Exploring the Conformational Landscape of G Protein Coupled Receptors

G蛋白偶联受体(GPCRs)是一种重要的跨膜信号蛋白，它 被许多细胞外配体激活，从小分子到整个蛋白质。主动型 GPCRs偶联到不同的细胞内转导蛋白，如G蛋白和拦阻蛋白，从而触发 不同的细胞反应。由于它们从根本上参与了多种信号转导过程， 许多疾病的起因是gpcr出现故障，在FDA批准的所有药物中，约有35%。 直接瞄准gpr。受体对配体和转导蛋白的混杂导致药物 旨在缓解症状的药物往往与一系列可能的副作用有关。一个详细的分子 因此，为了便于设计，了解配体结合、受体激活和信号传递是至关重要的 针对受体的高效药物，对下游信号通路具有更强的选择性。 为了发挥其作为变构和高度混杂的调节蛋白的作用，GPCRs依赖于高度的 结构灵活性，允许大规模构象变化，从而促进特定的识别 形状和大小各异的结合体。一个重要的、目前尚未探索的问题是，在多大程度上 单个受体片段的运动是耦合的，而不是独立运动。这一新范式 提示治疗药物可以被设计成只调节选定的构象亚集 而受体的其他部分不受影响。识别耦合网段的网络将 为药物研究确定新的目标，以更少的侧面促进功能选择性疗法的设计 效果。我们将通过调查两个功能截然不同的原型GPCR来验证这一假设 光受体视紫红质和多肽激活的1型血管紧张素II受体--新的应用 开发了位置定向自旋标记和电子顺磁共振(EPR)光谱技术。在……里面 目的(I)我们将确定不同受体构象的数目和拓扑。为此，我们将 使用各种不同的配体和施加静水压力来改变构象平衡 用双电子-电子共振(DER)绘制两个GPCRs的构象图谱 光谱学。在AIM(Ii)中，我们将开发一种改进的时间分辨EPR方法(TRED)来确定 活化能和节段运动之间的耦合。TRED将能够解析构象 随微秒时间分辨率和Angstrom空间敏感度变化。在AIM(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， 分别进行了分析。两种受体的比较将使我们能够识别偶联网络和过渡态 激活，要么在GPCRs中保守，要么是受体特有的，从而提示药物的靶点 设计。实现这一提议的目标将揭示分子药理学的一个新维度，即 有可能改善数百万患有急性或慢性病的人的生活。

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.