Yeast cells expressing modified G proteins and methods of use therefor
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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.
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Citations
42 Claims
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1-4. -4. (canceled)
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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.- View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27)
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25. (canceled)
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28. A method for identifying a compound which modulates a heterologous G protein coupled receptor, comprising:
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(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. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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Specification