Sugiyama Shinichiro, Kishida Shosei, Chayama Kazuaki, Koyama Shinya, Kikuchi Akira
Departments of Biochemistry and Medicine and Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551.
J Biochem. 2005 Mar;137(3):355-64. doi: 10.1093/jb/mvi044.
Epsin is a key molecule in receptor-mediated endocytosis. Epsin is phosphorylated and ubiquitinated, and these post-translational modifications are necessary for the regulation of endocytosis. Since human Epsin (hEpsin) has two ubiquitin-interacting motifs (UIMs), we investigated the roles of these UIMs in endocytosis. hEpsin formed a complex with ubiquitinated proteins but did not bind to monoubiquitin. Neither of the two UIMs of hEpsin alone was sufficient to form a complex with ubiquitinated proteins: both UIMs were necessary. Mutations of Asp209 and Asp210 to Ala in UIM (hEpsinDA) abolished the binding activity of hEpsin to ubiquitinated proteins. However, hEpsinDA interacted with Eps15, POB1, and AP-2, which are involved in receptor-mediated endocytosis, as efficiently as wild-type hEpsin. Expression of hEpsinDA in CHO-IR cells affected neither the binding of insulin to nor insulin-dependent autophosphorylation of its receptor. Expression of wild-type hEpsin inhibited the internalization of insulin, whereas that of hEpsinDA did not. These results suggest that the UIM motifs of hEpsin interact with proteins modified with ubiquitin, and that the complex formation is involved in insulin-dependent receptor endocytosis.
埃普辛是受体介导的内吞作用中的关键分子。埃普辛会发生磷酸化和泛素化，这些翻译后修饰对于内吞作用的调节是必需的。由于人类埃普辛（hEpsin）有两个泛素相互作用基序（UIM），我们研究了这些UIM在胞吞作用中的作用。hEpsin与泛素化蛋白形成复合物，但不与单泛素结合。hEpsin的两个UIM单独一个都不足以与泛素化蛋白形成复合物：两个UIM都是必需的。UIM中Asp209和Asp210突变为Ala（hEpsinDA）消除了hEpsin与泛素化蛋白的结合活性。然而，hEpsinDA与参与受体介导的内吞作用的Eps15、POB1和AP - 2的相互作用与野生型hEpsin一样有效。hEpsinDA在CHO - IR细胞中的表达既不影响胰岛素与其受体的结合，也不影响胰岛素依赖性的自身磷酸化。野生型hEpsin的表达抑制胰岛素的内化，而hEpsinDA的表达则没有。这些结果表明，hEpsin的UIM基序与泛素修饰的蛋白相互作用，并且复合物的形成参与胰岛素依赖性受体的内吞作用。
