses a mammalian, chimeric, and/or mutant G protein subunit.  
     
     
         20 . The yeast cell of  claim 19 , which expresses a mammalian, chimeric, and/or mutant Ga subunit.  
     
     
         21 . The yeast cell of  claim 20 , which expresses a GPA41-G ai3  subunit.  
     
     
         22 . The yeast cell of  claim 20 , which expresses a GPA41-G ai1  or GPA41-G ai2  subunit.  
     
     
         23 . The yeast cell of  claim 19 , which expresses a STE18-Gg2 subunit.  
     
     
         24 . The yeast cell of  claim 16 , which expresses a mammalian G as E10K subunit.  
     
     
         25 . The yeast cell of  claim 16 , which expresses a mammalian G as D229S subunit or a mammalian G as E10K+D229S subunit.  
     
     
         26 . The yeast cell of  claim 15 , which is a mutant cell having a pheromone response pathway that is desensitized at slower rate than a wild type strain under the same conditions of continuous stimulation of the pheromone response pathway.  
     
     
         27 . The yeast cell of  claim 15 , which has a ste14 mutation.  
     
     
         28 . The yeast cell of  claim 15 , which has a ste2 or ste3 mutation.  
     
     
         29 . The yeast cell of  claim 15 , wherein an endogenous pheromone gene is not functionally expressed in the yeast cell.  
     
     
         30 . The yeast cell of  claim 15 , wherein an endogenous FAR1 gene is not functionally expressed in the yeast cell.  
     
     
         31 . The yeast cell of  claim 15 , wherein an endogenous SST2 gene is not functionally expressed in the yeast cell.  
     
     
         32 . The yeast cell of  claim 15 , which further comprises a detectable or selectable marker that is activated by a pheromone response pathway of the yeast cell.  
     
     
         33 . The yeast cell of  claim 32 , wherein the selectable marker comprises a pheromone-responsive promoter operably linked to a selectable gene.  
     
     
         34 . The yeast cell of  claim 33 , wherein the pheromone-responsive promoter is the FUS1 promoter.  
     
     
         35 . The yeast cell of  claim 33 , wherein the marker gene is a HIS 3 gene or a LacZ gene.  
     
     
         36 . The yeast cell of  claim 15 , which further comprises a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the region of said receptor expressed in the cell membrane and wherein modulation of the signal transduction of said receptor by the heterologous polypeptide provides a detectable signal.  
     
     
         37 . The yeast cell of  claim 36 , wherein the heterologous polypeptide includes a signal sequence.  
     
     
         38 . The yeast cell of  claim 37 , wherein the signal sequence corresponds to a leader peptide of the  Saccharomyces cerevisiae  a factor or a-factor.  
     
     
         39 . The yeast cells of  claim 15 , which belong to the species  Saccharomyces cerevisiae.    
     
     
         40 . A method for identifying a modulator of a mammalian G-coupled protein receptor expressed by a yeast cell, comprising: 
 (i) contacting a mixture of yeast cells as claimed in  claim 15  with a test compound;    (ii) allowing cells within the mixture to generate a detectable signal; and    (iii) identifying the test compound as a modulator of said receptor expressed by the yeast cell.    
     
     
         41 . A method for identifying a modulator of a mammalian G-coupled protein receptor expressed by a yeast cell, comprising: 
 (i) contacting a mixture of yeast cells as claimed in claim  2  with a test compound for a period of time sufficient for generation of a detectable signal; and    (ii) determining if said test compound is a modulator of said receptor by detecting the presence or absence of a signal.    
     
     
         42 . A method of identifying a modulator of a mammalian G-coupled protein receptor expressed by a mammalian cell, comprising: 
 (i) contacting a mixture of mammalian cells as claimed in claim  5  with a test compound for a period of time sufficient for generation of a detectable signal; and    (ii) determining if said test compound is a modulator of said receptor by detecting the presence or absence of a signal.