Cell based signal generation
First Claim
1. A recombinant yeast cell comprising:
- a mutated Fus3 yeast protein comprising an amino acid substitution at positions 42, 180 or 182, or at positions 180 and 182 as compared to wild type Fus3, and wherein said yeast protein is operatively linked to a chimeric transcription factor;
a heterologous receptor selected from the group consisting of melatonin receptor 1a, galanin receptor 1, neurotensin receptor, adenosine receptor 2a, somatostatin receptor 2, and corticotropin releasing factor receptor 1, wherein said receptor is functionally integrated into a pheromone response pathway of the yeast cell; and
a gene which produces a detectable protein, wherein said yeast protein is responsive to said yeast pheromone response pathway of the cell; and
wherein the activity of said yeast protein is modulated indirectly upon stimulation of said pathway, and wherein upon modulation of said yeast protein, said chimeric transcription factor causes a detectable signal to be generated.
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Abstract
The present invention makes available a rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular protein, e.g., a receptor or ion channel. The subject assay enables rapid screening of large numbers of compounds to identify those which act as an agonist or antagonist to the bioactivity of the cellular protein. In particular, the assay of the invention makes use of a cell that harbors a protein that is responsive to a cellular signal transduction pathway. The protein is operatively linked to a polypeptide which causes a detectable signal to be generated upon stimulation of the pathway, e.g., when a compound interacts with and modulates the activity of a cellular receptor or ion channel of the cell. Thus, the cell provides a signal generation means comprising a novel fusion protein the expression of which is independent of stimulation/activation of the signal transduction pathway, but the activity of which is responsive to the signal transduction pathway.
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Citations
49 Claims
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1. A recombinant yeast cell comprising:
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a mutated Fus3 yeast protein comprising an amino acid substitution at positions 42, 180 or 182, or at positions 180 and 182 as compared to wild type Fus3, and wherein said yeast protein is operatively linked to a chimeric transcription factor; a heterologous receptor selected from the group consisting of melatonin receptor 1a, galanin receptor 1, neurotensin receptor, adenosine receptor 2a, somatostatin receptor 2, and corticotropin releasing factor receptor 1, wherein said receptor is functionally integrated into a pheromone response pathway of the yeast cell; and a gene which produces a detectable protein, wherein said yeast protein is responsive to said yeast pheromone response pathway of the cell; and
wherein the activity of said yeast protein is modulated indirectly upon stimulation of said pathway, and wherein upon modulation of said yeast protein, said chimeric transcription factor causes a detectable signal to be generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A recombinant yeast cell comprising:
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a mutated Fus3 yeast protein comprising an amino acid substitution at positions 42, 180 or 182, or at positions 180 and 182 as compared to wild type Fus3, wherein said amino acid substitution is selected from the group consisting of Lys42Arg, Thr180Val, Thr180Glu, Tyr182Val and Tyr182Asp and wherein said yeast protein is operatively linked to a chimeric transcription factor; a heterologous receptor selected from the group consisting of melatonin receptor 1a, galanin receptor 1, neurotensin receptor, adenosine receptor 2a, somatostatin receptor 2, and corticotropin releasing factor receptor 1, wherein said receptor is functionally integrated into a pheromone response pathway of the yeast cell; and a gene which produces a detectable protein, wherein said yeast protein is responsive to said yeast pheromone response pathway of the cell; and
wherein the activity of said yeast protein is modulated indirectly upon stimulation of said pathway, and wherein upon modulation of said yeast protein, said chimeric transcription factor causes a detectable signal to be generated.
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Specification