Gelsolin Associates with the N Terminus of Syntaxin 4 to Regulate Insulin Granule Exocytosis
Gelsolin Associates with the N Terminus of Syntaxin 4 to Regulate Insulin Granule Exocytosis
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DOI：
10.1210/me.2011-1112
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发表时间：
2012-01-01
影响因子：
--
通讯作者：
Thurmond, Debbie C.
中科院分区：
文献类型：
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作者：
Kalwat, Michael A.;Wiseman, Dean A.;Thurmond, Debbie C.
质膜可溶性N-乙基马来酰亚胺敏感因子附着受体（SNARE）蛋白syntaxin（Syn）4是双相胰岛素分泌所必需的，尽管它如何调节每个阶段仍不清楚。在筛选以确定新的Syn 4相互作用因子，钙激活的F-肌动蛋白切断蛋白凝溶胶蛋白被发现。凝溶胶蛋白先前已被认为是胰岛素分泌的正效应子，尽管缺乏该功能的分子机制。为此，我们的体外结合研究表明，Syn 4-凝溶胶蛋白相互作用是直接的，并由Syn 4的N-末端Ha结构域（氨基酸残基39-70）介导。Syn 4-凝溶胶蛋白复合物在基础条件下形成，并在急性葡萄糖或KCl刺激时解离;硝苯地平阻断解离。通过表达与绿色荧光蛋白（GFP）融合的Syn 4的N-末端Ha结构域（GFP 39 - 70）可以模拟促分泌素的解离作用。此外，GFP-39-70在分离的小鼠胰岛和克隆MIN 6 β细胞中的表达在不存在适当刺激的情况下启动胰岛素释放。与此一致，抑制性GFP-39-70肽也在没有刺激的情况下启动Syn 4活化。此外，尽管表达GFP-39-70肽的MIN 6 β细胞响应于KCl维持正常的钙内流，但KCl刺激的胰岛素分泌和胰岛素分泌的触发途径显著受损。总之，这些数据支持凝溶胶蛋白在胰岛素胞吐中的作用的机制模型：凝溶胶蛋白通过在不存在适当刺激的情况下与Syn 4直接缔合来夹持未经请求的可溶性N-乙基马来酰亚胺敏感因子附着受体（SNARE）调节的胞吐，其在刺激诱导的钙内流激活凝溶胶蛋白并诱导其从Syn 4解离以促进胰岛素胞吐后被缓解。（分子内分泌学26：128-141，2012）
The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin (Syn) 4 is required for biphasic insulin secretion, although how it regulates each phase remains unclear. In a screen to identify new Syn4-interacting factors, the calcium-activated F-actin-severing protein gelsolin was revealed. Gelsolin has been previously implicated as a positive effector of insulin secretion, although a molecular mechanism to underlie this function is lacking. Toward this, our in vitro binding studies showed the Syn4-gelsolin interaction to be direct and mediated by the N-terminal Ha domain (amino acid residues 39-70) of Syn4. Syn4-gelsolin complexes formed under basal conditions and dissociated upon acute glucose or KCl stimulation; nifedipine blocked dissociation. The dissociating action of secretagogues could be mimicked by expression of the N-terminal Ha domain of Syn4 fused to green fluorescent protein (GFP) (GFP39- 70). Furthermore, GFP-39-70 expression in isolated mouse islet and clonal MIN6 beta-cells initiated insulin release in the absence of appropriate stimuli. Consistent with this, the inhibitory GFP-39-70 peptide also initiated Syn4 activation in the absence of stimuli. Moreover, although MIN6 beta-cells expressing the GFP-39-70 peptide maintained normal calcium influx in response to KCl, KCl-stimulated insulin secretion and the triggering pathway of insulin secretion were significantly impaired. Taken together, these data support a mechanistic model for gelsolin's role in insulin exocytosis: gelsolin clamps unsolicited soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-regulated exocytosis through direct association with Syn4 in the absence of appropriate stimuli, which is relieved upon stimulus-induced calcium influx to activate gelsolin and induce its dissociation from Syn4 to facilitate insulin exocytosis. (Molecular Endocrinology 26: 128-141, 2012)