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

项目摘要 G蛋白偶联受体是传递细胞外信号的重要通道， 下游细胞内信号转导途径。它们在细胞间 因此，与基因家族的剪切幅度（>800个基因）一起的通信具有 使GPCR成为极好的治疗靶点。了解激素作用的机制 GPCR和了解药物如何调节它们的行为是一项重要的基础奋进 也是健康科学家的重要使命。这项正在进行的研究的主要目标 该项目旨在研究GPCR调控其主要信号伴侣G proteins.在这次更新中，我们将使用生物化学和生物物理方法来描绘 GPCR·G蛋白（R·G）相互作用的机制，试图解决G GPCR超家族特定成员的蛋白质同种型。我们将集中在一个狭窄的，但 GPCR（b2 AR、M2和M3 AChR、µOR和NTSR 1）的代表性集合及其与 不同的G蛋白亚型（Gs、Gi/o、Gq/11和G12/13）。我们的主要目标是深入了解 结构和动态基础的R·G特异性，通过确定这些家庭成员如何 偶联并激活特定的G蛋白同种型。在上一个融资周期中， 通过解决6种不同的R·G复合物的结构而取得突破：μ阿片受体（μOR）·Gi 1， 神经降压素受体亚型1（NTSR 1）·Gi 1，大麻素受体亚型1（CB 1）·Gi 1，毒蕈碱 M2 AChR·果阿、毒蕈碱M1 AChR·G11和胰高血糖素受体（GCGR）·Gs。这些结构 揭示了受体和G蛋白上的关键区域，我们怀疑这些区域赋予了受体和G蛋白 异构体选择性。在这次更新中，我们建议应用一系列生物化学和生物物理学 探讨R·G结构中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 中间国家。在这次更新中，我们建议检查这些中间状态和探测 它们对R·G特异性和对受体催化的核苷酸 交易所我们将利用尖端的方法，包括冷冻电子显微镜（CryoEM）， 双电子-电子共振（DEER）光谱，荧光共振能量转移，荧光光谱 荧光共振能量转移（FRET），单分子光谱（SMS）和干涉测量来研究这些R-G相互作用。我们 将研究R-G特异性的性质，潜在的机制是否可能在前 关联（可能通过中间状态），或者在耦合阶段。我们认为，与我们的 专业知识，创新试剂的产生，结合尖端的生物物理 这些方法和生成的强有力的初步数据使这一建议易于处理。

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.