Okamoto M, Schoch S, Südhof T C
Center for Basic Neuroscience, Howard Hughes Medical Institute, and the Department of Molecular Genetics, The University of Texas Southwestern Medical School, Dallas Texas 75235, USA.
J Biol Chem. 1999 Jun 25;274(26):18446-54. doi: 10.1074/jbc.274.26.18446.
In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.
在酵母双杂交筛选与SNAP-25结合并可能参与胞吐作用的蛋白质时，我们分离出一种名为EHSH1（含EH结构域/SH3结构域的蛋白质）的蛋白质。全长cDNA的克隆显示，EHSH1由一个带有两个EH结构域的N端区域、一个富含赖氨酸、亮氨酸、谷氨酸、精氨酸和谷氨酰胺的中央区域（KLERQ结构域）以及一个由五个SH3结构域组成的C端区域构成。第三个SH3结构域存在可变剪接。数据库搜索表明，EHSH1与非洲爪蟾和人类的intersectin以及人类的SH3P17非常相似。此外，我们鉴定出了编码EHSH1第二种异构体（称为EHSH2）的表达序列标签。EHSH1在脑中大量表达，在所有其他检测的组织中表达水平较低。在结合研究中，我们发现EHSH1的中央KLERQ结构域与重组或天然脑SNAP-25和SNAP-23结合。相比之下，C端SH3结构域与发动蛋白定量相互作用，发动蛋白是一种参与胞吞作用的蛋白质。发动蛋白强烈结合可变剪接的中央SH3结构域（SH3C）和两个C端SH3结构域（SH3D和SH3E），但不结合N端SH3结构域（SH3A和SH3B）。免疫沉淀证实，在脑中发动蛋白和SNAP-25都与EHSH1形成复合物。我们的数据表明，EHSH1/intersectin可能是一种新型衔接蛋白，它将内吞膜运输与胞吐作用联系起来。EHSH1中多个SH3结构域与发动蛋白结合的能力表明，EHSH1能够以一种受可变剪接调控的方式聚集多个发动蛋白分子。
