HCG-LH/CG Receptor Binding and Activation

描述（由申请人提供）：该项目的长期目标是在分子水平上描述：(a)负责异二聚体糖蛋白激素，人绒毛膜促性腺激素（hCG）与lutropin/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，LHR是G蛋白偶联受体超家族的成员；(b)配体依赖性和非配体依赖性LH受体激活机制；(c)受体激活Gs启动细胞内信号通路的机制。最近的几项进展提供了希望，说明生殖内分泌学中的这些基本问题是可以实现的。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目标。1. hCG和LHR结合决定因素的描述：这一目标代表了正在进行的研究的继续，以阐明野生型和工程hCG类似物与受体外结构域之间的结构和接触位点，并对所提出的外结构域模型进行了严格的测试，该模型假定由于一系列富含亮氨酸的重复而具有尖端形状。2. 受体激活和Gs偶联机制：本研究旨在探讨野生型和工程型细胞外hcg -外结构域复合物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Gs激活所需的LHR决定因素的阐明。蛋白质工程、生物物理研究和细胞/分子生物学技术的结合将被用来解决这些目标。该项目的成功将大大提高我们对男性和女性生殖内分泌基本机制的认识，并为LHR激动剂和拮抗剂的合理设计提供新的信息。

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.