Cell Biological Dissection of GLUT4 Trafficking Pathways

我们以前的研究表明，GLUT 4的胞吐和胞吞途径分别由GTP结合蛋白Rab 4和发动蛋白调节。在本研究中，我们研究了Rab 4与syntaxin 4的相互作用，syntaxin 4是一种与胰岛素诱导的含GLUT 4的囊泡与质膜的胞吐融合有关的t-SNARE蛋白。Rab 4和syntaxin 4从大鼠脂肪细胞的裂解物中共免疫沉淀。胰岛素预处理可减弱两种蛋白的相互作用，而GTPγS预处理可增强两种蛋白的相互作用。Rab 4的GT3缺陷突变体，但不是GTP结合缺陷突变体与突触融合蛋白4结合，表明这两种蛋白质之间的相互作用受Rab 4的鸟嘌呤核苷酸结合状态的调节。此外，我们发现，存在的munc-18 c，负调节SNARE复合物的形成，取代Rab 4从syntaxin 4，表明munc-18 c的解离从syntaxin 4是必需的Rab 4-syntaxin 4 interaction.We还发现，胰岛素募集GLUT 4从不同的车厢通过不同的交通途径与不同的微管依赖性在大鼠原代脂肪细胞。用诺考达唑破坏微管显示，胰岛素刺激的葡萄糖转运和GLUT 4易位被抑制约50%。此外，诺考达唑显著延迟了胰岛素刺激葡萄糖转运的时间进程。诺考达唑部分抑制胰岛素诱导的IRAP和VAMP-2易位，但对GLUT 1和VAMP-3无影响。因此，不依赖于微管的GLUT 4亚群似乎定位于内体再循环区室，并且独立亚群可能定位于更专门的区室。

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.