Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling
Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling
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DOI：
10.1074/jbc.m304346200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Lafer, EM
中科院分区：
文献类型：
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作者：
Morgan, JR;Prasad, K;Lafer, EM
虽然遗传和生化研究表明含有Eps15同源结构域的蛋白在网格蛋白介导的内吞作用中起作用，但这些蛋白的具体功能尚不清楚。Eps15存在于网格蛋白包被凹坑的生长边缘，这导致了它参与包被囊泡形成的假设。我们通过研究Eps15对网格蛋白组装的影响来评估这一假设。我们发现，虽然Eps15没有组装网格蛋白的内在能力，但它能有效地刺激网格蛋白接头蛋白AP180在生理ph下组装网格蛋白的能力。我们还确定了Eps15在鱿鱼AP180上的结合位点。这些位点包含一个NPF基序，从这些结合位点衍生的肽在体外抑制Eps15刺激网格蛋白组装的能力。此外，当注射到鱿鱼巨大的突触前神经末梢时，这些肽抑制突触囊泡内吞过程中网格蛋白包被的凹坑和包被的囊泡的形成。这与Eps15在体内调节网格蛋白外壳组装的假设一致，并表明Eps15同源结构域和NPF基序之间的相互作用参与了突触囊泡循环过程中网格蛋白包被囊泡的形成。
Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.