Dual pathways of internalization of the cholecystokinin receptor.
Dual pathways of internalization of the cholecystokinin receptor.
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DOI：
10.1083/jcb.128.6.1029
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发表时间：
1995-03
期刊：
影响因子：
--
通讯作者：
Miller LJ
中科院分区：
文献类型：
--
作者：
Roettger BF;Rentsch RU;Pinon D;Holicky E;Hadac E;Larkin JM;Miller LJ
受体分子在激动剂刺激的细胞反应的脱敏中起主要作用。对于G蛋白偶联受体，通过受体磷酸化、隔离和内化发生快速脱敏，但这些事件发生的细胞间隔及其相互关系尚不清楚。在这项工作中，我们集中在CCK受体，它已经被很好地表征了磷酸化。我们使用新的荧光和电子致密的CCK受体配体和抗体来探测携带CCK受体的CHO细胞系中的受体定位。在非刺激状态下，受体弥漫分布在质膜上。激动剂通过依赖和独立两种途径刺激内吞作用。前者占优势，导致内质体室和溶酶体室，并循环到质膜。不依赖于笼蛋白的突起导致质膜附近有一个光滑的囊泡室，类似于小凹，它不会将配体输送到细胞内更深的地方。钾的耗竭在很大程度上消除了依赖于胞质蛋白的内吞作用，同时不干扰激动剂刺激的受体进入质膜下平滑的囊泡室。这些细胞内吞事件可能与CCK受体磷酸化和去磷酸化的既定周期有关，这是我们先前描述的(Klueppelberg，U.G.，L.K.Gates，F.S.Gorelick和L.J.Miller)。1991年。J.Biol.化学。2403-2408；Lutz，M.P.，D.I.Pinon，L.K.盖茨，S.1993年。J.Biol.化学。268：12136-12142)。激动剂占用后受体磷酸化的快速开始和高峰与胞浆定位最相关，而受刺激的受体磷酸酶活性与受体在细胞内的滞留最相关。我们推测，靠近质膜的光滑的囊泡室对受体的快速再增敏起作用，而经典的网状蛋白介导的内吞途径是通过溶酶体降解下调受体的关键，以及较不迅速的再增敏。
Receptor molecules play a major role in the desensitization of agonist- stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.