McPherson P S, de Heuvel E, Phillie J, Wang W, Sengar A, Egan S
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Quebec, Canada.
Biochem Biophys Res Commun. 1998 Mar 27;244(3):701-5. doi: 10.1006/bbrc.1998.8331.
The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.
内吞蛋白Eps15包含三个EH结构域拷贝，这是一种被认为在蛋白质-蛋白质相互作用中起作用的蛋白质模块。通过使用Eps15 EH结构域融合蛋白进行覆盖分析，我们现已鉴定出一种95 kDa的蛋白质（p95），它是多种组织中主要的EH结构域结合伴侣。天冬酰胺-脯氨酸-苯丙氨酸（NPF）氨基酸构成了EH结构域结合基序的核心，并且在内吞蛋白突触素-170中发现了三个NPF重复序列。我们证实了先前的研究，表明突触素-170是一种EH结构域结合蛋白，并使用肽阻断实验证明这种相互作用是通过NPF重复序列介导的。有趣的是，相同的肽也阻断EH结构域与p95的结合。最后，我们表明p95在网格蛋白包被小泡上富集，提示该蛋白在内吞作用中发挥作用。这些数据支持了EH结构域-NPF基序相互作用在内吞作用中的重要作用。
