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: 2142564
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 30.43万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2142564
• 关键词: 3T3 cells; adipocytes; biological signal transduction; chemical association; complementary DNA; diabetes mellitus; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; genetic library; genetically modified animals; glucose transport; glucose transporter; hormone regulation /control mechanism; insulin receptor; laboratory mouse; molecular cloning; mutant; nucleic acid sequence; peptide hormone metabolism; phosphatidylinositols; phosphorylation; plasmids; protein tyrosine kinase

The overall goal is to elucidate at the molecular level the signaling pathways from the insulin receptor to the known cellular responses of insulin. Several pathways being with the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1); in one of these, IRS-1 then complexes with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). One specific aim of this proposal is to determine what metabolic effects of insulin require this activation. The experimental design is to express a catalytically inactive point mutant of the 110 kD subunit of PI 3-kinase in 3T3-L1 adipocytes and in the fat tissue of transgenic mice and then to measure a variety of insulin effects, including the stimulation of glucose transport. This mutant subunit should function as a dominant negative for insulin activation of PI 3- kinase, and thereby reveal what insulin actions require this activation. The two other specific aims of this proposal are to characterize two proteins that are likely to be direct substrates for the insulin receptor and may, like IRS-1, regulate PI 3-kinase. One is a 60 kD protein that has been purified from rat adipocytes (referred to as pp60); the other is a 170 kD protein that has been slightly characterized to date in hematopoietic cell lines (referred to as 4PS). Both undergo rapid tyrosine phosphorylation in response to insulin and, in their phosphotyrosine state, complex with PI 3-kinase (at least with the 85 kD subunit thereof). Specific aims are: (i) to clone and sequence the cDNA's encoding these (ii) to describe their tissue and subcellular distributions (iii) to determine the effect of the association with PI 3-kinase upon the activity of this enzyme, both in cell lysates and with the purified proteins (iv) to discover other functions of each, through identification of other associated proteins and possibly through assay for other activities suggested by the primary sequence. The results of these studies will guide subsequent efforts to delineate the functions of these two proteins in insulin signaling in vivo. The proposed research is of direct relevance to diabetes. For both non- insulin dependent diabetes (NIDDM) and insulin-independent diabetes (IDDM) a knowledge of the signaling pathways from the insulin receptor may serve as the basis for the design of better therapeutic agents and/regimes. Moreover, in the case of NIDDM, where the basic cause(s) are not yet known, this knowledge may provide the framework for identifying the cause(s).

总的目标是在分子水平上阐明信号 从胰岛素受体到已知的细胞反应的途径 胰岛素。与酪氨酸磷酸化有关的几个途径 胰岛素受体底物1(IRS-1)；在其中之一，IRS-1然后 磷脂酰肌醇3-激酶(PI)与磷脂酰肌醇的络合物并激活 3-激酶)。这项提案的一个具体目标是确定 胰岛素的代谢效应需要这种激活。实验性的 设计目的是表达110kD的催化失活点突变体 3T3-L1脂肪细胞和大鼠脂肪组织中PI-3-K亚单位的表达 转基因小鼠，然后测量各种胰岛素效应， 包括对葡萄糖转运的刺激。这个突变的亚基 应作为PI-3-胰岛素激活的显性负值 从而揭示胰岛素的哪些活动需要这种激活。 这项提案的另外两个具体目标是描述两个 可能是胰岛素受体直接底物的蛋白质 并可能与IRS-1一样，调节PI3-激酶。一种是60kD的蛋白质， 是从大鼠脂肪细胞(称为pp60)中提纯的；另一种是 一种170kD的蛋白质，到目前为止已经在 造血细胞系(简称4PS)。两人都经历了快速 胰岛素对酪氨酸磷酸化的反应，以及在他们的 磷酸酪氨酸态，与PI 3-激酶络合物(至少与85kD 其亚单位)。具体目标是：(I)克隆和测序 编码这些(Ii)以描述它们的组织和亚细胞分布 (Iii)确定与PI-3-激酶的联合作用对 该酶在细胞裂解产物和纯化产物中的活性 蛋白质(Iv)通过鉴定发现每种蛋白质的其他功能 以及可能通过对其他相关蛋白的检测 初级序列所建议的活动。这些研究的结果 研究将指导随后的努力，以描绘这些功能 体内胰岛素信号中的两种蛋白质。 这项拟议的研究与糖尿病有直接关系。对于两个非 胰岛素依赖型糖尿病(NIDDM)和胰岛素非依赖性糖尿病(IDDM) 了解胰岛素受体的信号通路可能会有所帮助。 作为设计更好的治疗剂和/或方案的基础。 此外，在NIDDM的情况下，其根本原因(S)还不是 已知，该知识可提供用于识别 事业(S)。