Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju, 61186, South Korea.
Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju, 61186, South Korea.
Biochem Biophys Res Commun. 2020 Dec 10;533(3):383-390. doi: 10.1016/j.bbrc.2020.09.030. Epub 2020 Sep 19.
Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and β-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and β-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/β-arrestins. For this we utilized the following: (i) mutant dopamine D receptor and β adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, β-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/β-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/β-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of β-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by β-arrestin ubiquitination is a critical cellular event that links GRK2 and β-arrestins to clathrin-mediated endocytosis.
网格蛋白介导的和小窝蛋白介导的内吞途径是 G 蛋白偶联受体 (GPCR) 内化的主要途径。GPCR 激酶 2 (GRK2) 和β-arrestin 是介导 GPCR 内吞作用的代表性蛋白。然而，GRK2 和β-arrestin 介导网格蛋白介导的和小窝蛋白介导的内吞作用的分子机制尚不清楚。在这项研究中，我们确定了介导网格蛋白/小窝蛋白 1 和 GRK2/β-arrestin 选择性相互作用的细胞成分和过程。为此，我们利用了以下方法：(i) 改变潜在的 GRK2 磷酸化位点的多巴胺 D 受体和β肾上腺素受体突变体，以及 (ii) 敲低网格蛋白、小窝蛋白 1、β-arrestin 或 Mdm2 表达的细胞。我们的结果表明，网格蛋白介导的内吞作用比小窝蛋白介导的内吞作用发生得更快。网格蛋白介导的内吞作用和网格蛋白与 GRK2/β-arrestin2 的相互作用发生在 GRK2 介导的受体磷酸化依赖性方式中。相比之下，小窝蛋白介导的内吞作用和小窝蛋白 1 与 GRK2/β-arrestin2 的相互作用与受体磷酸化状态无关。Mdm2 介导的β-arrestin 泛素化，这是一种受体磷酸化依赖性的事件，对于 arrestin 与网格蛋白的相互作用是必需的。因此，本研究表明，GRK2 介导的受体磷酸化伴随着β-arrestin 的泛素化，是将 GRK2 和β-arrestin 与网格蛋白介导的内吞作用联系起来的关键细胞事件。
