Methods and compositions for identifying receptor effectors
First Claim
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1. A mixture of recombinant yeast cells, each cell of which comprises:
- (i) a recombinant gene encoding a heterologous orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; and
(ii) a recombinant gene encoding a heterologous test polypeptide, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane,wherein collectively the mixture of cells expresses a library of said test polypeptides, and modulation of the signal transduction activity of the receptor by a heterologous test polypeptides within the library provides a detectable signal.
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Abstract
The present invention makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which induce or antagonize receptor bioactivity. The subject assay is particularly amenable for identifying surrogate ligands for orphan receptors.
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Citations
19 Claims
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1. A mixture of recombinant yeast cells, each cell of which comprises:
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(i) a recombinant gene encoding a heterologous orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; and (ii) a recombinant gene encoding a heterologous test polypeptide, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane, wherein collectively the mixture of cells expresses a library of said test polypeptides, and modulation of the signal transduction activity of the receptor by a heterologous test polypeptides within the library provides a detectable signal. - View Dependent Claims (18)
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2. A mixture of recombinant yeast cells, each cell of which comprises:
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(i) a heterologous orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; (ii) a recombinant gene encoding a heterologous test polypeptide, receptor, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane; and (iii) a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the receptor, wherein collectively the mixture of cells expresses a library of test polypeptides.
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3. A mixture of recombinant yeast cells, each cell of which comprises:
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(i) an orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; (ii) a recombinant gene encoding a heterologous test polypeptide and including a signal sequence for secretion, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane; and (iii) a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the receptor, wherein collectively the mixture of cells expresses a library of test polypeptides.
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4. A mixture of recombinant yeast cells, each cell of which comprises:
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(i) an orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; and (ii) a recombinant gene encoding a heterologous test polypeptide and including a signal sequence for secretion, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane, wherein collectively the mixture of cells expresses a library of test polypeptides, and modulation of the signal transduction activity of the receptor by a test polypeptide within the library provides a detectable signal. - View Dependent Claims (5, 6, 7, 8, 9)
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10. A recombinant yeast cell, comprising:
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(i) a recombinant gene encoding a heterologous G protein-coupled receptor protein wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by an extracellular signal; (ii) a recombinant gene encoding a heterologous test polypeptide, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane; and (iii) a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity or the receptor. - View Dependent Claims (11, 12, 13, 19)
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14. A mixture of recombinant yeast cells, each cell of which comprises:
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(i) a recombinant gene encoding a heterologous orphan G protein-coupled receptor wherein said receptor is expressed on the cell membrane of said cell such that signal transduction activity is modulated by interaction with an extracellular signal; (ii) a recombinant gene encoding a heterologous test polypeptide and including a signal sequence for secretion, wherein the test polypeptide is transported to a location allowing interaction with the receptor expressed on the cell membrane; and (iii) a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the receptor, wherein collectively the mixture of cells expresses a library of test polypeptides. - View Dependent Claims (15, 16, 17)
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Specification