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一个中心问题。这一相互竞争的申请寻求定义胰岛素的活性结构以及激素与胰岛素受体(IR)的a亚单位之间的接触点。接下来，我们将利用受体胞外结构域的最新晶体结构来测试胰岛素结合是否触发胞外结构域新的倒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获得改变的配基特异性。我们设想，改变特异性激素-受体对将使一种新的策略能够在转基因小鼠中研究组织特异性胰岛素信号。目的1通过“手性突变”研究胰岛素中的非标准构效关系：比较构象改变位点上相应的D-和L-氨基酸取代。这一策略利用胰岛素的化学合成来验证B链根据受体结合进行重组的假设。这一模型将通过对含有荧光供体和受体的胰岛素衍生物的时间分辨FRET研究来测试，这些荧光供体和受体连接了建议的构象变化部位。目的2试图通过两种方法确定激素-受体接触点：(A)基于对叠氮-苯丙氨酸胰岛素衍生物的位点特异性光交联；(B)通过扫描受体a亚单位的丙氨酸突变来恢复原本不活跃的胰岛素类似物之间的结合。照片产品的测绘将使用由D.F.Steiner(大学)设计的外域结构来完成。芝加哥大学)和Case Center for Protetics&MS的Tandem-MS。通过确定激素-受体的多个接触点，将构建胰岛素-胞外结构域复合体的分子模型。目的3研究胰岛素结合是否触发IR胞外区的构象变化。实验设计建立在最近自由胞外结构域的倒V晶体结构的基础上。通过新的蛋白质工程策略，我们将测试胰岛素结合时胞外区域张开的腿之间的空间关系是否发生改变。在这些研究中，DMA双螺旋将被用作“分子尺子”来测量最佳倒V胞外结构域中的腿间距。目标4试图定义荷尔蒙和受体的改变特异性对。其基本思想是使用一种非活性胰岛素类似物来进化一种受体变体，该受体变体仅与该类似物结合并对其反应，而不是野生型胰岛素。荧光标记突变胰岛素的化学合成将使在基于FACS的检测中筛选补偿受体突变成为可能。目标2的结果将指导a亚基的随机盒式突变。目标4不仅承诺阐明受体特异性的原理，还将在转基因小鼠中进行新的生理学研究。为此，“诱饵”胰岛素类似物将被选择具有其他天然的结构、稳定性和组装特性-因此适合于小鼠的药物给药。作为一个长期目标，我们设想在Kahn组织特异性IR基因敲除小鼠的背景下引入一个“自有标签”的胰岛素信号系统。为了演示原理证明，与C.R.Kahn(Joslin糖尿病中心，波士顿)合作，计划将这项使能技术分别应用于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).