Development of a cell-free system for study of insulin action

开发用于研究胰岛素作用的无细胞系统

• 批准号: 10557102
• 负责人: SHIBATA Hiroshi
• 金额: $ 5.57万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557102/
• 关键词: insulin; glucose transporter; GTP-binding protein; Rab4; syntaxin4; SNARE; ダイナミン

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 comple formation, displaced Rab4 from syntaxin 4, indicating that the dissociation of munc-18c from syntaxin4 is required for Rab4 to bind to syntaxin 4.We also clarified that the actin filaments plays a critical role in exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

在本研究中，我们研究了Rab 4与syntaxin 4的相互作用，其中syntaxin 4是一种t-SNARE蛋白，参与胰岛素诱导的含GLUT 4的囊泡与质膜的胞外融合。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SNARE复合物形成的负调控因子munc-18 c的存在，将Rab 4从syntaxin 4中置换出来，表明Rab 4与syntaxin 4结合需要munc-18 c的解离。我们还阐明了肌动蛋白丝在大鼠脂肪细胞GLUT 4的胞吐募集中起关键作用，但在GLUT 4的胞吞中不起关键作用。

项目成果

Shibata, H.: "Insulin signaling and Rab."Seitai no Kagaku. 50. 565-573 (1999)

Shibata, H.：“胰岛素信号传导和 Rab”。Seitai no Kagaku。

Ohnishi,H. et al: "Involvement of Rab4 in regulated exocytosis of rat pancreatic acini"Gastroenterology. 116. 943-952 (1999)

大西，H.

Omata, W. et al.: "Subcellular distribution of GLUT4 in chinese hamster ovary cells overexpressing mutant dynamin" Biochem.Biophys.Res.Comm.241. 401-406 (1997)

Omata, W. 等人：“过表达突变动力的中国仓鼠卵巢细胞中 GLUT4 的亚细胞分布”Biochem.Biophys.Res.Comm.241。

Omata,W. et al: "Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes."Biochem.J.. 346. 321-328 (2000)

大俣，W.

Omata, W., Shibat, H., Li, L., Takata, K. and Kojima, I.: "Actin filaments plays a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes."Biochem. J.. 346. 321-328 (2000)

Omata, W.、Shibat, H.、Li, L.、Takata, K. 和 Kojima, I.：“肌动蛋白丝在胰岛素诱导的胞吐募集中发挥着关键作用，但在分离的大鼠脂肪细胞中的 GLUT4 内吞作用中不起关键作用。”