How Insulin Binds to the Insulin Receptor

胰岛素如何与胰岛素受体结合

基本信息

批准号：
8003136
负责人：
MICHAEL Aaron WEISS
金额：
$ 4.43万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8003136
关键词：
Address Affinity Alanine American Amino Acid Substitution Binding Biochemical Biochemistry Biological Assay Biophysics Biotin Boston C-terminal Cassette Mutageneses Cell Separation Cells Chicago Clinical Collaborations Complex Coumarins Crystallization Cysteine-Rich Domain DNA Development Diabetes Mellitus Dose Drug Formulations Endocrinology Engineering Evolution Exhibits Experimental Designs Figs - dietary Fluorescein Fluoresceins Fluorescence Fluorescence Resonance Energy Transfer Genetic Grant Hepatocyte Homeostasis Hormonal Hormone Receptor Hormones Human In Vitro Insulin Insulin Receptor Insulin-Like-Growth Factor I Receptor Interdisciplinary Study Knock-out Knockout Mice Label Laboratories Leg Length Letters Libraries Ligand Binding Domain Ligands Limb structure Liver Longitudinal Studies Maps Mass Spectrum Analysis Measures Membrane Metabolic Metabolic syndrome Metabolism Methods Modeling Molecular Molecular Conformation Molecular Models Movement Mus Mutagenesis Mutation N-terminal Organ Pancreas Peptide Mapping Pharmacologic Substance Phenylalanine Phosphotransferases Physiological Positioning Attribute Property Protein Chemistry Protein Engineering Proteomics Pyrenes Rabies Railroads Reagent Regulation Relaxation Research Personnel Resolution Role Saccharomyces cerevisiae Scanning Screening procedure Side Signal Transduction Site Societies Specificity Streptavidin Structure Structure-Activity Relationship Surface System Tail Technology Testing Time Tissues Transgenic Mice Variant X-Ray Crystallography Yeasts alpha benzopyrone analog base blood glucose regulation chemical synthesis crosslink design design and construction foot frontier homeodomain in vivo innovation insulin Wakayama insulin signaling interest molecular modeling mutant new technology novel novel strategies programs receptor receptor binding research study restoration spatial relationship ward

项目摘要

DESCRIPTION (provided by applicant): How insulin binds to its receptor defines a central problem in molecular endocrinology. This competing application seeks to define the active structure of insulin and points of contact between the hormone and the a subunit of the insulin receptor (IR). We will next exploit the recent crystal structure of the receptor ectodomain to test whether insulin binding triggers reorganization of the ectodomain's novel inverted-V conformation. As a bridge between structure and function, in vitro evolution of the insulin receptor will be undertaken to obtain altered ligand specificity. We envisage that altered-specificity hormone-receptor pairs will enable a novel strategy to investigate tissue-specific insulin signaling in transgenic mice. Aim 1 focuses on non-standard structure-activity relationships in insulin through 'chiral mutagenesis': comparison of corresponding D- and L-amino-acid substitutions at proposed sites of conformational change. This strategy exploits chemical synthesis of insulin to test the hypothesis that the B-chain reorganizes on receptor binding. This model will be tested through time-resolved FRET studies of insulin derivatives containing a fluorescent donor and acceptor bridging proposed sites of conformational change. Aim 2 seeks to define points of hormone-receptor contact by two approaches: (a) site-specific photo-cross- linking based on para-azido-Phe insulin derivatives; and (b) restoration of binding between otherwise inactive insulin analogs by alanine scanning mutations in the receptor a subunit. Mapping of photo-products will be accomplished using ectodomain constructs designed by D. F. Steiner (Univ. of Chicago) and tandem-MS in the Case Center for Proteomics & Mass Spectrometry. By determining multiple points of hormone-receptor contact, a molecular model of the insulin-ectodomain complex will be constructed. Aim 3 investigates whether insulin binding triggers a conformational change in the IR ectodomain. Experimental design builds on the recent inverted-V crystal structure of the free ectodomain. Through novel protein engineering strategies, we will test whether the spatial relationship between the splayed legs of the ectodomain is altered on binding of insulin. In these studies the DMA double helix will be employed as a "molecular ruler" to measure leg spacing in an optimal inverted-V ectodomain conformation. Aim 4 seeks to define altered-specificity pairs of hormones and receptors. The essential idea is to employ an inactive insulin analog to evolve a receptor variant that binds and responds only to that analog and not wild-type insulin. Chemical synthesis of fluorescently labeled mutant insulins will enable screening for compensating receptor mutations in a FACS-based assay. Random-cassette mutagenesis of the a subunit will be guided by the results of Aim 2. Aim 4 promises not only to illuminate principles of receptor specificity, but also to enable novel physiological studies in transgenic mice. To this end, "bait" insulin analogs will be chosen to have otherwise native structures, stabilities, and assembly properties - therefore to be appropriate for pharmaceutical administration to mice. As a long-term objective, we envisage introduction of a "private-label" insulin signaling system in the background of a Kahn tissue-specific IR knock-out mouse. To demonstrate proof-of- principle, respective application of this enabling technology to the liver and pancreatic p cells of LIRKO and PIRKO mice is planned in collaboration with C. R. Kahn (Joslin Diabetes Center, Boston).

描述（由申请人提供）：胰岛素如何与其受体结合定义了分子内分泌学中的核心问题。该竞争性应用程序旨在定义胰岛素的活性结构以及激素与胰岛素受体（IR）的亚基之间的接触点。接下来，我们将利用受体外生域的最新晶体结构，以测试胰岛素结合是否会触发细胞结构域的新型倒置-V构象的重组。作为结构和功能之间的桥梁，将进行胰岛素受体的体外演化，以获得改变配体特异性。我们设想改变了特异性激素 - 受体对将实现一种新的策略，以研究转基因小鼠中组织特异性胰岛素信号传导。 AIM 1通过“手性诱变”重点关注胰岛素中的非标准结构活性关系：在构象变化所提出的位点的相应D-和L-氨基酸取代的比较。该策略利用化学合成胰岛素来检验B链在受体结合上重组的假设。该模型将通过含有荧光供体的胰岛素衍生物和受体桥接构象变化的位点的胰岛素衍生物的时间研究进行测试。 AIM 2试图通过两种方法来定义激素受体接触点：（a）基于para-azido-phe胰岛素衍生物的特定于位点的照片交叉链接；（b）通过受体A亚基中的丙氨酸扫描突变在其他无活跃的胰岛素类似物之间恢复结合。将使用由D. F. Steiner（芝加哥大学）设计的型域构建体和蛋白质组学中心和质谱中心中的串联MS设计的光产品映射。通过确定激素受体接触的多个点，将构建胰岛素 - 离谱域复合物的分子模型。 AIM 3研究了胰岛素结合是否会触发IR型构域的构象变化。实验设计建立在自由过型域的最新倒V晶体结构上。通过新颖的蛋白质工程策略，我们将测试胰岛素结合的外域域腿部腿部腿的空间关系是否改变。在这些研究中，DMA双螺旋将被用作“分子尺”，以测量最佳倒置V型域构象中的腿间距。 AIM 4试图定义激素和受体的特异性对变化。重要的想法是利用一种非活性胰岛素类似物来进化一种仅对该类似物而不是野生型胰岛素响应的受体变体。荧光标记的突变胰岛素的化学合成将筛选基于FACS的测定中的补偿受体突变。 A亚基的随机插曲诱变将以AIM 2的结果为指导。AIM4不仅有望阐明受体特异性的原理，而且还可以在转基因小鼠中实现新的生理研究。为此，“诱饵”胰岛素类似物将被选择具有原本的天然结构，稳定性和组装特性 - 因此适合于小鼠药物给药。作为一个长期目标，我们设想在KAHN组织特异性IR敲除小鼠的背景下引入“私有标签”胰岛素信号系统。为了证明原则，计划与C. R. Kahn（波士顿的乔斯林糖尿病中心）合作，计划将这种启示技术应用于Lirko和Pirko小鼠的肝脏和胰腺P细胞。