Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC)
Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC)
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DOI：
10.1021/pr0502626
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Klip, A
中科院分区：
文献类型：
--
作者：
Foster, LJ;Rudich, A;Klip, A
胰岛素调节的葡萄糖转运蛋白（GLUT4）响应胰岛素易位到质膜，以促进餐后葡萄糖进入脂肪和肌肉细胞的摄取。虽然导致这种易位的早期胰岛素受体信号传导步骤已经明确，但GLUT4运输的信号传导和调节的整合仍然难以捉摸。一些证据表明肌动蛋白细胞骨架和蛋白-蛋白相互作用在胰岛素调节GLUT4定位中的重要作用。在这里，我们应用细胞培养中氨基酸的稳定同位素标记（SILAC）来鉴定以胰岛素调节方式与GLUT4相互作用的蛋白质。myc标记的GLUT4 （GLUT4myc）在L6肌管中稳定表达，通过myc表位从分别生长在含有正常同位素丰度亮氨酸或氘化亮氨酸的培养基中培养的基底细胞和胰岛素刺激细胞的总膜中分离得到。用液相色谱/串联质谱法分析与GLUT4myc共沉淀的蛋白。在被量化的603种蛋白中，36种与GLUT4myc相互作用的胰岛素依赖性变化在任何方向上都超过1.5倍。一些细胞骨架相关蛋白在胰岛素处理细胞的免疫沉淀物中升高，而泛素-蛋白酶体降解系统的成分普遍减少。参与囊泡运输的蛋白质也表现出胰岛素调节的关联。在细胞骨架相关蛋白中，GLUT4免疫沉淀物中α -肌动蛋白-4的回收率在胰岛素作用下增加了2.1 +/- 0.5倍，免疫印迹法增加了2.9 +/- 0.8倍。共聚焦免疫荧光显微镜显示胰岛素引起GLUT4和α - actiin -4共定位。我们得出的结论是，胰岛素引起了不同蛋白质与GLUT4之间相互作用的变化，并且细胞骨架蛋白，特别是α -肌动蛋白-4，与转运蛋白相关联，可能促进其到达质膜。
The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.