Takano T, Honda Z, Sakanaka C, Izumi T, Kameyama K, Haga K, Haga T, Kurokawa K, Shimizu T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1994 Sep 2;269(35):22453-8.
The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.
血小板活化因子（PAF）受体与多种信号通路偶联，如磷脂酶C、磷脂酶A2的激活，丝裂原活化蛋白激酶的激活以及腺苷酸环化酶的抑制。PAF诱导的信号会因激动剂的重复或长期应用而减弱（同源脱敏）。为了研究激动剂诱导脱敏的潜在机制，我们构建了克隆的豚鼠PAF受体的突变形式，并在中国仓鼠卵巢细胞中稳定表达。表达野生型受体的细胞在PAF刺激下短暂激活磷脂酶C。即使在持续存在配体的情况下，细胞内肌醇1,4,5-三磷酸水平和细胞内Ca2+浓度在20秒内达到最高水平，并在几分钟内恢复到基础水平。相比之下，缺少羧基末端胞质尾巴的截短型PAF受体导致肌醇1,4,5-三磷酸和细胞内Ca2+浓度持续升高。在另一个突变体中也观察到类似的结果，其中羧基末端尾巴中的丝氨酸/苏氨酸残基被丙氨酸取代。与野生型受体相比，两种突变型PAF受体更有效地激活了其他信号（丝裂原活化蛋白激酶激酶、花生四烯酸释放和腺苷酸环化酶抑制）。因此，虽然PAF受体的羧基末端胞质尾巴对于多种信号的正向激活不是必需的，但它在激动剂通过磷酸受体诱导的信号衰减中起着关键作用。我们还注意到，PAF受体羧基末端尾巴的合成肽被重组β-肾上腺素能受体激酶1强烈磷酸化，表明它或其相关物可能参与PAF受体的磷酸化和同源脱敏。
