Assay for agents which alter G-protein coupled receptor activity
AssigneeMillennium Pharmaceuticals, Inc.
InventorsKevin T. Madden, Patrick R. Errada, Carlos J. Gimeno
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Abstract
The invention relates to a novel transformed yeast cell comprising a first heterologous nucleic acid sequence encoding a G-protein coupled receptor; and second nucleic acid sequence encoding the yeast Gα subunit protein Gpa2; and an optional third heterologous nucleic acid sequence encoding a reporter protein. The G-protein coupled receptor can bind ligand thereby altering the activity of Gpa2 and cellular levels of second messenger molecules and ultimately the activity of target genes. The transformed yeast cell can be used, for example, in in vitro high throughput assays to screen for agonists and antagonists of G-protein coupled receptor ligand activity.
Claims12
A transformed yeast cell responsive to G-protein coupled activation of a yeast Gα Gpa2 protein comprising a nucleic acid construct comprising a promoter selected from the group consisting of YMR323w, YBL100c, YDR187c, YEL045c, YBLO96c, YNL028w, YFR056c, PHO4, YCL046w, YPL281c, YJL045w, YGL046w, GLK1, YML058c-a, YJR114w, IDH2, YML035c-a, HSP12, HSP26, YNL134c and YML128c, wherein said promoter is operably linked to a heterologous first DNA sequence which is operably linked to a second DNA sequence, wherein said heterologous first DNA sequence encodes a mammalian G-protein coupled receptor, and wherein said second DNA sequence encodes a yeast Gα Gpa2 protein, such that expression of the first and second DNA sequences produces a fusion protein wherein the yeast Gα Gpa2 protein is linked to the mammalian G-protein coupled receptor, and wherein binding of a ligand to the mammalian G-protein coupled receptor results in alteration of cellular levels or activity of adenylate cyclase and/or cAMP.
A nucleic acid construct comprising a promoter selected from the group consisting of YMR323w, YBL100c, YDR187c, YEL045c, YBLO96c, YNL028w, YFR056c, PHO4, YCL046w, YPL281c, YJL045w, YGL046w, GLK1, YML058c-a, YJR114w, IDH2, YML035c-a, HSP12, HSP26, YNL134c and YML128c, wherein said promoter is operably linked to a heterologous first DNA sequence which is operably linked to a second DNA sequence, wherein said heterologous first DNA sequence encodes a mammalian G-protein coupled receptor, and wherein said second DNA sequence encodes a yeast Gα Gpa2 protein, such that expression of the first and second DNA sequences produces a fusion protein wherein the yeast Gα Gpa2 protein is linked to the mammalian G-protein coupled receptor.
A transformed yeast cell responsive to G-protein coupled activation of a yeast Gα Gpa2 protein comprising a nucleic acid construct comprising a promoter selected from the group consisting of YMR323w, YBL100c, YDR187c, YEL045c, YBLO96c, YNL028w, YFR056c, PHO4, YCL046w, YPL281c, YJL045w, YGL046w, GLK1, YML058c-a, YJR114w, IDH2, YML035c-a, HSP12, HSP26, YNL134c and YML128c, wherein said promoter is operably linked to a heterologous first DNA sequence which is operably linked to a second DNA sequence, wherein said heterologous first DNA sequence encodes a G-protein coupled receptor, and wherein said second DNA sequence encodes a yeast Gpa2 protein, such that expression of the first and second DNA sequences produces a fusion protein wherein the yeast Gpa2 protein is linked to the mammalian G protein coupled receptor.
Dependencies are inferred from the claim text.