Development of a cell-free system for study of insulin action

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GLUT4的囊泡与质膜的胞外融合。这两种蛋白之间的相互作用可被胰岛素抑制，但被GTPγS增强。Rab4的GTP酶缺陷突变体没有GTP结合缺陷突变体与Synaxin 4结合，这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的内吞中不起关键作用。

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 co-immunoprecipitated from the lysates of rat adipocytes. The interaction of the two proteins were attenuated by pretreatment of the cells with insulin but enhanced with GTPγS. 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 plays a critical role in exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.