Cell having amplified signal transduction pathway responses and uses therefor

• US 7,273,747 B2
• Filed: 10/08/2002
• Issued: 09/25/2007
• Est. Priority Date: 08/27/1998
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Manfredi, John P., Wu, Meng-Yu, Benton, Benjamin K.
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US10/267,074
• Publication Number:
  US 20030108999A1
• Time in Patent Office: 1,813 Days
• Field of Search: None
• US Class Current: 435/254.21
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

The invention provides recombinant cells that have been engineered such that ligand stimulation of a receptor expressed by the cells leads to amplified signal transduction responses. In one embodiment, the receptor-expressing cells have been engineered to carry a heterologous DNA construct comprising a gene encoding a protein that activates the signal transduction pathway, which gene is operatively linked to a promoter that is responsive to activation of the signal transduction pathway. Stimulation of the receptor by a ligand leads to expression of the heterologous DNA construct encoding the protein that activates the signal transduction pathway such that signals generated by ligand binding to the receptor are amplified. Preferred cells are yeast cells expressing heterologous G protein coupled receptors functionally coupled to the yeast pheromone response pathway and overexpressing Ste5p, Ste4p, Ste12p, Ste11p or a dominant truncation allele of Ste20 via a pheromone-responsive promoter. The invention further provides cells expressing a heterologous receptor, wherein an endogenous gene encoding a protein that negatively regulates an endogenous signal transduction pathway is mutated to render the protein nonfunctional such that signals generated by ligand binding to the receptor are amplified. The invention further provides cells expressing a heterologous receptor, wherein an endogenous gene encoding a protein that positively regulates an endogenous signal transduction pathway is mutated to a supersensitive form such that the sensitivity of the pathway to ligand stimulation is increases. Methods of using the cells of the invention to identify receptor modulators are also provided.

Claims

1. A recombinant yeast cell comprising:

(i) a heterologous G protein-coupled receptor that, upon ligand stimulation, activates the endogenous yeast pheromone response pathway; and

(ii) a heterologous DNA construct comprising a gene encoding STE5, which STE5 activates the yeast pheromone response pathway, which gene is operably linked to a promoter that is responsive to activation of the yeast pheromone response pathway, wherein stimulation of the receptor by a ligand leads to expression of the gene encoding STE5 that activates the yeast pheromone response pathway such that signals generated by ligand binding to the receptor are amplified.

2. The yeast cell of claim 1, wherein the promoter that is responsive to activation of the yeast pheromone response pathway is selected from the group consisting of:

FUS1, AGA1, FAR1, and FUS2.

3. The yeast cell of claim 1, further comprising an endogenous yeast gene encoding a phosphatase that negatively regulates the yeast pheromone system pathway, wherein said endogenous gene is mutated to render the protein nonfunctional.

4. The yeast cell of claim 3, wherein the endogenous gene encoding the phosphatase is selected from the group consisting of:

MSG5, PTP2, and PTP3.

5. The yeast cell of claim 1, which further comprises a reporter gene construct that produees a detectable signal upon activation of the yeast pheromone response pathway.

6. The yeast cell of claim 5, wherein the reporter gene is lacZ.

7. The yeast cell of claim 5, wherein the reporter gene construct that produces a detectable signal is a FUS-1 promoter operatively linked to lacZ.

8. The yeast cell of claim 1, wherein the heterologous DNA construct is carried by a high copy number plasmid.

9. The yeast cell of claim 1, wherein the heterologous DNA construct is carried by a low copy number plasmid.

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

11. An assay to identify compounds that modulate the activity of a receptor, comprising:

(i) providing a recombinant cell as claimed in claim 1, wherein a detectable signal is produced in the cell upon stimulation of the receptor;

(ii) contacting the cell with a test compound; and

(iii) identifying a compound which induces a change in the detectable signal in the cell, such a change indicating that the compound modulates the activity of the receptor.

12. The assay of claim 11, wherein the cell comprises a reporter gene construct that produces a detectable signal upon receptor stimulation.

13. The yeast cell of claim 1, wherein the promoter is a FUS1 promoter.

14. The yeast cell of claim 1, wherein the gene encodes a mutant form of the Ste5 protein.

15. The yeast cell of claim 14, wherein the mutant Ste5 protein has a T52M mutation or T52M, S18R mutations.