« Previous AbstractEffects of oxygen vacancy defect on microwave pyrolysis of biomass to produce high-quality syngas and bio-oil: Microwave absorption and in-situ catalytic    Next AbstractIdentification of residues that contribute to receptor activation through the analysis of compensatory mutations in the G protein-coupled alpha-factor receptor »

Mol Cell Biol

Title:		A microdomain formed by the extracellular ends of the transmembrane domains promotes activation of the G protein-coupled alpha-factor receptor
Author(s):		Lin JC; Duell K; Konopka JB;
Address:		"Graduate Program in Molecular and Cellular Biology, State University of New York, Stony Brook, New York 11794-5222, USA"
Journal Title:		Mol Cell Biol
Year:		2004
Volume:		24
Issue:		5
Page Number:		2041 - 2051
DOI:		10.1128/MCB.24.5.2041-2051.2004
ISSN/ISBN:		0270-7306 (Print) 1098-5549 (Electronic) 0270-7306 (Linking)
Abstract:		"The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs"
Keywords:		"Amino Acid Sequence Binding Sites Biotin/chemistry/metabolism Cell Division/physiology Cysteine/metabolism Genes, Reporter Ligands Mesylates/chemistry/metabolism Models, Molecular Mutation Phenotype Pheromones/metabolism Protein Binding *Protein Structure;"
Notes:		"MedlineLin, Jennifer C Duell, Ken Konopka, James B eng R01 GM055107/GM/NIGMS NIH HHS/ T32 CA009176/CA/NCI NIH HHS/ GM55107/GM/NIGMS NIH HHS/ T32CAO9176/CA/NCI NIH HHS/ Research Support, U.S. Gov't, P.H.S. 2004/02/18 Mol Cell Biol. 2004 Mar; 24(5):2041-51. doi: 10.1128/MCB.24.5.2041-2051.2004"