Yeast cells having mutations in stp22 and uses therefor

• US 6,355,473 B1
• Filed: 05/05/1999
• Issued: 03/12/2002
• Est. Priority Date: 05/06/1998
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Pharmaceutical Corporation
• Inventors: Ostanin, Kirill, Silverman, Lauren
• Primary Examiner(s): Eyler, Yvonne
• Assistant Examiner(s): BRANNOCK, MICHAEL T
• Application Number:
  US09/305,923
• Time in Patent Office: 1,042 Days
• Field of Search: 435/7.1, 435/254.11, 435/254.2, 435/254.21, 435/320.7, 435/69.1, 435/7.2, 435/7.31, 530/350, 536/23.4, 536/23.1, 436/501
• US Class Current: 506/10
• CPC Class Codes: C07K 14/39 from yeasts
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides yeast cells which comprise a mutation in an endogenous yeast stp22 gene and which express a heterologous G protein coupled receptor. 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. A recombinant yeast cell having an endogenous yeast pheromone system pathway which yeast cell comprises:

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

(ii) a mutation which renders an endogenous yeast Stp22 protein nonfunctional.

2. The yeast cell of claim 1, which yeast cell further comprises a heterologous or chimeric G protein subunit.

3. The yeast cell of claim 1, which yeast cell further comprises a reporter gene construct which produces a detectable signal upon stimulation of the yeast pheromone system pathway.

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

5. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is a mammalian G protein coupled receptor.

6. The yeast cell of claim 5, wherein the heterologous G protein coupled receptor is a human G protein coupled receptor.

7. The yeast cell of claim 1, 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.

8. The yeast cell of claim 1, 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.

9. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is expressed in the yeast cell using a yeast leader sequence.

10. The yeast cell of claim 9, wherein the yeast leader sequence is used to express a heterologous G protein coupled receptor that naturally lacks a leader sequence.

11. The yeast cell of claim 9, wherein the yeast leader sequence replaces a natural leader sequence of the heterologous G protein coupled receptor.

12. The yeast cell of claim 9, 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.

13. The yeast cell of claim 9, 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.

14. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is a human nociceptin receptor.

15. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is a human melanocortin receptor.

16. The yeast cell of claim 15, wherein the human melanocortin receptor is hMCR4 or hMCR5.

17. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is a human somatostatin receptor.

18. The yeast cell of claim 17, wherein the human somatostatin receptor is hSSTR2 or hSSTR3.

19. The yeast cell of claim 1, wherein the heterologous G protein coupled receptor is a human corticotropin releasing factor receptor.

20. The yeast cell of claim 19, wherein the human corticotropin releasing factor receptor is hCRFR1 or hCRFR2α

.

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

a) providing a yeast cell which comprise;

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

(ii) a mutation which renders an endogenous yeast Stp22 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 detectable signal indicates that the compound is a modulator of the heterologous G protein coupled receptor.

22. The assay of claim 21, wherein the yeast cell further comprises a reporter gene construct.

23. The assay of claim 21, wherein the test compound is from a library of non-peptidic organic molecules.