HCG-LH/CG Receptor Binding and Activation

描述(由申请人提供)：这个项目的长期目标是在分子水平上描绘：(A)负责异二聚体糖蛋白激素人绒毛膜促性腺激素(HCG)与G蛋白偶联受体超家族成员促黄体激素/绒毛膜促性腺激素受体(LHR)特异性、高亲和力结合的结构决定因素；(B)配体依赖和非配体依赖的促黄体生成素受体激活的机制；以及(C)受体激活促黄体生成素以启动细胞内信号通路的机制。最近的一些进展为阐明生殖内分泌学中的这些基本问题提供了现实的希望。基于激素和受体构象可塑性的新兴概念，本项目提出了两个具体的目标。1.hCG和LHR结合决定因素的描述：这是正在进行的研究的继续，目的是阐明hCG的野生型和工程类似物与受体胞外结构域之间的结构和接触部位，并对所提出的胞外结构域模型进行严格的测试，该模型假设由于一系列富含亮氨酸的重复而具有尖形。2.受体激活和Gs偶联的机制：研究野生型和工程化hCG-胞外结构域复合体激活受体跨膜部分的机制，导致LHR激活的跨膜螺旋和细胞内环的结构变化，以及阐明Gs激活所需的LHR决定因素。将利用蛋白质工程、生物物理研究和细胞/分子生物技术的组合来解决这些目标。该项目的成功将极大地加深我们对男性和女性生殖内分泌学基本机制的了解，并为合理设计LHR激动剂和拮抗剂提供新的信息。

DESCRIPTION (provided by applicant): The long-range goals of this project are delineation, at the molecular level, of: (a) the structural determinants responsible for specific, high-affinity binding of the heterodimeric glycoprotein hormone, human choriogonadotroin (hCG), to the lutropin/choriogonadotropin receptor (LHR), a member of the G protein-coupled receptor superfamily; (b) the mechanisms of ligand-dependent and ligand-independent LH receptor activation; and (c) the mechanism by which the receptor activates Gs to initiate intracellular signaling pathways. Several recent advances offer promise that elucidation of these fundamental issues in reproductive endocrinology are realistically achievable. Two specific aims, based upon emerging concepts of conformational plasticities of both the hormone and receptor, are proposed for this project. 1. Delineation of hCG and LHR binding determinants: This aim represents a continuation of ongoing studies to elucidate structures of and contact sites between wild type and engineered analogs of hCG and the receptor ectodomain, with a rigorous test of the proposed model of the ectodomain that is postulated to have a cusp shape due to a series of leucine-rich repeats. 2. Mechanisms of receptor activation and Gs coupling: This aim addresses the mechanisms by which wild type and engineered extracellular hCG-ectodomain complexes activate the transmembrane portion of the receptor, the resulting structural changes of the transmembrane helices and intracellular loops responsible for LHR activation, and elucidation of determinants in LHR required for Gs activation. A combination of protein engineering, biophysical investigations, and cell/molecular biological techniques will be utilized to address these aims. Success of this project will greatly enhance our understanding of fundamental mechanisms in male and female reproductive endocrinology and provide new information to assist rational design of LHR agonists and antagonists.