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The functional analysis of a novel adaptor protein binding to GLUT4

与 GLUT4 结合的新型接头蛋白的功能分析

基本信息

批准号：
15590939
负责人：
OKUYA Shigeru
金额：
$ 2.18万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590939/
关键词：
glucose transporter GLUT4 translocation recycling adaptor protein glucose uptake insulin adipocyte

项目摘要

PurposeIt has been supposed that a protein binding to GLUT4 could play an important role during the recycling of GLUT4 vesicle and insulin-induced GLUT4 translocation. We assumed the existence of GLUT4 "adaptor protein" in adipocytes, and we have identified a novel protein "p61" that interacts with GLUT4, and analyzed its function.MethodUsing the yeast two-hybrid system, we screened from rat adipocyte cDNA library, and have cloned p61 as an interesting protein that interacts with GLUT4. The intracellular localization of p61 in L6-GLUT4myc myotubes, stably expressing myc-tagged GLUT4, was examined by the immunohistochemistry using the myc tag antibody. The interaction of GLUT4 and p61 was confirmed by the yeast two-hybrid system, and also the immunoprecipitation. After overexpression of p61 in 3T3-L1 adipocytes, the GLUT4 translocation and the insulin-induced glucose uptake were examined.ResultsThe expression of p61 in adipocytes was confirmed at the mRNA level using Northern blotting. The p61 consists of 540 amino acids with the ANK (ankyrin) structure thought to be a protein-protein interaction motif. The p61 existed in perinuclear region in the L6-GLUT4myc myotubes as a result of the immunohistochemistry. Moreover, it was confirmed that the interaction of p61 and GLUT4 by yeast two-hybrid system and the immunoprecipitation. In addition, overexpression of p61 in 3T3-L1 adipocytes using adenoviral system, increased the amount of GLUT4 in membrane fraction after insulin stimulation, and also significantly facilitated insulin-induced glucose uptake.ConclusionIt is suggested that a novel ANK structure protein p61 facilitates GLUT4 translocation, and then activates insulin-induced glucose uptake.

目的推测一种与GLUT4结合的蛋白可能在GLUT4囊泡循环和胰岛素诱导的GLUT4易位过程中发挥重要作用。我们假设在脂肪细胞中存在GLUT4“接头蛋白”，并鉴定了一种与GLUT4相互作用的新蛋白“p61”，并分析了其功能。方法利用酵母双杂交系统，从大鼠脂肪细胞cDNA文库中筛选出与GLUT4相互作用的蛋白p61。使用myc标记抗体，免疫组织化学检测p61在稳定表达myc标记的GLUT4的L6-GLUT4myc肌管中的细胞内定位。通过酵母双杂交系统和免疫沉淀证实了GLUT4与p61的相互作用。在3T3-L1脂肪细胞中过表达p61后，检测GLUT4易位和胰岛素诱导的葡萄糖摄取。结果Northern印迹法证实p61在脂肪细胞mRNA水平上表达。p61由540个氨基酸组成，其ANK（锚蛋白）结构被认为是蛋白质-蛋白质相互作用的基序。免疫组化结果显示p61存在于L6-GLUT4myc肌管的核周区。此外，通过酵母双杂交系统和免疫沉淀证实了p61与GLUT4的相互作用。此外，通过腺病毒系统，p61在3T3-L1脂肪细胞中过表达，增加了胰岛素刺激后膜组分中GLUT4的量，也显著促进了胰岛素诱导的葡萄糖摄取。结论一种新的ANK结构蛋白p61促进GLUT4易位，进而激活胰岛素诱导的葡萄糖摄取。