Yeast cells expressing modified G proteins and methods of use therefor

• US 20050202403A1
• Filed: 10/15/2004
• Published: 09/15/2005
• Est. Priority Date: 03/31/1993
• Status: Active Grant

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Trueheart, Joshua, Broach, James R., Murphy, Andrew J.M., Klein, Christine A., Fowlkes, Dana M., Manfredi, John P., Paul, Jeremy I.
• Granted Patent: US 7,361,498 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/4722 G-proteins
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• 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 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 a factor leader sequence. Drug discovery assays using the subject yeast cells are also provided.

Claims

1-4. -4. (canceled)

5. 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 STE18.

6. The yeast cell of claim 5, 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.

7. The yeast cell of claim 5, wherein said first polypeptide is from yeast STE18 and said second polypeptide is from a heterologous G protein γ

subunit.

8. The yeast cell of claim 5, wherein said heterologous G protein subunit is mammalian.

9. The yeast cell of claim 8, wherein said heterologous G protein subunit is human.

10. The yeast cell of claim 9, wherein at least one of said first and second polypeptides comprises a naturally occurring amino acid sequence.

11. The yeast cell of claim 9, wherein at least one of said first and second polypeptides comprises a non-naturally occurring amino acid sequence.

12. The yeast cell of claim 5, further comprising a heterologous G protein coupled receptor, which receptor is functionally integrated into the yeast cell.

13. The yeast cell of claim 12, 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.

14. The yeast cell of claim 7, wherein said second polypeptide is derived from the human Gγ

2 subunit.

15. The yeast cell of claim 14, wherein said second polypeptide comprises the amino acid sequence Arg Glu Lys Lys Phe Phe (amino acids 19-24 of SEQ ID NO:

33).

16. The yeast cell of claim 15, wherein said second polypeptide comprises the sequence shown in SEQ ID NO:

33.

17. The yeast cell of claim 6, wherein said chimeric G protein subunit is selected from the group consisting of:

gpal (41)-Gα

i2;

gpal (41)-Gα

16; and

gpal (41)-Gα

s.

18. The yeast cell of claim 17, wherein the Gα

i2, Gα

16, or Gα

s portion of said chimeric G protein subunit comprises an amino acid substitution compared to wild type Gα

i2, Gα

16, or Gα

s.

19. The yeast cell of claim 5, further comprising 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. The yeast cell of claim 12, wherein an endogenous yeast pheromone system receptor protein is not produced in functional form.

22. The yeast cell of claim 12, further comprising an indicator gene that produces a detectable signal upon functional coupling of the heterologous G protein coupled receptor to the G protein.

23. The yeast cell of claim 5, wherein the cell is a Saccharomyces cerevisiae cell.

24. The yeast cell of claim 12, wherein said heterologous G protein coupled receptor is an orphan receptor.

26. An assay to identify compounds capable of modulating the dissociation of Gα

and Gβ

γ

, comprising the steps of;

(i) providing a yeast cell according to claim 12 or 25,

(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

Gβ

γ

.

27. The assay of claim 26, wherein said test compound is from a library of non-peptidic organic molecules.

25. (canceled)

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 gpal 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 Gα

i subunit.

32. The assay of claim 30, wherein said second polypeptide is from a mammalian Gα

16 subunit.

33. The assay of claim 30, wherein said second polypeptide is from a mammalian Gα

s subunit.

34. The assay of claim 28, wherein the first chimeric G protein subunit comprises a polypeptide from mammalian Gα

12, the second chimeric G protein subunit comprises a polypeptide from mammalian Gα

16, and the third chimeric G protein subunit comprises a polypeptide from mammalian Gα

s.

35. The assay of claim 34, wherein the second chimeric G protein subunit comprises Gα

16(S270P) and the third chimeric G protein subunit comprises Gα

s(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 STE18 and a second polypeptide from a mammalian G protein γ

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.