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

JOURNAL OF MOLECULAR BIOLOGY

卷:409

Article

Mathew, Elizabeth¹  Bajaj, Anshika¹  Connelly, Sara M.¹  Sargsyan, Hasmik^2,3  Ding, Fa-Xiang^2,3  Hajduczok, Alexander G.¹  Naider, Fred^2,3  Dumont, Mark E.¹ 

[1] Univ Rochester, Sch Med & Dent, Dept Biochem & Biophys, Rochester, NY 14642 USA

[2] CUNY, Dept Chem, Coll Staten Isl, New York, NY 10314 USA

[3] CUNY, Macromol Assemblies Inst, New York, NY 10314 USA

关键词: receptor activation;    ligand binding;    flow cytometry;    G protein signaling;    fluorescent ligands;   

DOI  :  10.1016/j.jmb.2011.03.059

来源: Elsevier

【 摘 要 】

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 alpha-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 alpha-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. (C) 2011 Elsevier Ltd. All rights reserved.

【 授权许可】

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