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Syntaxin4的相互作用，Syntaxin4是一种在胰岛素诱导的含GLUT4的蔬菜与质膜的T-SNARE蛋白。从大鼠脂肪细胞的裂解物中共免疫沉淀Rab4和Syntaxin4。通过对细胞用胰岛素进行预处理来减弱两种蛋白质的相互作用，但用GTPγS增强。 Rab4的GTPase定义突变体，而不是GTP结合有缺陷的突变体与语法素4结合，这表明两种蛋白质之间的相互作用是由Rab4的鸟嘌呤核苷酸结合状态调节的。此外，我们发现，MUNC-18C是SNARE复合物的负调节剂，从索法蛋白4中移动了Rab4，表明MUSC-18C与syntaxin4的解离是Rab4-Syntaxin 4相互作用所需的。我们还发现，通过不同的界限胰岛素从不同的界限中募集了不同的隔离率。微管对诺科唑的破坏表明，胰岛素刺激的葡萄糖转运和GLUT4易位抑制了约50％。另外，诺科唑的胰岛素刺激的时间顺序会显着延迟。诺科唑部分抑制了胰岛素诱导的IRAP和VAMP-2易位，但对GLUT1和VAMP-3没有影响。这是，微管独立的GLUT4亚群似乎本地化为内体回收室，并且独立的亚群可能定位于更专业的隔室。