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Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.


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 ?-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 ?-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.

SUBMITTER: Mathew E 

PROVIDER: S-EPMC3104124 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

Mathew Elizabeth E   Bajaj Anshika A   Connelly Sara M SM   Sargsyan Hasmik H   Ding Fa-Xiang FX   Hajduczok Alexander G AG   Naider Fred F   Dumont Mark E ME  

Journal of molecular biology 20110406 4


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 fluore  ...[more]

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