Chen Q, Konopka J B
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794, USA.
Mol Cell Biol. 1996 Jan;16(1):247-57. doi: 10.1128/MCB.16.1.247.
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.
α因子信息素受体激活一种G蛋白信号级联反应，刺激MATa酵母细胞进行接合。受体的胞质C末端对于G蛋白激活并非必需，而是作为一个调节结构域促进对α因子的适应性。通过突变潜在的磷酸化位点，研究了磷酸化在调节α因子受体中的作用。将受体C末端四个最远端的丝氨酸和苏氨酸残基突变为丙氨酸，导致对α因子的敏感性增加，以及从α因子脉冲中恢复的延迟。32PO4标记实验表明，丙氨酸替代突变降低了受体在体内的磷酸化水平。磷酸化显然改变了G蛋白激活的调节，因为远端磷酸化位点的突变并未显著改变受体数量或对配体的亲和力。此外，在一个未能进行配体刺激的内吞作用的受体突变体中，远端磷酸化位点的突变导致对α因子的敏感性增加，这表明磷酸化调节可以在细胞表面发生，并且独立于内吞作用。受体远端丝氨酸和苏氨酸残基的突变也导致α因子诱导的形态发生出现轻微缺陷，但该缺陷不如受体胞质C末端截短所导致的形态发生缺陷严重。C末端的这些远端残基在受体调节中起特殊作用，因为将接下来五个相邻的丝氨酸和苏氨酸残基突变为丙氨酸并不影响对α因子的敏感性。总之，这些结果表明磷酸化在调节α因子受体功能中起重要作用。
