Shpetner H S, Herskovits J S, Vallee R B
Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
J Biol Chem. 1996 Jan 5;271(1):13-6. doi: 10.1074/jbc.271.1.13.
Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, proline-rich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wild-type forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to nonclathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.
发动蛋白是一种GTP酶，在内吞作用的早期阶段发挥关键作用，调节网格蛋白包被小窝和非网格蛋白包被小窝从质膜上的脱离。虽然发动蛋白在体内发挥作用的配体尚不清楚，但发动蛋白在体外通过富含脯氨酸的碱性C末端结构域与几种含有Src同源3（SH3）结构域的蛋白质、微管和阴离子磷脂结合。为了开始鉴定发动蛋白在体内的结合伴侣，我们通过免疫荧光检查了发动蛋白的突变体和野生型形式与通过瞬时转染细胞超声处理制备的质膜的结合情况。发现野生型发动蛋白几乎只与含有网格蛋白的区域结合。通过去除编码候选SH3结构域结合位点的C末端结构域内的一个九氨基酸序列，与这些区域的结合被消除。结合不需要网格蛋白，并且在高离子强度和低离子强度下都能抵抗提取，这与与SH3结构域的相互作用一致。令人惊讶的是，我们还发现发动蛋白包含多个与不含网格蛋白的区域结合的区域，包括C末端结构域上游直接的一个13氨基酸序列。这些观察结果表明，一种含有SH3结构域的蛋白质参与将发动蛋白招募到包被小窝，并为SH3结构域在发动蛋白功能中的生物学作用提供了首个证据。
