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J Mol Biol

Title:		Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene
Author(s):		Sommers CM; Dumont ME;
Address:		"Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642, USA"
Journal Title:		J Mol Biol
Year:		1997
Volume:		266
Issue:		3
Page Number:		559 - 575
DOI:		10.1006/jmbi.1996.0816
ISSN/ISBN:		0022-2836 (Print) 0022-2836 (Linking)
Abstract:		"G protein coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a wide variety of extracellular signals. However, the structural basis for activation of this class of receptors by ligand binding is not well understood. We report here the use of a systematic genetic protocol for identifying interactions among the seven transmembrane helices of the GPCR responsible for cellular responses to the alpha-mating pheromone of the yeast Saccharomyces cerevisiae. Random mutations were introduced into the region of the STE2 gene encoding the third transmembrane segment of the alpha-factor receptor, followed by screening for loss of signaling. The limited spectrum of non-conservative mutations recovered, including removal of the only negatively charged side-chain in the transmembrane region, indicates that most substitutions in the third transmembrane segment do not affect receptor function. Three second-site intragenic suppressors of these initial mutations were isolated following mutagenesis of the remaining six transmembrane segments. One of these suppressors, Y266C in the sixth transmembrane segment, is allele specific and shows non-additivity of phenotypes indicative of a physical interaction between the third and sixth transmembrane regions of the receptor. A second suppressor, M218T in the fifth transmembrane segment, exhibits only partial allele specificity. A third suppressor, R58G, in the first transmembrane segment, suppresses a variety of starting alleles and appears to cause global stabilization of the receptor. Analysis of these suppressors and additional alleles can provide a database for modeling GPCR structure"
Keywords:		"Amino Acid Sequence Cell Membrane GTP-Binding Proteins/*genetics *Gene Expression Regulation, Fungal Molecular Sequence Data Mutagenesis, Site-Directed Receptors, Mating Factor Receptors, Peptide/*genetics Saccharomyces cerevisiae/genetics *Transcription;"
Notes:		"MedlineSommers, C M Dumont, M E eng Research Support, Non-U.S. Gov't Netherlands 1997/02/28 J Mol Biol. 1997 Feb 28; 266(3):559-75. doi: 10.1006/jmbi.1996.0816"