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Cell Biological Dissection of GLUT4 Trafficking Pathways

GLUT4 贩运途径的细胞生物学解析

基本信息

批准号：
13671175
负责人：
SHIBATA Hiroshi
金额：
$ 2.56万
依托单位：
Gunma University, Institute For Molecular and Cellular Regulation
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671175/
关键词：
insulin glucose transporter GTP-binding protein Rab4 syntaxin4 SNARE microtubules 微小管

项目摘要

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.

我们之前的研究表明，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 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 的囊泡与质膜的胞吐融合。 Rab4 和 Syntaxin4 从大鼠脂肪细胞裂解物中进行免疫共沉淀。用胰岛素预处理细胞会减弱两种蛋白质的相互作用，但用 GTPγS 会增强这种相互作用。 Rab4 的 GTPase 缺陷突变体（而非 GTP 结合缺陷突变体）与突触融合蛋白 4 结合，表明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munc-18c（SNARE复合物形成的负调节因子）的存在，将Rab4从突触蛋白4中取代，表明munc-18c从突触蛋白4上解离是Rab4-突触蛋白4相互作用所必需的。我们还发现，胰岛素通过不同的交通途径从不同的区室招募GLUT4，在大鼠原代脂肪细胞中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 易位被抑制约 50%。此外，诺考达唑显着延迟了胰岛素刺激葡萄糖转运的时程。诺考达唑部分抑制胰岛素诱导的 IRAP 和 VAMP-2 易位，但对 GLUT1 和 VAMP-3 没有影响。因此，不依赖于微管的 GLUT4 亚群似乎定位于内体回收区室，而独立的亚群可能定位于更特化的区室。