The Role of Intermediate Conformations in G Protein-coupled Receptor Signaling

项目摘要 G蛋白偶联受体（GPCR）可被部分激动剂激活，导致亚最大信号传导 与完全激动剂相比，副作用减少。越来越多的研究间接证明， 部分激动剂稳定非活性和完全活化状态之间的中间构象状态， 从完全活化状态降低的异源三聚体Gabg蛋白偶联活性。由于技术障碍 在描绘GPCR构象状态和填充单个中间状态以研究它们时， 单独地，对部分激动信号传导的机制理解是具有挑战性的。通过创建 构象偏向突变体，我们确定了五个腺苷A2A受体（A2AR）的构象状态，包括 两个非活动状态（S1和S2）、两个中间状态（S3和S4）和一个完全活动状态（S5），使用19F 核磁共振（NMR）光谱。这一结果是对以前研究的重大进步。 R291A突变体主要积累中间S4状态，而R291AR293A突变体主要积累中间S4状态。 填充中间S3状态和完全激活状态S5。这一发现使我们能够研究 这些中间体和它们的配合物。我们将使用这两个突变体来研究中间体的作用。 状态S3和S4以及它们与G蛋白的相互作用和随后的信号传导效应。 在目标1中，我们将描述中间态S3和S4是否以及如何与Gasbg相互作用。这些 表征将包括构象转变和中间态动力学的研究， Gasbg和配体对其跃迁和动力学的影响。在目标2中，我们将绘制构象 气体的状态，并确定其与A2AR的S3和S4状态相互作用的中间状态。在 目标3，我们将确定A2AR的中间态S3和S4是否诱导出胜任的Gasbg态 对于GTP水解，G蛋白解离，以及在没有S5状态的情况下对次最大信号传导的贡献， 涉案我们期望关联的中间态的构象和动力学特性的 从目的1和2产生的A2AR和Gas蛋白对构象偏向突变体的信号传导功效， 配体化学计量，在目标3中测量。 完成这个拟议的项目将促进我们的理解的作用，中间 构象在GPCR信号传导中的作用，导致理解受体活化的概念创新 超越了简单的两态模型，并可能指导基于GPCR和G蛋白的药物设计 对配体的构象反应。此外，构象偏向突变体将指导一种方法， 在解析中间体配合物结构方面取得了进展。

Project Summary G protein-coupled receptors (GPCRs) can be activated by partial agonists, resulting in submaximal signaling with reduced side effects, compared to the full agonist. Increased studies have indirectly demonstrated that partial agonists stabilize intermediate conformational states between the inactive and fully activated states with a reduced heterotrimeric Gabg protein coupling activity from the fully activated state. Due to the technical hurdles in delineating GPCR conformational states and populating individual intermediate states to study them individually, a mechanistical understanding of partial agonism signaling has been challenging. By creating conformation-biased mutants, we identified five adenosine A2A receptor (A2AR) conformational states, including two inactive states (S1 and S2), two intermediate states (S3 and S4), and a fully active state (S5), using 19F nuclear magnetic resonance (NMR) spectroscopy. This result is a significant advancement to previous research. The R291A mutant predominantly accumulates the intermediate S4 state while the R291AR293A mutant populates both intermediate S3 state and the full activated state S5. This finding enables us to study the roles of these intermediates and their complexes. We will use these two mutants to examine the roles of intermediate states S3 and S4 and their interactions with G proteins and consequent signaling effects. In Aim 1, we will characterize whether and how the intermediate states S3 and S4 interact with Gasbg. These characterizations will include the study of conformational transitions and dynamics of intermediate states and the effects of Gasbg and ligands on their transitions and dynamics. In Aim 2, we will map the conformational states of the Gas and determine its intermediate states that interact with the S3 and S4 states of the A2AR. In Aim 3, we will determine if the intermediate states S3 and S4 of the A2AR induce Gasbg states that are competent for GTP hydrolysis, G protein dissociation, and contribution to submaximal signaling without the S5 state being involved. We expect to correlate conformational and dynamic characteristics of the intermediate states of the A2AR and Gas protein created from Aims 1 and 2 to the signaling efficacies of conformation-biased mutants with ligand stoichiometries, measured in Aim 3. The completion of this proposed project will advance our understanding of the roles that intermediate conformations play in GPCR signaling, lead to a conceptual innovation in understanding receptor activation beyond a simple two-state model, and potentially guide drug design based on GPCR and G protein conformational responses to ligands. Moreover, the conformation-biased mutants will guide an approach development in resolving the structures of intermediate complexes.