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.

项目摘要 G蛋白偶联受体（GPCR）可以由部分激动剂激活，导致次最大信号传导与完整的激动剂相比，副作用减少。增加的研究间接表明局部激动剂稳定非活动状态和完全激活状态之间的中间构象状态从完全激活的状态下降低了异三聚体GABG蛋白偶联活性。由于技术障碍在描述GPCR构象状态和填充个体中间状态以研究它们时单独地，对部分激动剂信号的机械理解一直在挑战。通过创建构象偏置突变体，我们确定了五个腺苷A2A受体（A2AR）构象状态，包括使用19F 19F 核磁共振（NMR）光谱。这个结果是先前研究的重大进步。 R291a突变体主要积累中间S4态，而R291AR293A突变体填充中间S3状态和完整激活状态S5。这一发现使我们能够研究角色这些中间体及其复合物。我们将使用这两个突变体检查中间体的作用状态S3和S4及其与G蛋白的相互作用以及随之而来的信号效应。在AIM 1中，我们将表征中间状态S3和S4是否与GASBG相互作用。这些特征将包括研究中间状态的构象转变和动力学的研究煤气和配体对它们的过渡和动力学的影响。在AIM 2中，我们将绘制构象气体状态并确定其与A2AR的S3和S4状态相互作用的中间状态。在 AIM 3，我们将确定A2AR的中间状态S3和S4是否诱导GASBG状态对于GTP水解，G蛋白解离，以及对没有S5状态的次最大信号的贡献涉及。我们期望将中间状态的构象和动态特征相关联 A2AR和气体蛋白从目标1和2产生的构象偏置突变体的信号传导效果配体石化元素，以AIM 3进行测量。该提议的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除了简单的两态模型之外，还可以根据GPCR和G蛋白引导药物设计对配体的构象反应。此外，构象有偏的突变体将指导一种方法解决中间复合物的结构的发展。