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

中间构象在 G 蛋白偶联受体信号传导中的作用

基本信息

批准号：
10635763
负责人：
Libin Ye
金额：
$ 29.9万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10635763
关键词：
ADORA2A gene Affinity Agonist Binding Biological Assay Biophysics Cells Characteristics Communities Complex Coupling Cryoelectron Microscopy Cyclic AMP Data Development Dissociation Drug Design G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Gases Genetic Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hydrolysis In Vitro Individual Label Lead Ligands Link Maps Measures Mental Depression Modeling Molecular Conformation NMR Spectroscopy Nature Nuclear Magnetic Resonance Nucleotides Outcome Output Parkinson Disease Peptides Play Positioning Attribute Production Protein Conformation Proteins Receptor Activation Relaxation Research Resolution Role Signal Transduction Sodium Chloride Structure Testing Work conformational conversion drug discovery experimental study gas dynamics inhibitor innovation insight interfacial molecular dynamics mutant neuropsychiatric disorder pharmacologic protein activation radioligand receptor response side effect stoichiometry structural biology therapeutic target tool transmission process unnatural amino acids

项目摘要

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.

项目概要 G 蛋白偶联受体 (GPCR) 可以被部分激动剂激活，从而产生次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表明部分激动剂稳定非活性和完全激活状态之间的中间构象状态异源三聚体 Gabg 蛋白偶联活性从完全激活状态降低。由于技术障碍描绘 GPCR 构象状态并填充各个中间状态以对其进行研究就个人而言，对部分激动信号传导的机制理解一直具有挑战性。通过创建针对构象偏向突变体，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。这一结果相对于之前的研究来说是一个重大进步。 R291A突变体主要积累中间S4状态，而R291AR293A突变体填充中间 S3 状态和完全激活状态 S5。这一发现使我们能够研究这些角色这些中间体及其配合物。我们将使用这两个突变体来检查中间体的作用陈述了 S3 和 S4 及其与 G 蛋白的相互作用以及随之而来的信号传导效应。在目标 1 中，我们将描述中间状态 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 和配体对其转变和动力学的影响。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 的中间状态 S3 和 S4 是否诱导出有效的 Gasbg 状态用于 GTP 水解、G 蛋白解离以及在 S5 状态不存在的情况下对次最大信号传导的贡献涉及。我们期望将中间态的构象和动态特征关联起来从目标 1 和 2 创建的 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。该拟议项目的完成将加深我们对中介角色的理解构象在 GPCR 信号传导中发挥作用，导致理解受体激活的概念创新超越简单的两种状态模型，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解决中间配合物结构的发展。