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: 3243979
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 25.67万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3243979
• 关键词: 3T3 cells; adipocytes; antisense nucleic acid; biological signal transduction; complementary DNA; cyclic AMP; diabetes mellitus; insulin receptor; microtubules; molecular cloning; phosphodiesterases; phosphorylation; protein purification; protein signal sequence; protein tyrosine kinase; proteins; tissue /cell culture; transfection; transposon /insertion element

The overall goal is to elucidate at the molecular level the pathways of signaling from the insulin receptor to the known cellular responses of insulin. The insulin receptor is a protein tyrosine (tyr) kinase, and there is evidence to support the hypothesis that signaling proceeds by the phosphorylation of target proteins. Consequently, the general approach will be to purify and characterize those proteins that are rapidly phosphorylated on tyr in response to insulin and to determine the roles of these proteins in signal transduction from the insulin receptor. The project will mainly involve the use of the 3T3-L1 adipocyte, a cultured cell that is very insulin-responsive. In preliminary studies, the major insulin-elicited phosphotyrosyl (Ptyr) protein in this cell, one of 160 kDa (designated pp160), has been purified and the amino acid sequences of peptides from it obtained. Additionally, four other polypeptides, of 55, 44, 40 and 27 kDa, also phosphorylated on tyr in response to insulin, have been partially purified. Specific aims are: (i) to clone and sequence the cDNA encoding pp160, (ii) to determine the role of pp160 in insulin action in vivo, both by preventing its action, through overexpression upon transfection with DNA encoding it and through direct introduction or purified pp160, (iii) to characterize pp160 with regard to possible function, subcellular location, associated polypeptides, interaction with the insulin receptor, and sites of phosphorylation, and (iv) to purify completely each of the other four Ptyr polypeptides and investigate these as described for pp160 under aims i-iii. The proposed research is of direct relevance to diabetes. For both type I and II diabetes a knowledge of he signaling pathways from the insulin receptor may serve as the basis for the design of better therapeutic agents and/or regimes. Moreover, in the case of type II diabetes, where the basic cause(s) are not yet known, a modification in one of these proteins may prove to be a cause.

总体目标是在分子水平上阐明 从胰岛素受体到已知受体的信号通路 胰岛素的细胞反应。胰岛素受体是一种蛋白质 酪氨酸(Tyr)激酶，有证据支持 假设信号通过磷酸化途径进行 靶蛋白。因此，一般的方法将是 提纯和鉴定那些快速 酪氨酸上的磷酸化对胰岛素的反应并确定 这些蛋白在胰岛素信号转导中的作用 受体。该项目将主要涉及3T3-L1的使用 脂肪细胞，一种对胰岛素非常敏感的培养细胞。在……里面 初步研究，胰岛素诱导的主要磷酸酪氨酰 (PtyR)蛋白，160 kDa之一(命名为pp160)， 已被提纯，并测定了来自 它获得了。此外，另外四种多肽，55，44， 40 kDa和27 kDa，也是胰岛素对Tyr的磷酸化反应， 已被部分提纯。具体目标是：(I)克隆 并对编码pp160的cDNA进行测序，(Ii)以确定其作用 Pp160在体内的胰岛素作用，既通过阻止其 作用，通过DNA转染后的过度表达 对其进行编码并通过直接导入或纯化pp160， (Iii)就可能的功能对pp160进行表征， 亚细胞定位、相关多肽、相互作用 胰岛素受体和磷酸化部位，以及(Iv)至 将其他四种Ptyr多肽分别完全提纯 按照AIMS I-III中pp160的描述进行调查。 这项拟议的研究与糖尿病有直接关系。为 I型和II型糖尿病都了解他的信号通路 可以作为设计胰岛素受体的基础 更好的治疗剂和/或治疗方案。此外，在 II型糖尿病病例，其根本病因(S)尚不清楚 已知，其中一种蛋白质的修饰可能被证明是一种 因为。