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， 通过从小分子到整个蛋白质的众多细胞外配体激活。积极的 GPCR夫妇到不同的细胞内换能蛋白，例如G蛋白和逮捕蛋白，从而触发 潜水细胞反应。由于它们基本参与了多种信号转导过程 许多疾病起源于故障GPCR，大约有35％的FDA批准药物 直接靶向GPCR。对配体和换能器蛋白的受体滥交需要药物 旨在减轻症状通常与一系列可能的副作用有关。详细的分子 因此，了解配体结合，受体激活和信号传递是关键的，以促进设计 对下游信号通路的选择性增强的受体特异性和高效药物的速度。 为了履行其作为变构和高度混杂的调节蛋白的作用，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 具有独特形状和大小的绑定伙伴。一个重要且目前出乎意料的问题是在多大程度上 单个接收器段的运动耦合，而不是独立移动。这个新的范式 表明可以设计治疗药物仅调节构象的选定子集 更改，同时使接收器的其他部分不受影响。识别耦合段的网络将 定义用于药物研究的新目标，并促进功能选择性治疗的设计 效果。我们将测试这一假设，研究两个非常不同功能的典型GPCR- 光受体视紫红蛋白（Rho）和肽激活1型血管紧张素II受体（AT1R） - 使用新的 开发了定向定向的自旋标记和电子顺磁共振（EPR）光谱技术。 目的（i）我们将确定不同受体构象的数量和拓扑。为此，我们将 使用各种不同的配体和静静力压力移动会议等效。 使用双电子电子共振（DEER）绘制两个GPCR的会议景观 光谱法。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 分段运动之间的激活能和耦合。 TRED将能够解决构象 随着微秒时间分辨率和埃斯特罗姆空间灵敏度的变化。在目标（iii）中，我们将确定 RHO和AT1R激活期间的节段耦合，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状态 激活，它们要么在GPCR或接收器特异性中配置，因此表明药物的目标位点 设计。完成该提案的目的将揭示分子药理学的新维度，这是 有可能改善急性或慢性疾病的数百万生活的生活。