Molecular Biology of Insulin- Receptor Interactions

描述（由申请人提供）：糖尿病是美国的主要健康问题，II型疾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。在任何一种情况下，了解胰岛素作用初始阶段的分子基础，受体结合和酪氨酸激酶激活，对于开发用于治疗该疾病的改进药物至关重要。然而，尽管超过25年的深入研究，这仍然是难以捉摸的。因此，这项建议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。这导致了许多假设模型的形成，以解释胰岛素受体相互作用的复杂性。本实验室使用系统的突变分析方法鉴定并表征了其中一个受体结合位点的功能表位。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，并评估胰岛素结合的假设模型。

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.