Epsin binds to clathrin by associating directly with the clathrin-terminal domain - Evidence for cooperative binding through two discrete sites
Epsin binds to clathrin by associating directly with the clathrin-terminal domain - Evidence for cooperative binding through two discrete sites
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DOI：
10.1074/jbc.275.9.6479
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Traub, LM
中科院分区：
文献类型：
--
作者：
Drake, MT;Downs, MA;Traub, LM
Epsin是最近发现的一种蛋白质，它似乎在网状蛋白介导的内吞作用中发挥着重要作用。Epsin 1的中心区，即所谓的DPW结构域，通过直接与α亚基的球状附属物结合，与异四聚体AP-2适配器复合体结合。我们已经发现，epsin 1的这个中心部分也与clathrin有关。与网状蛋白的相互作用是直接的，而不是由epsin结合的AP-2介导的。丙氨酸扫描突变表明，胞苷的结合依赖于位于epsin 1DPW结构域内的序列(LMD)-L-257-LADV。该序列与接头β亚基、两面体蛋白和β-arrestin中已知的cathrin结合序列有关，促进了epsin 1与cathrin重链末端结构域的结合，出人意料地通过逐渐删除DPW三联体而保持LMDLADV序列不变，抑制了AP-2与GST-epsin DPW融合蛋白的结合，减少了与AP-2平行的cathrin的结合。由于AP-2复合体的β亚基也含有一个网状蛋白结合位点，因此与可溶性网状蛋白的最佳结合似乎依赖于至少两个不同的网状蛋白结合位点的存在，我们发现位于epsin 1羧基末端的第二个网状蛋白结合序列-L-480也直接与网状蛋白相互作用。LMDLADV和LVDLD序列在网状蛋白募集分析中协同作用，表明它们结合到网状蛋白末端结构域上的不同位置。在几个后生动物类epsin分子中相似的cathrin结合序列的进化保守性表明，在一个被cathrin覆盖的发育芽中建立多个蛋白质-蛋白质接触的能力是epsin功能的一个重要方面。
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 (LMD)-L-257-LADV 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 (LVDLD)-L-480, 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.