Hishinuma S, Naiki A, Tsuga H, Young J M
Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
J Neurochem. 1998 Dec;71(6):2626-33. doi: 10.1046/j.1471-4159.1998.71062626.x.
We investigated the regulation by intracellular Ca2+ of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca2+/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H1-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of Gq protein-coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.
我们研究了细胞内Ca2+对激动剂诱导的人U373 MG星形细胞瘤细胞中Gq蛋白偶联组胺H1受体隔离的调节作用。组胺诱导的H1受体从细胞表面膜的隔离表现为完整细胞上可被亲水性H1受体拮抗剂匹多宁作用的[3H]美吡拉敏结合位点的丧失。[3H]美吡拉敏对匹多宁敏感的结合变化与通过蔗糖密度梯度离心检测到的H1受体亚细胞分布变化相对应。组胺诱导的H1受体隔离在高渗培养基中不发生，已知高渗培养基中网格蛋白介导的内吞作用受到抑制，但在无细胞外Ca2+或存在钙调蛋白拮抗剂W-7和氯米达唑时显著加速。蛋白激酶C抑制剂（H-7和GF109203X）、Ca2+/钙调蛋白依赖性蛋白激酶II（KN-62）或蛋白磷酸酶2B（FK506）均未改变H1受体隔离的时间进程。这些结果首次证明，激动剂诱导的、网格蛋白介导的Gq蛋白偶联受体隔离被Ca2+/钙调蛋白短暂抑制，结果是受体在激动剂刺激的早期阶段仍保留在细胞表面膜上。
