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:
- 金额:$ 4.43万
- 依托单位:
- 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:
- 关键词:AddressAffinityAlanineAmericanAmino Acid SubstitutionBindingBiochemicalBiochemistryBiological AssayBiophysicsBiotinBostonC-terminalCassette MutagenesesCell SeparationCellsChicagoClinicalCollaborationsComplexCoumarinsCrystallizationCysteine-Rich DomainDNADevelopmentDiabetes MellitusDoseDrug FormulationsEndocrinologyEngineeringEvolutionExhibitsExperimental DesignsFigs - dietaryFluoresceinFluoresceinsFluorescenceFluorescence Resonance Energy TransferGeneticGrantHepatocyteHomeostasisHormonalHormone ReceptorHormonesHumanIn VitroInsulinInsulin ReceptorInsulin-Like-Growth Factor I ReceptorInterdisciplinary StudyKnock-outKnockout MiceLabelLaboratoriesLegLengthLettersLibrariesLigand Binding DomainLigandsLimb structureLiverLongitudinal StudiesMapsMass Spectrum AnalysisMeasuresMembraneMetabolicMetabolic syndromeMetabolismMethodsModelingMolecularMolecular ConformationMolecular ModelsMovementMusMutagenesisMutationN-terminalOrganPancreasPeptide MappingPharmacologic SubstancePhenylalaninePhosphotransferasesPhysiologicalPositioning AttributePropertyProtein ChemistryProtein EngineeringProteomicsPyrenesRabiesRailroadsReagentRegulationRelaxationResearch PersonnelResolutionRoleSaccharomyces cerevisiaeScanningScreening procedureSideSignal TransductionSiteSocietiesSpecificityStreptavidinStructureStructure-Activity RelationshipSurfaceSystemTailTechnologyTestingTimeTissuesTransgenic MiceVariantX-Ray CrystallographyYeastsalpha benzopyroneanalogbaseblood glucose regulationchemical synthesiscrosslinkdesigndesign and constructionfootfrontierhomeodomainin vivoinnovationinsulin Wakayamainsulin signalinginterestmolecular modelingmutantnew technologynovelnovel strategiesprogramsreceptorreceptor bindingresearch studyrestorationspatial relationshipward
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的中心问题。该竞争性申请旨在定义胰岛素的活性结构以及激素与胰岛素受体 (IR) α 亚基之间的接触点。接下来,我们将利用受体胞外域的最新晶体结构来测试胰岛素结合是否会触发胞外域的新型倒V构象的重组。作为结构和功能之间的桥梁,胰岛素受体的体外进化将被进行以获得改变的配体特异性。我们设想改变的特异性激素受体对将启用一种新策略来研究转基因小鼠中组织特异性胰岛素信号传导。目标 1 通过“手性诱变”关注胰岛素中的非标准结构-活性关系:比较提议的构象变化位点上相应的 D- 和 L- 氨基酸取代。该策略利用胰岛素的化学合成来检验 B 链在受体结合时重组的假设。该模型将通过胰岛素衍生物的时间分辨 FRET 研究进行测试,该衍生物含有桥接构象变化的拟议位点的荧光供体和受体。目标 2 试图通过两种方法定义激素受体接触点:(a) 基于对叠氮基-Phe 胰岛素衍生物的位点特异性光交联; (b)通过丙氨酸扫描受体a亚基中的突变来恢复原本无活性的胰岛素类似物之间的结合。照片产物的映射将使用 D. F. Steiner(芝加哥大学)设计的胞外域结构和蛋白质组学和质谱案例中心的串联 MS 来完成。通过确定激素-受体接触的多个点,将构建胰岛素-胞外域复合物的分子模型。目标 3 研究胰岛素结合是否会触发 IR 胞外域的构象变化。实验设计建立在最近的自由胞外域倒V晶体结构的基础上。通过新颖的蛋白质工程策略,我们将测试胞外域张开的腿之间的空间关系是否因胰岛素的结合而改变。在这些研究中,DMA 双螺旋将被用作“分子尺”来测量最佳倒置 V 胞外域构象中的腿间距。目标 4 试图定义改变特异性的激素和受体对。其基本思想是利用无活性的胰岛素类似物来进化出一种受体变体,该变体仅与该类似物结合并做出反应,而不是野生型胰岛素。荧光标记突变胰岛素的化学合成将能够在基于 FACS 的测定中筛选补偿性受体突变。 a 亚基的随机盒诱变将由目标 2 的结果指导。目标 4 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进行新的生理学研究。为此,将选择具有天然结构、稳定性和组装特性的“诱饵”胰岛素类似物,因此适合对小鼠进行药物给药。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“自有品牌”胰岛素信号系统。为了进行原理验证,计划与 C. R. Kahn(波士顿乔斯林糖尿病中心)合作,分别将该技术应用于 LIRKO 和 PIRKO 小鼠的肝脏和胰腺 p 细胞。
项目成果
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MICHAEL Aaron WEISS其他文献
MICHAEL Aaron WEISS的其他文献
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