The mechanisms of insulin-regulated exocytosis of the GLUT4-containing vesicles

在本研究中，我们研究了Rab4与syntaxin4的相互作用，syntaxin4是一种t-SNARE蛋白，参与胰岛素诱导的含有glut4的囊泡与质膜的胞外融合。从大鼠脂肪细胞裂解物中共免疫沉淀Rab4和syntaxin4。胰岛素预处理能减弱两种蛋白的相互作用，而GTPgS则能增强两者的相互作用。Rab4的GTPase缺陷突变体与syntaxin 4结合，而不是gtp结合缺陷突变体，这表明这两种蛋白之间的相互作用受到Rab4鸟嘌呤核苷酸结合状态的调节。此外，我们发现SNARE复合物形成的负调节因子munc-18c的存在使Rab4从syntaxin4中移位，这表明Rab4需要从syntaxin4中解离munc-18c才能与syntaxin4结合。我们还澄清了肌动蛋白丝在离体大鼠脂肪细胞的胞外募集中起关键作用，但在GLUT4的内吞作用中不起作用。

In the present study, we investigated the interaction of Rab4 with syntaxin4, a t-SNARE protein implicated in the insulin-induced exocytic fusion of the GLUT4-containing vesicle with the plasma membrane. Rab4 and syntaxin4 were coimmunoprecipitated from the lysates of rat adipocytes. The interaction of the two proteins were attenuated by pretreatment of the cells with insulin but enhanced with GTPgS.A GTPase deficient mutant of Rab4, but not a GTP-binding defective mutant was bound to syntaxin 4, suggesting that the interaction between the two proteins were regulated by the guanine-nucleotide-binding state of Rab4. In addition, we found that the presence of munc-18c, a negative regulator of the SNARE comple formation, displaced Rab4 from syntaxin 4, indicating that the dissociation of munc-18c from syntaxin4 is required for Rab4 to bind to syntaxin 4.We also clarified that the actin filaments play a critical role in exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.