Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.
The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794, USA.
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Konopka
D001665:Binding Sites / Q000235:genetics; D003227:Conjugation, Genetic / Q000187:drug effects; D004271:DNA, Fungal / Q000235:genetics; D004705:Endocytosis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008024:Ligands; D000072235:Mating Factor; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D010675:Pheromones / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D014157:Transcription Factors
D004271:DNA, Fungal; D008024:Ligands; D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D019204:GTP-Binding Proteins