Molecular Biology of Insulin- Receptor Interactions

胰岛素受体相互作用的分子生物学

• 批准号: 6969914
• 负责人: JONATHAN WHITTAKER
• 金额: $ 29.88万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6969914
• 关键词: CHO cells; affinity chromatography; alanine; binding sites; diabetes mellitus; enzyme linked immunosorbent assay; genetic regulatory element; growth factor receptors; hormone regulation /control mechanism; hybridomas; insulin receptor; intermolecular interaction; molecular cloning; molecular pathology; protein isoforms; protein purification; protein tyrosine kinase; receptor binding; regulatory gene; site directed mutagenesis; surface plasmon resonance; western blottings

DESCRIPTION (provided by applicant): Diabetes mellitus is a major health problem in the U.S. and the Type II disease will soon reach epidemic proportions. Type I is due to absolute insulin deficiency, whereas Type II is due to impairment of insulin action. In either case an understanding of the molecular basis of the initial stages of insulin action, receptor binding and tyrosine kinase activation, will be essential for the development of improved agents for the treatment of the disease. However, despite more than 25 years intensive study, this remains elusive. Thus the longterm objective of this proposal is to elucidate the molecular mechanisms underlying the formation of the insulin-receptor complex. The weight of evidence suggests that both insulin and the receptor have two distinct binding sites. This has lead to the formulation of a number of hypothetical models to explain complexities of the insulin-receptor interaction. Studies in this laboratory using systematic, mutational analysis have identified and characterized the functional epitope of one of these receptor binding sites. This strategy will now be used to localize and characterize the functional epitope of the other ligand binding site and also to evaluate proposed hypothetical models of insulin binding. The Specific Aims of the proposed studies are: 1) Identification of receptor domains and sub-domains contributing to the second receptor ligand binding site; 2) Localization of the functional epitope of the second ligand binding site by alanine scanning mutagenesis; 3) Evaluation of the kinetics of insulin and analog binding to alanine mutants of determinants of the functional epitope; 4) Evaluation of the effects of alanine receptor mutations on negative cooperativity of insulin binding; and 5) Comparison of the functional epitopes of the second ligand binding sites of A and B isoforms of the insulin receptor.

描述（由申请人提供）：糖尿病是美国的一个主要健康问题，II型糖尿病将很快达到流行病的程度。I型是由于绝对胰岛素缺乏，而II型是由于胰岛素作用受损。在任一情况下，了解胰岛素作用、受体结合和酪氨酸激酶活化的初始阶段的分子基础对于开发用于治疗该疾病的改进剂将是必不可少的。然而，尽管有超过25年的深入研究，这仍然难以捉摸。因此，本提案的长期目标是阐明胰岛素受体复合物形成的分子机制。大量证据表明胰岛素和受体都有两个不同的结合位点。这导致了一些假设模型的制定，以解释胰岛素受体相互作用的复杂性。本实验室使用系统突变分析的研究已经鉴定并表征了这些受体结合位点之一的功能表位。该策略现在将用于定位和表征其他配体结合位点的功能表位，并评估所提出的胰岛素结合的假设模型。 拟议研究的具体目标是： 1)鉴定有助于第二受体配体结合位点的受体结构域和亚结构域; 2)丙氨酸扫描诱变法定位第二配体结合位点的功能表位; 3)胰岛素和类似物与功能表位决定簇的丙氨酸突变体结合的动力学评价 4)评估丙氨酸受体突变对胰岛素结合的负协同性的影响;和5）胰岛素受体的A和B同种型的第二配体结合位点的功能表位的比较。