PHOSPHOTYROSYL PROTEINS IN INSULIN RECEPTOR SIGNALING

胰岛素受体信号转导中的磷酸酪酰蛋白

• 批准号: 6011667
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 37.03万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6011667
• 关键词: adipocytes; biological signal transduction; cell type; enzyme activity; enzyme mechanism; gene deletion mutation; gene targeting; genetically modified animals; glucose transport; glycogen synthase; hormone regulation /control mechanism; human tissue; immunocytochemistry; immunoprecipitation; insulin receptor; laboratory mouse; lipolysis; messenger RNA; molecular cloning; mutant; nucleic acid sequence; phenotype; phosphatidylinositol 3 kinase; phosphorylation; protein protein interaction; protein structure function; protein tyrosine kinase; yeast two hybrid system

The overall goal of this proposal is to elucidate at the molecular level signaling pathways from the insulin receptor. Many of these pathways begin with tyrosine phosphorylation of the insulin receptor substrates (IRS's) by the activated insulin receptor. The phosphorylated IRS's then function as docking/effector proteins for SH2 domain-containing signaling proteins. The IRS's thus have a key role in signaling from the insulin receptor. Until recently, only two members of the IRS family, IRS-1 and IRS-2, were known. In the past three years we have discovered and begun the characterization of two additional members of the IRS family, IRS-3 and IRS-4. IRS-3 and IRS-4 are most abundant in adipocytes and human embryonic kidney 293 cells, respectively; and we have determined the SH2 domain proteins associated with them in these cell types. In addition, we have generated mice with targeted disruption of the IRS-3 and IRS-4 genes; initial analyses of these knockout (ko) mice have not revealed abnormalities in glucose homeostasis or otherwise. One specific aim of this proposal is to determine the physiological roles of IRS-3 and IRS-4. This will be achieved by further characterization of the IRS-3 and IRS-4 ko mice. Investigation of the IRS-3 ko mice will focus on the effects of IRS-3 deletion on insulin signaling in adipocytes. In addition, in order to detect overlapping and/or compensatory roles for the IRS's, mice lacking two IRS's will be generated and characterized. The following three double ko's will be examined in our laboratory: IRS-1,4; IRS-2 and either 3 or 4; and IRS-3,4. The other double ko's will be examined in the laboratories of collaborators. The second specific aim of this proposal is to discover and characterize novel proteins that are components of signaling pathways involving IRS-3 or IRS-4. This will be done by searching for proteins that interact with IRS-3 and IRS-4. Already we have found a group of eight proteins that are associated with IRS-4 isolated by immunoprecipitation. These will be characterized. In addition, proteins associated with IRS-3 will be found by screening expression libraries with 32P-labeled IRS-3 and by screening with various domains of IRS-3 as bait in a yeast two-hybrid system.

该提案的总体目标是在分子水平上阐明胰岛素受体的信号传导途径。这些途径中的许多开始于活化的胰岛素受体对胰岛素受体底物（IRS）的酪氨酸磷酸化。 磷酸化的IRS然后作为含SH 2结构域的信号传导蛋白的对接/效应蛋白发挥作用。 IRS因此在胰岛素受体的信号传导中起关键作用。 直到最近，IRS家族中只有两个成员，IRS-1和IRS-2，是已知的。 在过去的三年里，我们发现并开始表征IRS家族的另外两个成员，IRS-3和IRS-4。 IRS-3和IRS-4分别在脂肪细胞和人胚肾293细胞中最丰富;我们已经确定了这些细胞类型中与它们相关的SH 2结构域蛋白。 此外，我们已经产生了IRS-3和IRS-4基因有针对性的破坏小鼠;这些敲除（ko）小鼠的初步分析没有显示葡萄糖稳态或其他方面的异常。该提案的一个具体目标是确定IRS-3和IRS-4的生理作用。 这将通过IRS-3和IRS-4 ko小鼠的进一步表征来实现。IRS-3 ko小鼠的研究将集中于IRS-3缺失对脂肪细胞中胰岛素信号传导的影响。 此外，为了检测IRS的重叠和/或补偿作用，将产生和表征缺乏两种IRS的小鼠。 以下三个双ko将在我们的实验室进行检查：IRS-1，4; IRS-2和3或4;以及IRS-3，4。其他的双ko将在合作者的实验室中进行检查。 该提案的第二个具体目标是发现和表征作为涉及IRS-3或IRS-4的信号通路的组分的新蛋白质。 这将通过寻找与IRS-3和IRS-4相互作用的蛋白质来完成。我们已经发现了一组八种蛋白质，它们与通过免疫沉淀分离的IRS-4相关。 这些将被描述。 此外，通过用32 P标记的IRS-3筛选表达库以及在酵母双杂交系统中用IRS-3的各个结构域作为诱饵进行筛选，将发现与IRS-3相关的蛋白质。