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Mol Cell Biol

Title:		"The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization"
Author(s):		Stefan CJ; Blumer KJ;
Address:		"Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110"
Journal Title:		Mol Cell Biol
Year:		1994
Volume:		14
Issue:		5
Page Number:		3339 - 3349
DOI:		10.1128/mcb.14.5.3339-3349.1994
ISSN/ISBN:		0270-7306 (Print) 1098-5549 (Electronic) 0270-7306 (Linking)
Abstract:		"To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization"
Keywords:		"Alleles Amino Acid Sequence Genes, Fungal Genotype Kinetics Mating Factor Models, Structural Molecular Sequence Data Mutagenesis, Site-Directed Peptides/pharmacology Pheromones/pharmacology Plasmids *Protein Structure, Secondary Receptors, Mating Factor R;"
Notes:		"MedlineStefan, C J Blumer, K J eng MC_UU_12018/6/MRC_/Medical Research Council/United Kingdom GM44592/GM/NIGMS NIH HHS/ GM08036/GM/NIGMS NIH HHS/ Comparative Study Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1994/05/01 Mol Cell Biol. 1994 May; 14(5):3339-49. doi: 10.1128/mcb.14.5.3339-3349.1994"