Proteomic characterization of insulin signaling targets

描述(申请人提供)：胰岛素刺激控制有丝分裂和代谢输出的多个信号通路。在脂肪和肌肉中，至少有一个信号通路导致GLUT4葡萄糖转运体的重新分布。胰岛素需要将GLUT4移动到质膜以增强葡萄糖摄取。参与这一过程的胰岛素信号通路(S)作用于包含GLUT4和TUG的蛋白质复合体，TUG是一种在未受刺激的细胞中保留GLUT4的蛋白质。胰岛素刺激TUG和GLUT4的解离，重新分配GLUT4，增加葡萄糖摄取。 我们假设TUG是GLUT4上形成的大型蛋白质复合体的第一个被鉴定的成分，并且这个复合体在没有胰岛素的情况下可以稳定地组装。胰岛素作用于改变该复合体的一个或多个组分的翻译后修饰，导致其分解并动员葡萄糖转运体。对这种蛋白质复合体以及胰岛素如何作用于它的详细描述将是理解胰岛素代谢作用的关键。 在这里，我们建议使用蛋白质组学方法来分析包含TUG和GLUT4的蛋白质复合体。在目标1中，我们将研究这种复合体在未受刺激的细胞中的组成。我们将使用几种生化方法来纯化存在于复合体中的蛋白质，并将使用串联质谱学来鉴定这些蛋白质。在目标2中，我们将使用蛋白质组学技术来研究胰岛素如何改变复合体中蛋白质的翻译后修饰，以及它如何改变蛋白质复合体的整体组成。我们预计，我们获得的数据将有助于理解胰岛素调节血糖稳态的分子机制。此外，这项工作可能对胰岛素信号和蛋白质靶向有更广泛的影响。

DESCRIPTION (provided by applicant): Insulin stimulates multiple signaling pathways controlling mitogenic and metabolic outputs. In fat and muscle, at least one of these signaling pathways leads to the redistribution of GLUT4 glucose transporters. Movement of GLUT4 to the plasma membrane is required for insulin to enhance glucose uptake. The insulin signaling pathway(s) involved in this process impinge on a protein complex containing GLUT4 and TUG, a protein that retains GLUT4 intracellularly in unstimulated cells. Insulin stimulates the dissociation of TUG and GLUT4 to redistribute GLUT4 and augment glucose uptake. We hypothesize that TUG is the first identified component of a large protein complex that is formed on GLUT4, and that this complex is stably assembled in the absence of insulin. Insulin acts to alter the posttranslational modification of one or more components of this complex, causing its disassembly and mobilizing the glucose transporter. A detailed characterization of this protein complex and of how insulin acts on it will be essential to understanding insulin's metabolic actions. Here, we propose to use proteomic approaches to analyze the protein complex containing TUG and GLUT4. In Aim 1, we will study the composition of this complex in unstimulated cells. We will use several biochemical methods to purify proteins present in the complex, and will identify these using tandem mass spectrometry. In Aim 2, we will use proteomic techniques to study how insulin alters the posttranslational modification of proteins in the complex, as well as how it alters the overall composition of the protein complex. We anticipate that the data we obtain will contribute to understanding of the molecular mechanisms by which insulin regulates glucose homeostasis. Additionally, this work may have broader implications for insulin signaling and for protein targeting.