Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

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 environme...

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Bibliographic Details
Published in:Journal of molecular biology 2011-06, Vol.409 (4), p.513-528
Main Authors: Mathew, Elizabeth, Bajaj, Anshika, Connelly, Sara M., Sargsyan, Hasmik, Ding, Fa-Xiang, Hajduczok, Alexander G., Naider, Fred, Dumont, Mark E.
Format: Article
Language:English
Subjects:
A-factor
agonists
Amino Acid Sequence
Antagonists
Antibodies
Cell surface
Flow cytometry
Flow Cytometry - methods
Fluorescence
Fluorescent Dyes - chemistry
fluorescent ligands
fungal antagonists
G protein signaling
G protein-coupled receptors
ligand binding
Ligands
Molecular Sequence Data
Mutation
Probes
Protein Binding
Protein Conformation
rapid methods
receptor activation
receptors
Receptors, Mating Factor - agonists
Receptors, Mating Factor - antagonists & inhibitors
Receptors, Mating Factor - genetics
Receptors, Mating Factor - metabolism
Saccharomyces cerevisiae - cytology
Saccharomyces cerevisiae Proteins - agonists
Saccharomyces cerevisiae Proteins - antagonists & inhibitors
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
yeasts
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Summary: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. [Display omitted]
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2011.03.059