Laporte S A, Oakley R H, Holt J A, Barak L S, Caron M G
Howard Hughes Medical Institute Laboratories and the Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2000 Jul 28;275(30):23120-6. doi: 10.1074/jbc.M002581200.
Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.
β-抑制蛋白是一种胞质蛋白，可调节G蛋白偶联受体（GPCRs）的信号传导和内化。尽管受体偶联的终止需要β-抑制蛋白与激动剂激活的受体结合，但GPCR的内吞作用涉及受体-β-抑制蛋白复合物与其他内吞蛋白（如衔接蛋白2（AP-2）和网格蛋白）之间的协同相互作用。网格蛋白与β-抑制蛋白C末端尾巴中的一个保守基序相互作用；然而，β-抑制蛋白中与AP-2结合的特定分子决定因素尚未确定。此外，β-抑制蛋白与AP-2和网格蛋白相互作用对GPCR靶向网格蛋白包被小泡的各自贡献尚未明确。在这里，我们鉴定了β-抑制蛋白2 C末端的特定精氨酸残基（Arg(394)和Arg(396)），它们介导β-抑制蛋白与AP-2的结合，并在体外表明，β-抑制蛋白1和2中的这些结构域与AP-2的相互作用同样良好，且与网格蛋白结合无关。我们通过荧光显微镜在HEK 293细胞中证明，缺乏β-抑制蛋白-网格蛋白结合基序的β(2)-肾上腺素能受体-β-抑制蛋白复合物仍靶向网格蛋白包被小窝。与之形成鲜明对比的是，缺乏β-抑制蛋白/AP-2相互作用的受体-β-抑制蛋白复合物不能有效地在质膜的点状区域中分隔开来。这些结果表明，受体-β-抑制蛋白复合物与AP-2而非网格蛋白的结合是β(2)-肾上腺素能受体最初靶向网格蛋白包被小窝所必需的。
