Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin
Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin
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DOI：
10.1074/jbc.273.49.32966
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发表时间：
1998-12-04
影响因子：
4.8
通讯作者：
Tornehl, CK
中科院分区：
文献类型：
--
作者：
Chapman, ER;Desai, RC;Tornehl, CK
生化和遗传学研究表明，synaptotagmin I在突触囊泡胞吐过程中作为Ca2+传感器，在胞吞过程中作为网格蛋白接头复合物AP-2的膜受体。这些功能涉及两个保守结构域C2A和C2B与效应蛋白的相互作用。C2B结构域介导Ca2+触发的synaptotagmin寡聚化，结合AP-2，对synaptotagmin与Ca2+通道的相互作用很重要。在这里，我们报告了这些是保守的生化特性：Ca2+促进突触塔蛋白I与突触塔蛋白III和TV的异齐聚化，并且所有三种突触塔蛋白同工型结合了n型Ca2+通道α 1B亚基的突触区。利用嵌合和截断的C2结构域，我们定义了C2B的一个共同区域，该区域介导寡聚化和AP-2结合，在该区域内，鉴定了两个相邻的赖氨酸残基，它们对synaptotagmin寡聚化、AP-2和synprint结合至关重要。竞争实验表明，synprint片段是synaptotagmin寡聚化的有效抑制剂，也阻断了synaptotagmin与AP-2的结合。在C2B的结构模型中，共同的效应结合位点定位于一个假定的Ca2+结合环和一个由两条β链形成的凹区，这些研究提供了关于C2B靶蛋白识别的第一个结构信息，并为功能研究提供了选择性破坏synaptotagmin-effector相互作用的手段。
Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and TV, and all three synaptotagmin isoforms bound the synprint region of the alpha 1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding, Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands, These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.