Yeast cells expressing modified g proteins and methods of use therefor

Abstract

The present invention pertains to novel yeast cells which are useful for the expression of heterologous G protein coupled receptors. The yeast cells of the present invention can be used in screening assays which can be used to screen for modulators of G protein coupled receptors. Specifically, the invention provides novel yeast cells which express a heterologous G protein coupled receptor and mutant and/or chimeric G protein subunit molecules which serve to functionally integrate the heterologous into the pheromone signaling pathway of the yeast cell. The invention also provides for the expression of heterologous G protein coupled receptors which are functionally integrated into the yeast cell membrane using a yeast α factor leader sequence. Drug discovery assays using the subject yeast cells are also provided.

1 - 18 . (canceled) 
     
     
         19 . A yeast cell comprising a first chimeric G protein subunit, said chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a heterologous G protein subunit, wherein said first polypeptide is selected from the group consisting of:
 a polypeptide comprising about 40 amino acids from the amino terminus of yeast GPA1; and   a polypeptide from yeast STE 18; and   a second chimeric G protein subunit, said second chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a mammalian G protein subunit, wherein said second chimeric G protein subunit is different from said first chimeric G protein subunit.   
     
     
         20 . The yeast cell of  claim 19 , wherein said second polypeptide of said second chimeric G protein subunit is from a protein selected from the group consisting of: a mammalian Gα subunit, a mammalian Gβ subunit, and a mammalian Gγ subunit. 
     
     
         21 - 25 . (canceled) 
     
     
         26 . An assay to identify compounds capable of modulating the dissociation of Gα and Gβγ, comprising the steps of:
 (i) providing a yeast cell comprising:
 (a) a heterologous G Protein coupled receptor, which receptor is functionally integrated into the yeast cell; and 
 a chimeric G Protein subunit, said chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a heterologous G protein subunit, wherein said first polypeptide is selected from the group consisting of a polypeptide comprising about 40 amino acids from the amino terminus of yeast GPA1 and a polypeptide from yeast STE 18; or 
 (b) (i) a heterologous G protein coupled receptor;
 (ii) a non-naturally occurring G protein subunit which comprises a sequence from a heterologous G protein subunit in which at least one amino acid substitution has been introduced compared to the wild type sequence; such that expression of said non-naturally occurring G protein subunit functionally integrates said heterologous G protein coupled receptor into the yeast cell pheromone signaling pathway; and 
 (iii) an indicator gene that produces a detectable signal upon functional coupling of the heterologous G Protein coupled receptor to the G protein; 
 
   (ii) contacting the yeast with a test compound; and   (iii) identifying compounds which induce a change in a detectable signal in the yeast cell, wherein said detectable signal indicates dissociation of Gα and Gβγ.   
     
     
         27 . The assay of  claim 26 , wherein said test compound is from a library of non-peptidic organic molecules. 
     
     
         28 . A method for identifying a compound which modulates a heterologous G protein coupled receptor, comprising:
 (i) providing a first, second, third, and fourth yeast cell, each cell comprising:
 (a) a G protein, wherein:
 1) the first yeast cell comprises a first chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a mammalian G protein subunit; 
 2) the second yeast cell comprises a second chimeric G protein subunit comprising a first polypeptide, derived from a yeast G protein subunit and a second polypeptide from a mammalian G protein subunit, said second chimeric G protein subunit being different from said first chimeric G protein subunit; 
 3) the third yeast cell comprises a third chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a mammalian G protein subunit, said third chimeric G protein subunit being different from said first and second chimeric G protein subunits; and 
 4) the fourth yeast cell comprises an endogenous yeast gpa1 G protein subunit; 
 
 (b) an expressible gene construct encoding a heterologous G protein coupled receptor (GPCR) which couples to the yeast pheromone system pathway; and 
 (c) an indicator gene that produces a change in a detectable signal upon functional coupling of the heterologous GPCR with the G protein; 
   (ii) contacting the first, second, third, and fourth yeast cells with a test compound; and   (iii) determining whether the test compound induces a change in a detectable signal in at least one of the first, second, third, or fourth yeast cells to thereby identify a compound which modulates a heterologous GPCR.   
     
     
         29 . The assay of  claim 28 , wherein at least one of said first, second, or third yeast cells comprises a fourth chimeric G protein subunit, said fourth chimeric G protein subunit being different from the first, second, or third chimeric G protein subunit expressed by the first, second, or third yeast cell, respectively. 
     
     
         30 . The assay of  claim 28 , wherein at least one of said first, second, or third chimeric G proteins comprises a first polypeptide from yeast GPA1 and a second polypeptide from a mammalian G protein α-subunit. 
     
     
         31 . The assay of  claim 30 , wherein said second polypeptide is from a mammalian Gαi subunit. 
     
     
         32 . The assay of  claim 30 , wherein said second polypeptide is from a mammalian Gα16 subunit. 
     
     
         33 . The assay of  claim 30 , wherein said second polypeptide is from a mammalian Gαs subunit. 
     
     
         34 . The assay of  claim 28 , wherein the first chimeric G protein subunit comprises a polypeptide from mammalian Gα12, the second chimeric G protein subunit comprises a polypeptide from mammalian Gα16, and the third chimeric G protein subunit comprises a polypeptide from mammalian Gαs. 
     
     
         35 . The assay of  claim 34 , wherein the second chimeric G protein subunit comprises Gα16(S270P) and the third chimeric G protein subunit comprises Gαs(D229S). 
     
     
         36 . The assay of  claim 28 , wherein each of said first, second, and third yeast cells further comprises a fourth chimeric G protein subunit, said fourth chimeric G protein subunit comprising a first polypeptide from yeast STE 18 and a second polypeptide from a mammalian G protein y subunit. 
     
     
         37 . The assay of  claim 28 , wherein the first, second, third, and fourth yeast cells are contacted with each member of a library of test compounds. 
     
     
         38 . The assay of  claim 36 , wherein each member of said library is a non-peptidic organic molecule. 
     
     
         39 . The assay of  claim 28 , wherein said first, second, third, and fourth yeast cells are  Saccharomyces cerevisiae  cells. 
     
     
         40 . The assay of  claim 28 , wherein the indicator gene that gives rise to a detectable signal is selected from the group consisting of: β galactosidase, alkaline phosphatase, horseradish peroxidase, exoglucanase, luciferase, BAR1, PHO5, green fluorescent protein, and chloramphenicol acetyl transferase. 
     
     
         41 . The assay of  claim 28 , wherein the indicator gene that gives rise to a detectable signal is selected from the group consisting of: HIS 3, β galactosidase, and green fluorescent protein. 
     
     
         42 . The assay of  claim 28 , wherein said heterologous G protein coupled receptor is an orphan receptor.