Regulation of the G-protein-coupled α-factor pheromone receptor by phosphorylation

The α-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 α-factor. The role of phosphorylation in regulating the α-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 α-factor and a delay in recovering from a pulse of α-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 α-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 α-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 α-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating α-factor receptor function.