Structural Determinants of Heterotrimeric G-protein Nucleotide Cycling

描述(申请人提供)：G蛋白偶联受体(GPCRs)通过用G？亚单位。GPCRs的这种鸟嘌呤核苷酸交换因子(GPCRs)活性是G蛋白周期的初始步骤，并决定了各种细胞内信号通路的启动，这些信号通路控制着对细胞外信号的关键生理反应。在过去的十年中，G蛋白核苷酸循环中的几个步骤的结构基础已经被弄清楚，包括G？并通过RGS结构域加速这种水解(‘GAP活性’)；然而，受体介导的G蛋白激活以及RGS蛋白促进信号启动的确切结构决定因素仍未完全确定。由于GPCRs代表了一系列丰富的药物靶点，更彻底地了解它们激活细胞内信号的机制应该会为药物发现提供有价值的进一步途径。目前，已经提出了几个不同的(有些相互冲突的)模型来解释激活的GPCRs和G蛋白异源三聚体之间的通信，这导致了鸟嘌呤核苷酸交换所需的结构变化。这项研究致力于高分辨率地阐明通过核苷酸交换激活异三聚体G蛋白的结构细节。目标1是解决G？通过蛋白质结晶学的快速交换G？我们最近从拟南芥和线虫基因组中鉴定出的亚基，以及附加的G？GDP释放增强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、哺乳动物细胞GIRK电流、DictyostelialcAMP反应)将被用来确定RGS蛋白促进GPCR/异源三聚体信号启动动力学的非间隙作用的结构决定因素。第二个目的依赖于我们最近的结晶学证据，即G？I1突变体G202a的快速水解型源于对通常由RGS结构域稳定的GTP水解过渡态的模仿。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结晶学来解析受体催化的核苷酸交换的结构决定因素。后一个目标将通过我们发现的G？I亚家族全环基金多肽KB-752来促进，它可以替代G？介导的开关区改变；我们最近使用KB-752建立了与其G-蛋白靶标结合的受体环的第一个晶体结构--与G？I1结合的多巴胺D2受体IC3环肽D2N。从这些追求中获得的高分辨率结构模型将在生化和细胞研究中得到验证，根据结构细节预测的点突变可以取消或增强核苷酸交换或开关受体/G蛋白偶联的特异性。这一研究计划的成功将导致对GPCR/G蛋白偶联、激动剂诱导激活和RGS蛋白易化的精确结构决定因素的新理解。与公共卫生相关：被称为G蛋白偶联受体的蛋白质家族代表着当前药物治疗的最大单一靶点，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确切分子细节却鲜为人知。因此，这项研究旨在建立和验证结构模型，描述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.