Cell Biological Dissection of GLUT4 Trafficking Pathways

我们以前的研究表明，GLUT4的胞吐和内吞途径分别受GTP结合蛋白Rab4和Dynamin的调节。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。用免疫共沉淀法从大鼠脂肪细胞裂解产物中分离出Rab4和Synaxin4。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，SNARE复合体形成的负调控因子MUNC-18c的存在取代了Rab4与Synaxin 4的结合，这表明MUNC-18c与Synaxin 4的相互作用需要MUNC-18c的解离。我们还发现，在大鼠原代脂肪细胞中，胰岛素通过不同的交通通路从不同的区段招募GLUT4，并具有不同的微管依赖性。用诺考达唑破坏微管表明，胰岛素刺激的葡萄糖转运和GLUT4转位被抑制了约50%。此外，诺康唑显著延缓胰岛素刺激葡萄糖转运的时程。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，微管非依赖性的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业的室。

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.