Structure and dynamics of G protein coupled receptor-G protein complexes

G蛋白偶联受体-G蛋白复合物的结构和动力学

• 批准号: 10656566
• 负责人: Brian K Kobilka
• 金额: $ 47.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656566
• 关键词: Address; Alzheimer' s Disease; Behavior; Behavior Disorders; Binding; Biochemical; Cardiovascular Diseases; Cell physiology; Cells; Collection; Communication; Complement; Complex; Couples; Coupling; Cryoelectron Microscopy; Data; Diabetes Mellitus; Disease; Drug Modulation; Drug Targeting; Electrons; Family; Family member; Funding; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G13 Protein; GTP-Binding Proteins; Gases; Gene Family; Generations; Genes; Glucagon Receptor; Goals; Health; Hormones; Human Genome; Inflammation; Interferometry; Lung diseases; Mediating; Membrane Proteins; Mission; Molecular; Molecular Conformation; Muscarinics; N-terminal; NPBWR1 gene; Nature; Neurotensin Receptors; Neurotransmitters; Nucleotides; Obesity; Opioid Receptor; Pathway interactions; Pharmacotherapy; Physiology; Play; Process; Protein Family; Protein Isoforms; Reagent; Regulation; Research; Resolution; Role; Scientist; Signal Transduction; Signal Transduction Pathway; Site; Specificity; Spectrum Analysis; Structure; Surveys; arrestin 1; arrestin 2; arrestin3; base; beta-2 Adrenergic Receptors; biophysical techniques; cannabinoid receptor; drug development; experimental study; extracellular; innovation; insight; intercellular communication; member; next generation; novel; programs; protein activation; protein complex; protein structure; receptor; response; single-molecule FRET; targeted treatment; therapeutic target

Project Summary G protein-coupled receptors are important conduits to relay extracellular signals to downstream intracellular signal transduction pathways. Their central role in intercellular communication together with the shear magnitude of the gene family (>800 genes) have therefore made GPCRs superb therapeutic targets. Understanding the mechanism of hormone action on GPCRs and understanding how drugs modulate their behavior is an important fundamental endeavor but also an important mission for health scientists. The primary goal of this ongoing research program is to study the mechanism of GPCR regulation of their primary signaling partners, G proteins. In this renewal we will use biochemical and biophysical approaches to delineate the mechanism of GPCR·G protein (R·G) interactions to try to resolve the extraordinary selectivity of G protein isoforms for specific members of the GPCR superfamily. We will focus on a narrow but representative collection of GPCRs (b2AR, M2 & M3AChR, µOR and NTSR1) and their coupling to different G protein isoforms (Gs, Gi/o, Gq/11 and G12/13). Our major goal is to gain insight into the structural and dynamic bases underlying R·G specificity by determining how these family members couple to and activate specific G protein isoforms. In the previous funding cycle we made several breakthroughs by solving the structures of 6 different R·G complexes: µ opioid receptor (µOR)·Gi1, neurotensin receptor subtype 1 (NTSR1)·Gi1, cannabinoid receptor subtype 1 (CB1)·Gi1, muscarinic M2AChR·GoA, muscarinic M1AChR·G11, and glucagon receptor (GCGR)·Gs. These structures reveal key regions on the receptors and G proteins that we suspect confers receptor and G protein isoform selectivity. In this renewal we propose to apply a spectrum of biochemical and biophysical approaches to interrogate the interaction sites revealed in the R·G structures. In addition, our recent studies suggests various conformational states of the R·G complex, strongly suggesting the existence of intermediate states. In this renewal we propose to examine these intermediate states and probe their potential to contribute toward R·G specificity, and toward receptor-catalyzed nucleotide exchange. We will utilize cutting-edge approaches including cryo-electron microscopy (CryoEM), double electron-electron resonance (DEER) spectroscopy, fluorescence resonance energy transfer (FRET), single molecule spectroscopy (SMS) and interferometry to study these R-G interactions. We will study the nature of the R-G specificity, whether the underlying mechanism may be at the pre- association (perhaps through an intermediate state), or at the coupling stage. We feel that with our expertise, the generation of innovative reagents, the incorporation of cutting edge biophysical approaches and the generation of strong preliminary data together make this proposal tractable.

项目摘要 G蛋白偶联受体是将细胞外信号传递到的重要导管 下游细胞内信号转导途径。它们在细胞间的中心作用 因此，与基因家族（> 800个基因）的剪切幅度进行了交流 使GPCR出色的治疗靶标。了解骑马动作的机制 GPCR和了解药物如何调节其行为是重要的基本努力 这也是健康科学家的重要任务。这项正在进行的研究的主要目标 程序是研究其主要信号伴侣的GPCR调节机制 蛋白质。在这种续约中，我们将使用生化和生物物理方法来描述 GPCR·G蛋白（R·G）相互作用的机制试图解决G的非凡选择性 GPCR超家族特定成员的蛋白质同工型。我们将专注于一个狭窄但 GPCR（B2AR，M2和M3ACHR，µOR和NTSR1）的代表性收集及其耦合到 不同的G蛋白同工型（GS，GI/O，GQ/11和G12/13）。我们的主要目标是深入了解 通过确定这些家庭成员的方式，结构和动态基础是r·g的特异性 对并激活特定的G蛋白同工型。在上一个资金周期中，我们做了几个 通过求解6种不同的R·G复合物的结构：µ阿片受体（µOR）·Gi1， 神经素素受体亚型1（NTSR1）·Gi1，大麻素受体亚型1（CB1）·Gi1，毒蕈碱 M2ACHR·果阿，毒蕈碱M1ACHR·G11和胰高血糖素受体（GCGR）·GS。这些结构 揭示我们怀疑受体和G蛋白的关键区域 同工型选择性。在此续约中，我们建议应用一系列生化和生物物理 询问R·G结构中揭示的相互作用位点的方法。此外，我们最近 研究表明R·G复合物的各种构象状态，强烈表明存在 中间状态。在此续约中，我们建议检查这些中间状态并证明 它们有助于r·g特异性和受体催化的核苷酸的潜力 交换。我们将利用尖端方法，包括冷冻电子显微镜（冷冻）， 双电子电子共振（鹿）光谱，荧光共振能量转移 （FRET），单分子光谱（SMS）和研究这些R-G相互作用的干扰。我们 将研究R-G特异性的性质，是否存在基本机制 关联（也许是通过中间状态），或在耦合阶段。我们感觉到我们的 专业知识，创新试剂的产生，尖端生物物理的结合 方法和强大的初步数据共同产生了这一建议。