REGULATION OF THE G-PROTEIN-COUPLED ALPHA-FACTOR PHEROMONE RECEPTOR BY PHOSPHORYLATION

The alpha-factor pheromone receptor activates a G protein signaling ca scade that stimulates MATa yeast cells to undergo conjugation. The cyt oplasmic C terminus of the receptor is not necessary for G protein act ivation but instead acts as a regulatory domain that promotes adaptati on to alpha-factor. The role of phosphorylation in regulating the alph a-factor receptor was examined by mutating potential phosphorylation s ites. Mutation of the four most distal serine and threonine residues i n the receptor C terminus to alanine caused increased sensitivity to a lpha-factor and a delay in recovering from a pulse of alpha-factor. (P O4)-P-32 labeling experiments demonstrated that the alanine substituti on mutations decreased the in vivo phosphorylation of the receptor. Ph osphorylation apparently alters the regulation of G protein activation , since neither receptor number nor affinity for ligand was significan tly altered by mutation of the distal phosphorylation sites. Furthermo re, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased s ensitivity to alpha-factor, which suggests that regulation by phosphor ylation can occur at the cell surface and is independent of endocytosi s. Mutation of the distal serine and threonine residues of the recepto r also caused a slight defect in alpha-factor-induced morphogenesis, b ut 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 residu es to alanine did not affect the sensitivity to alpha-factor. Altogeth er, these results indicate that phosphorylation plays an important rol e in regulating alpha-factor receptor function.