Ishii I, Saito E, Izumi T, Ui M, Shimizu T
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113, Japan.
J Biol Chem. 1998 Apr 17;273(16):9878-85. doi: 10.1074/jbc.273.16.9878.
Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.
在转染的中国仓鼠卵巢细胞中对血小板活化因子（PAF）受体的激动剂诱导的隔离、再循环和再敏化进行了表征。将细胞暴露于PAF会导致受体迅速隔离到细胞内区室，并使对PAF的反应脱敏。用干扰网格蛋白介导途径的预处理可抑制这种隔离。随后用受体拮抗剂洗涤去除PAF会导致隔离的受体迅速再循环到细胞表面，同时对PAF重新敏感。为了评估在这些过程中受体细胞质尾部磷酸化的潜在作用，构建了尾部被截断或替换以缺乏丝氨酸/苏氨酸残基的突变受体。PAF能快速且强烈地使野生型受体磷酸化，但突变体则不能。每个突变体的最大隔离程度低于野生型，并且其中一个替换突变体未表现出隔离。此外，隔离缺陷型突变体在激动剂刺激后显示出脱敏的证据，但在激动剂去除后未显示再敏化。因此，激动剂诱导的细胞质尾部磷酸化有助于受体隔离，但不是其必需条件，并且隔离/再循环在受体再敏化中似乎很重要。
