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

项目摘要 G蛋白偶联受体(GPCRs)可被部分激动剂激活，导致亚最大信号转导 与完全的激动剂相比，副作用更少。越来越多的研究间接证明 部分激动剂稳定非活化态和完全活化态之间的中间构象状态 完全激活状态下异三聚体GABG蛋白偶联活性降低。由于技术上的障碍 在描绘GPCR构象状态和填充单个中间状态以研究它们时 就个人而言，对部分激动性信号的机械理解一直是具有挑战性的。通过创建 构象偏向突变体，我们鉴定了五种腺苷A2a受体(A2AR)构象状态，包括 使用19F的两个非活动状态(S1和S2)、两个中间状态(S3和S4)以及完全活动状态(S5 核磁共振(核磁共振)光谱。这一结果对以前的研究是一个重大的进步。 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络合物。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及其与G蛋白的相互作用和由此产生的信号效应。 在目标1中，我们将表征中间态S3和S4是否以及如何与Gasbg相互作用。这些 表征将包括研究构象转变和中间态的动力学和 Gasbg及其配体对其跃迁和动力学的影响。在目标2中，我们将绘制构象图 气体的状态，并确定与A2AR的S3和S4状态相互作用的中间状态。在……里面 目的3，我们将确定A2AR的中间态S3和S4是否诱导有能力的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的贡献 牵涉其中。我们期望将中间态的构象和动力学特征关联起来 AIMS 1和AIMS 2产生的A2AR和GAS蛋白对构象偏向突变体的信号转导作用 配基化学计量学，在目标3中测量。 这个拟议项目的完成将促进我们对中间角色的理解 构象在GPCR信号中发挥作用，导致理解受体激活的概念创新 超越了简单的两态模型，并有可能指导基于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 拆分中间体配合物结构的研究进展。

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.