Cell Biological Dissection of GLUT4 Trafficking Pathways

我们前期的研究表明，GLUT4的胞外通路和内吞通路分别受gtp结合蛋白、Rab4和动力蛋白的调控。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。经胰岛素预处理后，两种蛋白的相互作用减弱，而经gtp - γ s处理后，两者的相互作用增强。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-syntaxin 4相互作用需要munc-18c从syntaxin4中解离。我们还发现，在大鼠原发性脂肪细胞中，胰岛素通过不同的运输途径从不同的区室募集GLUT4，并具有不同的微管依赖性。诺可达唑破坏微管显示胰岛素刺激的葡萄糖转运和GLUT4易位被抑制约50%。此外，诺可达唑显著延缓了胰岛素刺激葡萄糖转运的时间过程。诺可达唑部分抑制胰岛素诱导的IRAP和VAMP-2易位，但对GLUT1和VAMP-3无影响。因此，与微管无关的GLUT4亚群似乎定位于内体循环室，而独立的亚群可能定位于更专门的室。

Our previous studies indicated that the exocytotic and endocytotic pathways for GLUT4 are regulated by GTP-binding proteins, Rab4 and dynamin, respectively. 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 complex formation, displaced Rab4 from syntaxin 4, indicating that the dissociation of munc-18c from syntaxin4 is required for the Rab4-syntaxin 4 interaction.We also found that insulin recruits GLUT4 from distinct compartments via distinct traffic pathways with differential microtubule dependence in rat primary adipocytes. Disruption of the microtubules with nocodazole revealed that insulinstimulated glucose transport and GLUT4 translocation were inhibited by about 50%. In addition, the time-course of the insulin stimulation of glucose transport was significantly delayed with nocodazole. Nocodazole partially inhibited insulin-induced translocation of IRAP and VAMP-2 but without effect on GLUT1 and VAMP-3. Thus, the microtubule-independent GLUT4 subpopulation seems to localize to the endosomal recycling compartment, and the independent subpopulation are likely to localize to more specialized compartment.