Wigge P, Köhler K, Vallis Y, Doyle C A, Owen D, Hunt S P, McMahon H T
Neurobiology Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Mol Biol Cell. 1997 Oct;8(10):2003-15. doi: 10.1091/mbc.8.10.2003.
Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.
发动蛋白结合蛋白（Amph）是一种含有src同源3结构域的蛋白质，因其与发动蛋白的相互作用而参与突触小泡的内吞作用。在对发动蛋白结合蛋白家族新成员的筛选中，我们鉴定出了发动蛋白结合蛋白2（Amph2），它是一种与先前鉴定的发动蛋白结合蛋白1（Amph1）有49%同源性的异构体。这种异构体的亚细胞分布与Amph1相似，二者在神经末梢中均高度富集。与Amph1一样，去极化时Amph2的快速去磷酸化支持了其在神经末梢内吞作用中的作用。重要的是，这两种异构体可以从大脑中作为等摩尔复合物进行共免疫沉淀，表明这两种异构体协同发挥作用。通过对脑提取物的交联分析确定，Amph1 - Amph2复合物是一种220至250 kDa的异二聚体。单独转染Amph1或Amph2的COS细胞显示转铁蛋白摄取大幅减少，但两种蛋白的共表达可挽救这一缺陷，支持了异二聚体在网格蛋白介导的内吞作用中的作用。尽管两种异构体的src同源3结构域均与发动蛋白相互作用，但异二聚体在体外可与多个发动蛋白分子结合并激活发动蛋白的GTP酶活性。我们提出，正是发动蛋白结合蛋白异二聚体驱动发动蛋白被募集到内吞神经末梢的网格蛋白包被小窝中。
