Drake M T, Downs M A, Traub L M
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2000 Mar 3;275(9):6479-89. doi: 10.1074/jbc.275.9.6479.
Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.
埃普辛是一种最近发现的蛋白质，它似乎在网格蛋白介导的内吞作用中发挥重要作用。埃普辛1的中心区域，即所谓的DPW结构域，通过直接与α亚基的球状附属物结合，与异源四聚体AP - 2衔接蛋白复合物结合。我们发现埃普辛1的这个中心部分也与网格蛋白相关联。与网格蛋白的相互作用是直接的，并非由与埃普辛结合的AP - 2介导。丙氨酸扫描诱变表明，网格蛋白结合取决于位于埃普辛1 DPW结构域内的序列（257）LMDLADV。该序列与衔接蛋白β亚基、发动蛋白和β - 抑制蛋白中已知的网格蛋白结合序列相关，促进了埃普辛1与网格蛋白重链末端结构域的结合。出乎意料的是，通过逐步删除DPW三联体但保留LMDLADV序列完整来抑制AP - 2与GST - 埃普辛DPW融合蛋白的结合，会使网格蛋白与AP - 2的结合同时减少。由于AP - 2复合物的β亚基也含有一个网格蛋白结合位点，与可溶性网格蛋白的最佳结合似乎取决于至少两个不同的网格蛋白结合位点的存在，并且我们表明位于埃普辛1羧基末端片段内的第二个网格蛋白结合序列（480）LVDLD也直接与网格蛋白相互作用。LMDLADV和LVDLD序列在网格蛋白募集试验中协同作用，表明它们与网格蛋白末端结构域上的不同位点结合。几种后生动物埃普辛样分子中相似网格蛋白结合序列的进化保守性表明，在正在形成的网格蛋白包被小泡内建立多个蛋白质 - 蛋白质接触的能力是埃普辛功能的一个重要方面。
