Structural Determinants of Heterotrimeric G-protein Nucleotide Cycling

异源三聚体 G 蛋白核苷酸循环的结构决定因素

• 批准号: 7523807
• 负责人: David P. Siderovski
• 金额: $ 21.59万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7523807
• 关键词: Acceleration; Affect; Agonist; Animal Model; Binding; Biochemical; Biochemical Process; Bipolar Disorder; Caenorhabditis elegans; Cells; Communication; Complex; Condition; Conflict (Psychology); Coupled; Coupling; Crystallization; Crystallography; Cues; Cyclic AMP; Data; Dictyostelium; Dopamine D2 Receptor; Drug Delivery Systems; Drug effect disorder; Etiology; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Proteins; Genome; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Hydrolysis; Kinetics; Knowledge; Lead; Light; Mammalian Cell; Mediating; Medicine; Mental Depression; Modeling; Molecular; Molecular Target; Mutation; Nucleotides; Pathway interactions; Peptides; Phage Display; Pharmacotherapy; Phenotype; Pheromone; Physiological; Protein Family; Protein Subunits; Proteins; Public Health; RGS Domain; RGS Proteins; Reaction; Relative (related person); Research; Resolution; Schizophrenia; Signal Pathway; Signal Transduction; Specificity; Structural Models; Structure; System; Therapeutic Agents; Yeasts; base; design; drug discovery; extracellular; mimicry; mouse Gdi2 protein; mutant; novel; programs; protein activation; receptor; reconstitution; response; structural biology; success

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.

描述（由申请人提供）：G蛋白偶联受体（GPCR）通过将GTP换成G上的GDP，用作异三聚体G蛋白的催化活化剂？亚基。 GPCRS的这种鸟嘌呤核苷酸交换因子（GEF）活性是G蛋白周期中的第一步，并确定了控制细胞外提示的关键生理反应的各种细胞内信号通路的发作。在过去的十年中，已经明确了G蛋白核苷酸周期中几个步骤的结构基础，包括G？ RGS域对这种水解（“间隙活性”）的加速度；然而，受体介导的G蛋白激活的确切结构决定因素以及RGS蛋白对信号发作的促进，仍未完全定义。由于GPCR代表了一组丰富的药物靶标，因此对它们激活细胞内信号传导机制的更透彻理解应为药物发现提供有价值的进一步途径。当前，已经提出了几种不同的（且有些冲突的）模型来解释激活的GPCR和G蛋白异三聚体之间的通信，从而导致鸟嘌呤核苷酸交换所需的结构变化。这项研究工作集中在通过核苷酸交换的杂点G蛋白激活基础的结构细节的高分辨率阐明。目标1是解决G中核苷酸交换的结构决定因素？通过快速交换G的蛋白质晶体学？我们最近从A. thaliana和C. exemans的基因组中确定的亚基，以及其他G？ GDP释放增强或以稳定的无核苷酸状态存在的突变体。在AIM 2中，将使用三个互补的细胞系统（酵母信息素信号传导，哺乳动物细胞毛孔电流，dictyostelium CAMP响应）来确定RGS蛋白质的非间隙作用的结构决定因素，这些决定因素促进了促进GPCR/Heterotrimer信号发起的RGS蛋白质。第二个目标依赖于我们最近的晶体学证据，即G？i1突变体G202a的快速水解表型源于GTP水解过渡态的模仿，通常由RGS域稳定。 AIM 3是通过功能受体环的蛋白质晶体学通过与异构三聚体G蛋白亚基结合的功能受体环肽的蛋白质晶体学交换的结构决定因素。我们发现了G？i亚科GEF肽KB-752，这将促进后一个目标，该肽是G ?? - 介导的开关区域变化的替代物；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 - 多巴胺D2受体IC3环肽D2N与G？i1结合。从这些追求中得出的高分辨率结构模型将在从结构细节预测的点突变体的生化和细胞研究中得到验证，从结构细节到废除或增强核苷酸交换或开关受体/G蛋白偶联特异性。该研究计划的成功将导致对GPCR/G蛋白耦合，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：称为G蛋白偶联受体的蛋白质家族是当前药物疗法靶标的最大的单个靶标，包括控制精神分裂症，双相情感障碍和抑郁症的关键药物。尽管对于这些药物的作用至关重要，但这些受体蛋白激活细胞内部生化过程的精确分子细节却鲜为人知。因此，这项研究针对建立和验证结构模型，以描述受体如何激活其耦合G蛋白的细节，而这样的新知识为药物发现和设计提供了宝贵的进一步途径。