G Protein-Coupled Receptors - Structure and Regulation

G 蛋白偶联受体 - 结构和调控

• 批准号: 7836597
• 负责人: Elliott M Ross
• 金额: $ 15.7万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7836597
• 关键词: Accounting; Agonist; Behavior; Biochemical; Cells; Cellular Structures; Cholinergic Receptors; Competitive Binding; Complex; Computer Simulation; Coupling; Cultured Cells; Detection; Disease; Drug Prescriptions; Equilibrium; Event; Excision; Experimental Designs; Funding; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; GTPase-Activating Proteins; Goals; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Hormones; Hour; Human; Individual; Kinetics; Ligands; Mediating; Methods; Molecular; Molecular Conformation; Monitor; Muscarinic Acetylcholine Receptor; Muscarinics; Neurotransmitters; Output; Pharmaceutical Preparations; Phospholipase C; Physiological; Positioning Attribute; Process; Protein Binding; Protein Conformation; Protein Subunits; Proteins; Reaction; Recovery; Regulation; Relative (related person); Reporter; Request for Proposals; Research; Signal Transduction; Signaling Molecule; Specificity; Speed; Stimulus; Structure; Sum; System; Testing; Time; Time Study; base; programs; protein activation; protein protein interaction; public health relevance; reaction rate; receptor; receptor binding; receptor coupling; reconstitution; research study; response; scaffold; sensor; synergism

DESCRIPTION (provided by applicant): We propose to continue our studies of the biochemical and biophysical mechanisms used by G protein signaling modules to organize, integrate and transmit information. The diversity, number and ubiquity of G protein-coupled receptors (GPCRs) account for their involvement in a huge number of diseases and for their being efficacious targets of numerous drugs. While all G protein modules contain the same basic components GPCR, heterotrimeric G protein, effector protein, GTPase-activating protein (GAP) and follow the same cycle of activation and deactivation, their detailed behaviors vary enormously. We propose to discover in quantitative terms how the underlying protein-protein interactions within a G protein module determine the amplitude, timing and specificity of its output. The dynamics of G protein signaling reflects multiple regulatory interactions among Ga and G¿? subunits, GPCR, GAP and effector. We will use kinetic and equilibrium methods to monitor the GTPase cycle reactions, conformational states of the proteins and protein-protein binding events. Our three specific aims are inter-related by this focus. 1. We will study the independent effects of receptors, GAPs and G¿? on the rates of G protein activation and deactivation, and the relation of these rates to the steady-state activation of G protein and effector. Experiments will utilize a reconstituted system of purified proteins at known concentrations, including fluorescent sensors of protein conformation and protein-protein binding. We will use a quantitative computational model of these events to guide experimental design and analysis, and to help interpret the complex behaviors of this system. Key findings will be qualitatively tested in cultured cells. We will focus on the mechanism(s) whereby GAPs modulate the speed of response to receptor input without significantly inhibiting signal output. 2. Receptor conformation is regulated by both agonist and G proteins, but the relationship between agonist binding, receptor conformation and G protein regulation remains unclear. We will use fluorescent reporters within ¿1 muscarinic receptors to study their conformational changes in response to agonists, Ga and G¿? subunits. This study should identify the equilibrium poise and reaction rates among the receptor's conformational states and their energetic coupling to ligands and G proteins. 3. G¿? subunits are required for normal stimulation of G protein activation by receptors and regulate many effector proteins. G¿? also inhibits GAP activity and, at high concentrations, inhibits receptor-mediated G protein activation. We will determine the mechanisms that coordinate these effects, focusing on the kinetics of the cooperative and competitive binding interactions among the proteins involved. PUBLIC HEALTH RELEVANCE: G proteins and G protein-coupled receptors mediate regulation of all human cells in response to hormones, neurotransmitters and diverse other signaling molecules. G protein signaling systems are therefore involved in a huge number of disease processes and are estimated to be the targets of over half of the approved prescription drugs. This project studies how the timing and intensity of G protein signaling is controlled at the molecular level. The additional studies proposed for this revision are aimed at understanding how signals from distinct G proteins in a cell potentiate each other.

描述（由适用提供）：我们建议继续研究G蛋白信号传导模块用于组织，整合和传输信息的生化和生物物理机制。 G蛋白偶联受体（GPCR）的多样性，数量和无处不在，解释了它们参与大量疾病的参与以及它们是众多药物的有效靶标。虽然所有G蛋白模块都包含相同的基本成分GPCR，异构体G蛋白，效应蛋白，GTPase激活蛋白（GAP），并遵循相同的激活和失活循环，但它们的详细行为却大不相同。我们建议用定量术语发现G蛋白模块中的潜在蛋白质 - 蛋白质相互作用如何确定其输出的放大器，时机和特异性。 G蛋白信号传导的动力学反映了GA和G g的多种调节相互作用？亚基，GPCR，间隙和效应子。我们将使用动力学和等效方法来监测GTPase循环反应，蛋白质的构象状态和蛋白质 - 蛋白质结合事件。我们的三个特定目标与这个重点相关。 1。我们将研究接收者，差距和g¿的独立影响？关于G蛋白激活和失活的速率，以及这些速率与G蛋白和效应子的稳态激活的关系。实验将在已知浓度（包括蛋白质构象和蛋白质 - 蛋白质结合的荧光传感器）下利用重建的纯化蛋白质系统。我们将使用这些事件的定量计算模型来指导实验设计和分析，并帮助解释该系统的复杂行为。主要发现将在培养的细胞中进行定性测试。我们将重点关注机制，从而使差距调节对受体输入的响应速度而不显着抑制信号输出。 2。受体组成受激动剂和G蛋白的调节，但是激动剂结合，受体构象和G蛋白调节之间的关系尚不清楚。我们将在````1个毒蕈碱受体''中使用荧光记者研究其构象变化，以响应激动剂，GA和G？？亚基。这项研究应确定接收器构象状态之间的平衡毒物和反应速率以及它们与配体和G蛋白的能量耦合。 3。G⿿？通过受体正常刺激G蛋白激活并调节许多效应蛋白需要亚基。 g？？还抑制间隙活性，并在高浓度下抑制受体介导的G蛋白激活。我们将确定协调这些效果的机制，重点是所涉及的蛋白质之间合作和竞争性结合相互作用的动力学。 公共卫生相关性：G蛋白和G蛋白偶联受体对所有人类细胞的培养基调节，以响应激素，神经递质和其他信号分子。因此，G蛋白信号系统参与了大量疾病过程，估计是批准的处方药的一半以上的靶标。该项目研究了如何在分子水平控制G蛋白信号传导的时间和强度。为此修订提出的其他研究旨在了解细胞电位中不同G蛋白的信号如何相互潜力。