Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene

JOURNAL OF MOLECULAR BIOLOGY

卷:266

Article

Sommers, CM ; Dumont, ME

关键词: G protein coupled receptor;    intragenic suppressor;    alpha-factor receptor;    mating pheromone;    membrane protein;   

DOI  :  10.1006/jmbi.1996.0816

来源: Elsevier

【 摘 要 】

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. (C) 1997 Academic Press Limited.

【 授权许可】

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