How Insulin Binds to the Insulin Receptor

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核心问题。这个竞争性的应用程序试图定义胰岛素的活性结构和激素和胰岛素受体（IR）的一个亚基之间的接触点。接下来，我们将利用受体外结构域的最新晶体结构来测试胰岛素结合是否会触发外结构域的新型倒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。我们设想特异性改变的激素受体对将使研究转基因小鼠组织特异性胰岛素信号传导的新策略成为可能。目的1侧重于通过“手性突变”研究胰岛素中非标准的结构-活性关系：在构象变化的建议位点上比较相应的D-氨基酸和l -氨基酸取代。这种策略利用胰岛素的化学合成来检验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和受体桥接构象变化提议位点的胰岛素衍生物的时间分辨FRET研究进行测试。目的2试图通过两种方法定义激素受体接触点：(a)基于对叠氮多-苯二酚胰岛素衍生物的位点特异性光交联；(b)通过受体a亚基的丙氨酸扫描突变，恢复非活性胰岛素类似物之间的结合。光产物的绘制将使用D. F. Steiner（芝加哥大学）设计的外畴结构和蛋白质组学与质谱分析案例中心的串联质谱来完成。通过确定激素-受体接触的多个点，胰岛素-外结构域复合物的分子模型将被构建。目的3研究胰岛素结合是否触发IR外畴的构象变化。实验设计建立在最近自由外畴的倒v晶体结构上。通过新的蛋白质工程策略，我们将测试在胰岛素结合的情况下，外畴的分叉腿之间的空间关系是否被改变。在这些研究中，DMA双螺旋将被用作“分子尺子”来测量最佳反v外畴构象中的腿间距。目的4旨在定义特异性改变的激素和受体对。其基本思想是利用一种不活跃的胰岛素类似物来进化一种受体变体，这种受体变体只与这种类似物结合并产生反应，而不是对野生型胰岛素产生反应。化学合成的荧光标记突变胰岛素将使筛选补偿受体突变在一个基于facs的分析。a亚基的随机盒诱变将以Aim 2的结果为指导。目的4不仅阐明了受体特异性的原理，而且还使转基因小鼠的新的生理研究成为可能。为此，“诱饵”胰岛素类似物将被选择具有其他天然结构，稳定性和组装特性-因此适合给药给药小鼠。作为长期目标，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。为了证明原理，我们计划与C. R. Kahn（波士顿乔斯林糖尿病中心）合作，将这项技术分别应用于LIRKO和PIRKO小鼠的肝脏和胰腺p细胞。

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).