Pancreatic β-cell protein granuphilin binds Rab3 and Munc-18 and controls exocytosis
Pancreatic β-cell protein granuphilin binds Rab3 and Munc-18 and controls exocytosis
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DOI：
10.1091/mbc.02-02-0025
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发表时间：
2002-06-01
影响因子：
3.3
通讯作者：
Regazzi, R
中科院分区：
文献类型：
--
作者：
Coppola, T;Frantz, C;Regazzi, R
嗜粒蛋白/Slp-4是在胰腺β细胞和脑垂体中表达的突触结合蛋白样蛋白家族的成员。我们通过共聚焦显微镜显示，颗粒蛋白-a和-B与位于胰腺β细胞外周的含胰岛素分泌颗粒共定位。在胰岛素分泌细胞系中过表达的颗粒蛋白引起了刺激诱导的胞吐的深刻抑制。发现颗粒蛋白结合胰腺β细胞分泌机制的两种组分，即小GTP结合蛋白Rab 3和可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）结合蛋白Munc-18。与Rab 3的相互作用只发生在GTP结合形式的蛋白质中，并通过GTP酶效应结构域中的点突变来阻止。使用颗粒蛋白-b突变体的结构-功能研究表明，Rab 3结合的完全丧失与抑制胞吐的能力降低有关。然而，单独的颗粒蛋白/Rab 3复合物不足以介导胞吐作用的减少，这表明存在额外的结合伴侣。总之，我们的观察表明，颗粒蛋白在胰腺P细胞胞吐中起重要作用。鉴于假定的作用，Munc-18分泌囊泡对接，我们的数据表明，颗粒蛋白也可能参与这一过程。
Granuphilin/Slp-4 is a member of the synaptotagmin-like protein family expressed in pancreatic beta-cells and in the pituitary gland. We show by confocal microscopy that both granuphilin-a and -b colocalize with insulin-containing secretory granules positioned at the periphery of pancreatic beta-cells. Overexpression of granuphilins in insulin-secreting cell lines caused a profound inhibition of stimulus-induced exocytosis. Granuphilins were found to bind to two components of the secretory machinery of pancreatic beta-cells, the small GTP-binding protein Rab3 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-binding protein Munc-18. The interaction with Rab3 occurred only with the GTP-bound form of the protein and was prevented by a point mutation in the effector domain of the GTPase. Structure-function studies using granuphilin-b mutants revealed that complete loss of Rab3 binding is associated with a reduction in the capacity to inhibit exocytosis. However, the granuphilin/Rab3 complex alone is not sufficient to mediate the decrease of exocytosis, suggesting the existence of additional binding partners. Taken together, our observations indicate that granuphilins play an important role in pancreatic P-cell exocytosis. In view of the postulated role of Munc-18 in secretory vesicle docking, our data suggest that granuphilins may also be involved in this process.