Mathew E, et al. (2011) Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p. J Mol Biol 409(4):513-28
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 a-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 a-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.
|Status: Published||Type: Journal Article | Research Support, N.I.H., Extramural||PubMed ID: 21477594|
Topics addressed in this paper
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|Protein Physical Properties|
|Protein Sequence Features|
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