Yeast cells having mutations in Cav1 and uses therefor

• US 6,251,605 B1
• Filed: 10/25/1999
• Issued: 06/26/2001
• Est. Priority Date: 10/27/1998
• Status: Expired due to Term

• Current Assignee: Cadus Pharmaceutical Corporation
• Original Assignee: Cadus Pharmaceutical Corporation
• Inventors: Ostanin, Kirill, Silverman, Lauren
• Primary Examiner(s): Schwartzman, Robert A.
• Application Number:
  US09/426,332
• Time in Patent Office: 610 Days
• Field of Search: 435/6, 435/254.2, 435/254.21, 435/255.1, 435/255.2
• US Class Current: 506/10
• CPC Class Codes: C07K 14/395 from Saccharomyces

Abstract

The invention provides isolated yeast cells which comprise a mutation in an endogenous yeast CAV1 gene, which exhibit increased signaling via the pheromone response pathway. In a preferred embodiment, the cav1 mutant yeast cells of the invention also express a heterologous G protein coupled receptor that functionally couples to the pheromone response pathway. The instant yeast cells display enhanced sensitivity to ligand induced stimulation of heterologous G protein coupled receptors and, therefore, show improved properties in drug screening assays.

Claims

1. An isolated yeast cell comprising a mutation in a nucleic acid encoding an endogenous yeast Cav1 protein which renders said endogenous yeast Cav1 protein nonfunctional.

2. The yeast cell of claim 1, which further comprises a reporter gene construct which produces a detectable signal upon stimulation of an endogenous yeast pheromone response pathway of the yeast cell.

3. The yeast cell of claim 1, which is a Saccharomyces cerevisiae cell.

4. A recombinant yeast cell having an endogenous yeast pheromone response pathway, which yeast cell comprises:

(i) a heterologous G protein coupled receptor which functionally couples to the endogenous yeast pheromone response pathway; and

(ii) a mutation in a nucleic acid encoding an endogenous yeast Cav1 protein which renders said endogenous yeast Cav1 protein nonfunctional.

5. The yeast cell of claim 4, which further comprises a heterologous or chimeric G protein subunit.

6. The yeast cell of claim 4, which further comprises a reporter gene construct which produces a detectable signal upon stimulation of the yeast pheromone response pathway.

7. The yeast cell of claim 4, which is a Saccharomyces cerevisiae cell.

8. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is a mammalian G protein coupled receptor.

9. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is expressed in the yeast cell using a native leader sequence of the heterologous G protein coupled receptor.

10. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor naturally lacks a leader sequence and is expressed in the yeast cell in its native form without a leader sequence.

11. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is expressed in the yeast cell using a yeast leader sequence.

12. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is a human nociceptin receptor.

13. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is a human melanocortin receptor.

14. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is a human somatostatin receptor.

15. The yeast cell of claim 4, wherein the heterologous G protein coupled receptor is a human neuropeptide Y1 receptor or a human neuropeptide Y2 receptor.

16. The yeast cell of claim 8, wherein the heterologous G protein coupled receptor is a human G protein coupled receptor.

17. The yeast cell of claim 11, wherein the yeast leader sequence is used to express a heterologous G protein coupled receptor that naturally lacks a leader sequence.

18. The yeast cell of claim 11, wherein the yeast leader sequence replaces a natural leader sequence of the heterologous G protein coupled receptor.

19. The yeast cell of claim 11, wherein the yeast leader sequence is linked to the heterologous G protein coupled receptor in addition to a natural leader sequence of the heterologous G protein coupled receptor.

20. The yeast cell of claim 11, wherein the heterologous G protein coupled receptor is expressed in the yeast cell using a yeast leader sequence other than an α

-factor leader sequence.

21. The yeast cell of claim 11, wherein the heterologous G protein coupled receptor is expressed in the yeast cell using an α

-factor leader sequence.

22. The yeast cell of claim 13, wherein the human melanocortin receptor is hMCR4 or hMCR5.

23. The yeast cell of claim 14, wherein the human somatostatin receptor is hSSTR2 or hSSTR3.

24. A method of identifying compounds which modulate a G protein coupled receptor, comprising the steps of:

a) providing a yeast cell which comprises;

i) a heterologous G protein coupled receptor which fumctionally couples to the yeast pheromone response pathway; and

ii) a mutation in a nucleic acid encoding an endogenous yeast Cav1 protein which renders said endogenous yeast Cav1 protein nonfunctional;

b) contacting the yeast cell with a compound

c) identifying compounds which induce a change in a detectable signal in the yeast cell,

wherein said change in the detectable signal indicates that the compound is a modulator of the heterologous G protein coupled receptor.

25. The method of claim 24, wherein the yeast cell further comprises a reporter gene construct.

26. The method of claim 24, wherein the compound is from a library of non-peptidic organic molecules.