title: 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 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mathew
authors.forename: Anshika
authors.initials: A
authors.lastname: Bajaj
authors.forename: Sara M
authors.initials: SM
authors.lastname: Connelly
authors.forename: Hasmik
authors.initials: H
authors.lastname: Sargsyan
authors.forename: Fa-Xiang
authors.initials: FX
authors.lastname: Ding
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Hajduczok
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dumont
mesh_terms: D000595:Amino Acid Sequence; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D051964:Receptors, Mating Factor / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology; D029701:Saccharomyces cerevisiae Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins