Exploring the Conformational Landscape of G Protein Coupled Receptors

探索 G 蛋白偶联受体的构象景观

• 批准号: 10376344
• 负责人: Axel Peter Matthias Elgeti
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376344
• 关键词: Acute Disease; Angiotensin II Receptor; Arrestins; Binding; Chronic Disease; Complement; Coupled; Coupling; Data; Development; Dimensions; Disease; Drug Design; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; FDA approved; Free Energy; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Heterogeneity; Hydrostatic Pressure; Individual; Investigation; Kinetics; Laboratories; Lasers; Ligand Binding; Ligands; Light; Lighting; Maps; Measurement; Methods; Modeling; Molecular; Molecular Conformation; Motion; Muramidase; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacology; Photoreceptors; Physiological; Process; Property; Proteins; Reaction; Receptor Activation; Receptor Signaling; Receptor, Angiotensin, Type 1; Relaxation; Reporting; Research; Resolution; Rhodopsin; Role; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Site; Spectrum Analysis; Spin Labels; Techniques; Technology; Temperature; Testing; Therapeutic; Thermodynamics; Time; Transducers; base; design; extracellular; flexibility; genetic regulatory protein; improved; insight; millisecond; new therapeutic target; novel; pressure; reaction rate; receptor; reduce symptoms; response; side effect; small molecule; temporal measurement; time use

Project Summary G protein coupled receptors (GPCRs) are important transmembrane signaling proteins, which are activated by a multitude of extracellular ligands ranging from small molecules to entire proteins. Active GPCRs couple to different intracellular transducer proteins, such as G proteins and arrestins, thereby triggering diverse cellular responses. Due to their fundamental involvement in a multitude of signal transduction processes, many diseases have their origin in a malfunctioning GPCR, and approximately 35% of all FDA-approved drugs directly target a GPCR. Receptor promiscuity towards ligands and transducer proteins entails that drugs designed to alleviate symptoms are often associated with a range of possible side effects. A detailed molecular understanding of ligand binding, receptor activation and signal transfer is thus pivotal in order to facilitate design of receptor-specific and highly efficacious drugs with enhanced selectivity for downstream signaling pathways. To fulfil their role as allosteric and highly promiscuous regulatory proteins, GPCRs rely on a high degree of structural flexibility, which allows large scale conformational changes thus facilitating specific recognition by binding partners of distinct shape and size. An important and currently unexplored question is to what extent the motions of individual receptor segments are coupled, as opposed to moving independently. This new paradigm suggests that therapeutic drugs could be designed that modulate only a selected subset of conformational changes, while leaving other parts of the receptor unaffected. Identifying networks of coupled segments will define new targets for drug research and facilitate design of functionally selective therapeutics with fewer side effects. We will be testing this hypothesis investigating two prototypical GPCRs of very different function – the light receptor rhodopsin (Rho) and the peptide-activated type 1 angiotensin II receptor (AT1R) - using newly developed site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic techniques. In Aim (I) we will determine the number and topology of different receptor conformations. For this purpose we will shift the conformational equilibria using a variety of different ligands and the application of hydrostatic pressure to map the conformational landscape of both GPCRs using Double Electron-Electron Resonance (DEER) spectroscopy. In Aim (II) we will develop an improved time-resolved EPR method (“TRED”) to determine activation energies and coupling between segmental motions. TRED will be capable of resolving conformational changes with microsecond time resolution and Angstrom spatial sensitivity. In Aim (III) we will determine segmental coupling during Rho and AT1R activation, triggered by flash illumination and pressure jump, respectively. Comparison of the two receptors will allow us to identify coupling networks and transition states of activation, which are either conserved among GPCRs or receptor-specific, thus suggesting target sites for drug design. Accomplishing the aims of this proposal will reveal a new dimension of molecular pharmacology, which has the potential to improve the lives of millions suffering from acute or chronic disease.

项目摘要 G 蛋白偶联受体 (GPCR) 是重要的跨膜信号蛋白，它 被从小分子到整个蛋白质的多种细胞外配体激活。积极的 GPCR 与不同的细胞内转导蛋白（例如 G 蛋白和视紫红质抑制蛋白）偶联，从而触发 不同的细胞反应。由于它们基本参与多种信号转导过程， 许多疾病的根源是 GPCR 功能失常，FDA 批准的所有药物中约有 35% 直接靶向GPCR。受体对配体和转导蛋白的混杂需要药物 旨在缓解症状的药物通常会带来一系列可能的副作用。详细的分子 因此，了解配体结合、受体激活和信号传递对于促进设计至关重要 受体特异性且高效的药物，对下游信号通路具有增强的选择性。 为了履行其作为变构和高度混杂调节蛋白的作用，GPCR 依赖于高度 结构灵活性，允许大规模构象变化，从而促进特定识别 不同形状和大小的结合伙伴。一个重要且目前尚未探索的问题是，在多大程度上 各个受体片段的运动是耦合的，而不是独立运动。这个新范式 表明可以设计治疗药物来仅调节选定的构象子集 变化，同时受体的其他部分不受影响。识别耦合段的网络将 确定药物研究的新靶点，并促进设计具有更少副作用的功能选择性疗法 影响。我们将测试这个假设，研究两个功能截然不同的原型 GPCR—— 光受体视紫红质 (Rho) 和肽激活 1 型血管紧张素 II 受体 (AT1R) - 新使用 开发了定点自旋标记和电子顺磁共振（EPR）光谱技术。在 目标 (I) 我们将确定不同受体构象的数量和拓扑结构。为此，我们将 使用各种不同的配体和静水压力的应用来改变构象平衡 使用双电子共振 (DEER) 绘制两个 GPCR 的构象图 光谱学。在目标 (II) 中，我们将开发一种改进的时间分辨 EPR 方法（“TRED”）来确定 激活能和分段运动之间的耦合。 TRED 将能够解决构象问题 随着微秒时间分辨率和埃空间灵敏度的变化。在目标 (III) 中，我们将确定 Rho 和 AT1R 激活期间的分段耦合，由闪光照明和压力跳跃触发， 分别。两种受体的比较将使我们能够识别耦合网络和过渡态 激活，其在 GPCR 中保守或受体特异性，从而表明药物的靶位点 设计。实现该提案的目标将揭示分子药理学的新维度， 有潜力改善数百万人患有急性或慢性疾病的生活。