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是分子内分泌学的一个中心问题。这一竞争性应用试图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，我们将利用最近的受体胞外域的晶体结构来测试胰岛素结合是否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，将进行胰岛素受体的体外进化以获得改变的配体特异性。我们设想，改变特异性胰岛素受体对将使一种新的策略，在转基因小鼠中研究组织特异性胰岛素信号。目的1着重于通过“手性诱变”在胰岛素中的非标准结构-活性关系：在建议的构象变化位点处的相应D-和L-氨基酸取代的比较。该策略利用胰岛素的化学合成来测试B链在受体结合时重组的假设。该模型将通过时间分辨FRET研究胰岛素衍生物含有荧光供体和受体桥接建议的构象变化的网站进行测试。目的2试图通过两种方法来定义胰岛素-受体接触点：（a）基于对叠氮基-苯丙氨酸胰岛素衍生物的位点特异性光交联;和（B）通过受体a亚基中的丙氨酸扫描突变来恢复否则无活性的胰岛素类似物之间的结合。将使用由D. F. Steiner（芝加哥大学）和蛋白质组学与质谱案例中心的串联MS。通过确定胰岛素-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。目的3研究胰岛素结合是否引发IR胞外域的构象变化。实验设计建立在最近的倒V型晶体结构的游离胞外域。通过新的蛋白质工程策略，我们将测试胞外域的张开腿之间的空间关系是否在胰岛素结合时改变。在这些研究中，DMA双螺旋将被用作“分子标尺”来测量最佳倒V胞外域构象中的腿间距。目标4试图定义激素和受体的特异性改变对。其基本思想是利用一种无活性的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产生反应，而不与野生型胰岛素结合并产生反应。荧光标记的突变胰岛素的化学合成将使得能够在基于FACS的测定中筛选补偿受体突变。α亚基的随机盒式诱变将由目标2的结果指导。目的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，“诱饵”胰岛素类似物将被选择为具有其他天然结构、稳定性和组装性质-因此适合于对小鼠的药物施用。作为一个长期的目标，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“私有标签”胰岛素信号传导系统。为了证明原理验证，计划与C. R. Kahn（波士顿Joslin糖尿病中心）。

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).