Structural Determinants of Heterotrimeric G-protein Nucleotide Cycling

描述（由申请人提供）：G蛋白偶联受体（GPCR）作为异源三聚体G蛋白的催化激活剂，通过将GTP交换为G蛋白上的结合GDP。亚单位GPCR的这种鸟嘌呤核苷酸交换因子（GEF）活性是G蛋白循环的初始步骤，并决定了各种细胞内信号传导途径的开始，这些途径控制着对细胞外信号的关键生理反应。在过去的十年中，G蛋白核苷酸循环的几个步骤的结构基础已经清楚，包括G蛋白的内在GTP水解？以及RGS结构域对这种水解（“GAP活性”）的加速作用;然而，作为受体介导的G蛋白活化的基础的精确结构决定因素以及RGS蛋白对信号起始的促进作用仍然没有完全确定。由于GPCR代表了一组丰富的药物靶点，因此对其激活细胞内信号传导机制的更深入理解应该为药物发现提供有价值的进一步途径。目前，已经提出了几种不同的（有些冲突）模型来解释激活的GPCR和G蛋白异源三聚体之间的通信，导致鸟嘌呤核苷酸交换所需的结构变化。这项研究工作的重点是通过核苷酸交换的异源三聚体G蛋白激活的结构细节的高分辨率阐明。目的1是解决G？通过蛋白质晶体学的快速交换G？我们最近从A. thaliana和C. elegans，以及额外的G？具有增强的GDP释放或倾向于以稳定的无核苷酸状态存在的突变体。在目标2中，三个互补的细胞系统（酵母信息素信号传导，哺乳动物细胞GIRK电流，Dictyosteoprotein cAMP反应）将被用来确定RGS蛋白的非GAP行动，促进GPCR/异源三聚体信号开始动力学的结构决定因素。这第二个目标依赖于我们最近的晶体学证据，快速水解表型的G？i1突变体G202 A源于对通常由RGS结构域稳定的GTP水解过渡态的模拟。目的3是通过与异源三聚体G蛋白亚基结合的功能性受体环肽的蛋白质晶体学来解析受体催化的核苷酸交换的结构决定因素。这后一个目标将促进我们发现的G？i亚家族GEF肽，KB-752，其作为G？介导的开关区域变化;我们最近使用KB-752建立了与其G蛋白靶结合的受体环的第一个晶体结构-多巴胺D2受体IC 3环肽D2 N与G蛋白结合？i1.高分辨率的结构模型来自这些追求将在生化和细胞研究的点突变预测的结构细节，废除或增强核苷酸交换或开关受体/G-蛋白偶联特异性进行验证。这项研究计划的成功将导致对GPCR/G蛋白偶联、激动剂诱导的激活和RGS蛋白促进的精确结构决定因素的新理解。公共卫生相关性：被称为G蛋白偶联受体的蛋白质家族代表了当前药物治疗的最大单一目标，包括控制精神分裂症，双相情感障碍和抑郁症的关键药物。虽然对这些药物的作用至关重要，但这些受体蛋白激活细胞内生化过程的精确分子细节却知之甚少。因此，这项研究旨在建立和验证结构模型，这些模型描述了受体如何激活其偶联G蛋白的细节，这些新知识为药物发现和设计提供了有价值的进一步途径。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) serve as catalytic activators of heterotrimeric G-proteins by exchanging GTP for the bound GDP on the G? subunit. This guanine nucleotide exchange factor (GEF) activity of GPCRs is the initial step in the G-protein cycle and determines the onset of various intracellular signaling pathways that govern critical physiological responses to extracellular cues. The structural basis for several steps in the G-protein nucleotide cycle have been made clear over the past decade, including intrinsic GTP hydrolysis by G? and acceleration of this hydrolysis (`GAP activity') by RGS domains; however, the precise structural determinants underlying receptor-mediated G-protein activation, and facilitation of signal onset by RGS proteins, remain incompletely defined. As GPCRs represent a rich set of drug targets, more thorough understanding of their mechanism of activating intracellular signaling should provide valuable further avenues for drug discovery. Currently, several distinct (and somewhat conflicting) models have been proposed to explain the communication between activated GPCRs and G-protein heterotrimers that leads to the structural changes required for guanine nucleotide exchange. This research effort is focused on a high-resolution elucidation of the structural details underlying heterotrimeric G-protein activation via nucleotide exchange. Aim 1 is to resolve the structural determinants of nucleotide exchange within G? via protein crystallography of fast-exchanging G? subunits we recently identified from the genomes of A. thaliana and C. elegans, as well as additional G? mutants with enhanced GDP release or propensity to exist in a stable, nucleotide-free state. In Aim 2, three complementary cellular systems (yeast pheromone signaling, mammalian cell GIRK currents, Dictyostelium cAMP responses) will be used to ascertain the structural determinants underlying non-GAP actions of RGS proteins that facilitate GPCR/heterotrimer signal onset kinetics. This second aim relies on our recent crystallographic evidence that the fast-hydrolyzing phenotype of the G?i1 mutant G202A arises from mimicry of the transition state for GTP hydrolysis normally stabilized by RGS domains. Aim 3 is to resolve the structural determinants of receptor-catalyzed nucleotide exchange via protein crystallography of functional receptor loop peptides bound to heterotrimeric G-protein subunits. This latter aim will be facilitated by our discovery of a G?i subfamily GEF peptide, KB-752, which acts as a surrogate for G??-mediated switch region changes; we have recently used KB-752 to establish the first crystal structure of a receptor loop bound to its G- protein target - the dopamine D2-receptor ic3 loop peptide D2N bound to G?i1. High-resolution structural models derived from these pursuits will be validated in biochemical and cellular studies of point mutants predicted from structural details to abrogate or enhance nucleotide exchange or switch receptor/G-protein coupling specificity. Success of this research program will lead to a new understanding of the precise structural determinants of GPCR/G-protein coupling, agonist-induced activation, and RGS protein facilitation. The Public Health Relevance: The family of proteins known as G protein-coupled receptors represent the largest single fraction of targets for current drug therapies, including key medicines that control schizophrenia, bipolar disorder, and depression. While critically important for these drugs' actions, the precise molecular details by which these receptor proteins activate biochemical processes inside cells is poorly understood. This research is thus directed towards building and validating structural models that describe the details of how receptors activate their coupled G-proteins, with such new knowledge providing valuable further avenues for drug discovery and design.