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: 3243978
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 24.77万
• 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/3243978
• 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的 胰岛素的细胞反应。 胰岛素受体是一种 酪氨酸（tyr）激酶，有证据支持 假设信号传导通过磷酸化 靶蛋白。 因此，一般做法是 纯化和鉴定那些快速 磷酸化的酪氨酸，并确定 这些蛋白质在胰岛素信号转导中的作用 受体的 该项目将主要涉及使用3 T3-L1 脂肪细胞，一种对胰岛素非常敏感的培养细胞。 在 初步研究，胰岛素引起的主要磷酸酪氨酸 （Ptyr）蛋白，160 kDa之一（命名为pp 160）， 已经被纯化，并且来自 它获得了。 此外，还发现了另外四种多肽，分别为55，44， 40和27 kDa，也在对胰岛素的反应中在tyr上磷酸化， 已经被部分净化了。 具体目标是：（一）克隆 并对编码pp 160的cDNA进行测序，（ii）以确定 pp 160在体内胰岛素作用中的作用， 作用，通过DNA转染后的过表达 编码它并通过直接引入或纯化的pp 160， (iii)为了表征PP 160的可能功能， 亚细胞定位，相关多肽，相互作用 胰岛素受体和磷酸化位点，和（iv） 完全纯化其他四种Ptyr多肽中的每一种， 按照目标一至三对pp 160所作的说明调查这些问题。 这项研究与糖尿病直接相关。 为 I型和II型糖尿病都需要了解信号通路 可以作为设计的基础 更好的治疗剂和/或方案。 而且在 II型糖尿病的病例，其中基本原因尚不明确 已知，这些蛋白质之一的修饰可能被证明是 事业