Yeast cells expressing modified G proteins and methods of use therefor
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. A yeast cell comprising 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.
2. The yeast cell of claim 1 , wherein the cell is a Saccharomyces cerevisiae cell.
3. The yeast cell of claim 1 , wherein said first polypeptide comprises about 40 amino acids from the amino terminus of yeast GPA1 and said second polypeptide is from a heterologous G protein α subunit.
4. The yeast cell of claim 3 , wherein said chimeric G protein subunit is selected from the group consisting of: gpal (41)-Gi2; gpal (41)-G16; and gpal (41)-Gs.
5. The yeast cell of claim 4 , wherein the Gαi2, Gα16, or Gαs portion of said chimeric G protein subunit comprises an amino acid substitution compared to wild type Gi2, G16, or Gs.
6. The yeast cell of claim 1 , wherein said heterologous G protein subunit is mammalian.
7. The yeast cell of claim 6 , wherein said heterologous G protein subunit is human.
8. The yeast cell of claim 7 , wherein at least one of said first and second polypeptides comprises a naturally occurring amino acid sequence.
9. The yeast cell of claim 7 , wherein at least one of said first and second polypeptides comprises a non-naturally occurring amino acid sequence.
10. The yeast cell of claim 1 , further comprising a heterologous G protein coupled receptor, which receptor is functionally integrated into the yeast cell.
11. The yeast cell of claim 10 , wherein said chimeric G protein subunit demonstrates enhanced coupling to the heterologous G protein coupled receptor when compared to that demonstrated by an endogenous yeast G protein subunit.
12. The yeast cell of claim 10 , wherein an endogenous yeast pheromone system receptor protein is not produced in functional form.
13. The yeast cell of claim 10 , further comprising an indicator gene that produces a detectable signal upon functional coupling of the heterologous G protein coupled receptor to the G protein.
14. The yeast cell of claim 10 , wherein said heterologous G protein coupled receptor is an orphan receptor.