β-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor
β-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor
复制标题
DOI：
10.1124/mol.59.2.239
复制
发表时间：
2001-02-01
影响因子：
3.6
通讯作者：
Hunyady, L
中科院分区：
文献类型：
--
作者：
Gáborik, Z;Szaszák, M;Hunyady, L
激动剂诱导 G 蛋白偶联受体 (GPCR) 内化的主要机制是通过网格蛋白包被的囊泡进行 β-抑制蛋白和动力依赖性内吞作用。然而，最近的报告表明，一些 GPCR（以人胚胎肾 (HEK) 293 细胞中表达的 AT(1) 血管紧张素受体为例）通过不依赖于 β-抑制蛋白和动力蛋白的机制，并且可能通过不依赖于网格蛋白的途径内化。在这项研究中，中国仓鼠卵巢 (CHO) 细胞中表达的大鼠 AT(1A) 受体的激动剂诱导的内吞作用在高渗蔗糖处理期间被网格蛋白消耗所消除，并且不受菲律宾蛋白通过小凹抑制内吞作用的影响。此外，内化的荧光素缀合的血管紧张素 II 出现在内体中，如与转铁蛋白的共定位所证明的。 β-arrestin1（V53D）和β-arrestin1（1-349）的过度表达对CHO细胞中放射性碘化血管紧张素II的内吞作用产生显性负抑制作用。 dynamin-1 和 dynamin-2 的 GTPase 缺陷型 (K44A) 突变体形式，以及磷酸肌醇结合受损的 pleckstrin 同源结构域突变体 (K535A) dynamin-2，也抑制 CHO 细胞中 AT(1) 受体的内吞作用。在 COS-7 和 HEK 293 细胞中也获得了类似的结果。使用荧光素缀合的血管紧张素 II 进行的共聚焦显微镜显示，dynamin-1 (K44A) 和 dynamin-2 (K44A) 亚型的过表达同样会抑制 CHO 细胞中激动剂诱导的 AT(1) 受体内吞作用。对 AT(1) 受体内吞作用的血管紧张素 II 浓度依赖性的研究表明，在较高的激动剂浓度下，其速率常数降低，并且显性负性动力构建体的抑制作用被消除。这些数据证明了在生理血管紧张素II浓度下通过网格蛋白包被的囊泡对AT(1)受体进行β-抑制蛋白和动力依赖性内吞作用的重要性。
The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta -arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT(1) angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta -arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT(1A) receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of beta -arrestin1( V53D) and beta -arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1( K44A) and dynamin-2( K44A) isoforms likewise inhibited agonist-induced AT(1) receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT(1) receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of beta -arrestin- and dynamin-dependent endocytosis of the AT(1) receptor via clathrin-coated vesicles at physiological angiotensin II concentrations.