INTERACTION BETWEEN INSULIN RECEPTORS AND CELL PROTEINS

胰岛素受体和细胞蛋白之间的相互作用

• 批准号: 2140629
• 负责人: Morris F. White
• 金额: $ 22.93万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140629
• 关键词: SDS polyacrylamide gel electrophoresis; bioassay; carbon; cell biology; cell membrane; complementary DNA; endocytosis; enzyme activity; epidermal growth factor; immunoprecipitation; insulin; insulin receptor; insulinlike growth factor; intermolecular interaction; laboratory rabbit; low density lipoprotein; mutant; oncogenes; phosphorylation; platelet derived growth factor; protein sequence; protein structure; protein tyrosine kinase; proteins; radiotracer; site directed mutagenesis; western blottings

This is the second revision of the competitive renewal of DK38712 entitled "Interaction between insulin receptors and cell proteins". This proposal focuses on the function of the insulin receptor tyrosyl kinase (IR) and the mechanisms of insulin signal transmission. This work is scientifically and clinically important because diabetes is a contemporary health problem that affects about 2% of the world population. Whereas 10% of these individuals suffer from an absolute lack of insulin, most are diabetic because their cells do not respond fully to normal or elevated amounts of circulating insulin. The biochemical problems causing insulin resistance in these Type II diabetics are not well understood. In a few cases, mutations of the IR appear to be the culprit; however, rational treatments for the majority of diabetics requires a knowledge of IR function and regulation, and the molecular details of IR signal transmission. The experiments outlined in this proposal will define structural features of the juxtamembrane region of the IR that are essential for receptor function and signal transmission. The juxtamembrane region of the IR contains about 23 amino acids and is located immediately after the transmembrane spanning region at the inner face of the plasma membrane. Although considerable amino acid sequence identity exists among tyrosyl kinases, the juxtamembrane regions are frequently unique and may be partially responsible for the specificity of signal transmission. Mutations in the juxtamembrane region have a minimal effect on autophosphorylation and kinase activity of the IR, but significantly impair its biological activity and endocytosis, and markedly reduce insulin-stimulated tyrosine phosphorylation of endogenous substrates. Thus, the juxtamembrane region appears to be essential for specific interactions between the IR and cellular proteins that are involved in these responses. Site-directed mutagenesis of the cDNA of the IR will be carried out to alter specific amino acids in the juxtamembrane region to define some essential features for IR function and signaling: (1) determine the role of Tyr960 and Tyr953 in the juxtamembrane region for the interaction between the IR and cellular proteins; (2) determine the requirement for a predicted beta-turn near these tyrosine residues by altering the amino acid sequence in the NPXY960 and GPXY953 motifs to sequences that predict or do not predict beta-turns; (3) assess the specificity of the juxtamembrane region for biological activity, endocytosis and substrate phosphorylation by replacing the IR juxtamembrane region with the corresponding sequence from the receptors for insulin-like growth factor-1, epidermal growth factor, platelet derived growth factor, c-ros and LDL; and (4) determine whether overexpression of the IR juxtamembrane region in a truncated IR molecule competitively inhibits normal IR function and signaling. These four classes of mutant IR molecules will be studied for biological activity, "affinity" for the cellular endocytosis system, their ability to interact with and phosphorylate the major IR substrate IRS-1(pp185), and their ability to activate the phosphatidylinositol 3'-kinase. These experiments will provide new information regarding the molecular basis for interactions between the IR and cellular protein, and provide a model to understand the mechanism of insulin receptor signal transmission.

这是DK38712的竞争更新的第二次修订 “胰岛素受体和细胞蛋白之间的相互作用”。 这个建议 着重于胰岛素受体酪酶激酶（IR）和 胰岛素信号传播的机制。 这项工作是科学的， 临床上很重要，因为糖尿病是当代健康问题 影响了大约2％的世界人口。 这些人中有10％ 患有绝对缺乏胰岛素，大多数是糖尿病的，因为他们 细胞对循环的正常或升高量不完全响应 胰岛素。 在这些类型中引起胰岛素抵抗的生化问题 II糖尿病患者尚不清楚。 在少数情况下，IR的突变 似乎是罪魁祸首；但是，大多数的理性治疗 糖尿病患者需要了解IR功能和调节，以及 IR信号传输的分子细节。 概述的实验 该提案将定义近膜区域的结构特征 IR的受体功能和信号传递至关重要的IR。 IR的近膜区域包含约23个氨基酸，IS 位于跨膜跨越区域之后立即位于内部 质膜的面部。 虽然相当大的氨基酸序列 在酪酶激酶中存在识别，近去膜区域是 通常是独一无二的，可能是部分负责的 信号传输。 近膜区域的突变具有最小的 对IR的自磷酸化和激酶活性的影响，但 显着损害其生物学活性和内吞作用，并明显 减少胰岛素刺激的内源性酪氨酸磷酸化 基材。 因此，近去膜区域似乎对 IR和细胞蛋白之间的特定相互作用 参与了这些回应。 地点定向诱变的cDNA IR将进行以更改近去膜中的特定氨基酸 定义IR功能和信号传导的一些基本特征的区域： （1）确定Tyr960和Tyr953在近膜区域的作用 IR和细胞蛋白之间的相互作用； （2）确定 要求通过这些酪氨酸残基附近预测的β-转弯 将NPXY960和GPXY953基序中的氨基酸序列更改为 预测或不预测β-转变的序列； （3）评估 近膜区域的特异性生物活性， 通过替换IR置膜的内吞作用和底物磷酸化 区域具有来自胰岛素样受体的相应序列 生长因子1，表皮生长因子，血小板得出的生长因子， C-ROS和LDL; （4）确定IR的过表达是否 截短的IR分子中的近膜区域有竞争力抑制 正常的IR功能和信号传导。 这四类突变体IR 将研究分子以进行生物学活性，“亲和力” 细胞内吞作用系统，它们与之相互作用的能力和 磷酸化主要的IR底物IRS-1（PP185）及其能力 激活磷脂酰肌醇3'-激酶。 这些实验会 提供有关相互作用分子基础的新信息 在IR和细胞蛋白之间，并提供了一个模型来了解 胰岛素受体信号传递的机理。