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

在本研究中，我们研究了Rab4与Synaxin4的相互作用，Synaxin4是一种t-SNARE蛋白，参与了胰岛素诱导的含有GLUT4的囊泡与质膜的胞外融合。免疫共沉淀法从大鼠脂肪细胞裂解产物中检测到Rab4和Synaxin4。这两种蛋白之间的相互作用可被胰岛素抑制，但被GTPgS增强。Rab4的GTP酶缺陷突变体，而不是GTP结合缺陷突变体，与Synaxin 4结合，表明两种蛋白之间的相互作用受Rab4的鸟氨酸-核苷酸结合状态的调节。此外，我们还发现，圈套复合体形成的负调节因子MUNC-18c的存在取代了Rab4与Synaxin 4的结合，这表明在分离的大鼠脂肪细胞中，需要将MUNC-18c从Synaxin 4解离出来，才能与Synaxin 4结合。我们还阐明了肌动蛋白细丝在GLUT4的胞吐募集中起关键作用，而在GLUT4的内吞中不起作用。

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.