Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p |
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Authors: | Mathew Elizabeth Bajaj Anshika Connelly Sara M Sargsyan Hasmik Ding Fa-Xiang Hajduczok Alexander G Naider Fred Dumont Mark E |
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Institution: | 1Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA 2Department of Chemistry, College of Staten Island and, Macromolecular Assemblies Institute, City University of New York, New York, NY 10314, USA |
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Abstract: | We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor. |
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Keywords: | GPCR (G protein coupled receptor) NBD (7-nitrobenz-2-oxa-1 3-diazol-4-yl) |
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