Exploring the Conformational Landscape of G Protein Coupled Receptors

G蛋白偶联受体（GPCRs）是重要的跨膜信号蛋白， 被从小分子到整个蛋白质的大量细胞外配体激活。活性 GPCR与不同的细胞内转导蛋白偶联，如G蛋白和抑制蛋白，从而触发细胞内信号转导。 不同的细胞反应。由于它们基本上参与多种信号转导过程， 许多疾病都起源于GPCR的故障，大约35%的FDA批准的药物 直接瞄准气相化学还原反应受体对配体和转导蛋白的混杂性使得药物 旨在缓解症状的药物往往与一系列可能的副作用有关。详细的分子 因此，理解配体结合、受体活化和信号传递是促进设计的关键。 受体特异性和高效药物，对下游信号通路具有增强的选择性。 为了实现其作为变构和高度混杂的调节蛋白的作用，GPCR依赖于高度的 结构灵活性，允许大规模构象变化，从而促进特异性识别， 不同形状和大小的结合伙伴。一个重要的和目前尚未探讨的问题是， 与独立运动相反，各个接收器段的运动是耦合的。这种新范式 表明治疗药物可以被设计成仅调节构象的选定子集， 变化，而不影响受体的其他部分。识别耦合段的网络将 为药物研究确定新的靶点，并促进设计具有更少副作用的功能选择性治疗药物 方面的影响.我们将测试这一假设，研究两种功能非常不同的原型GPCR- 光受体视紫红质（Rho）和肽激活的1型血管紧张素II受体（AT 1 R）-使用新的 开发了定点自旋标记和电子顺磁共振（EPR）光谱技术。在 目的（I）确定不同受体构象的数目和拓扑结构。为此，我们将 使用各种不同的配体和应用流体静压力来改变构象平衡 使用双电子-电子共振（DEER）绘制两种GPCR的构象图 谱在目的（II）中，我们将发展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 改变微秒时间分辨率和埃空间灵敏度。在Aim（III）中，我们将确定 Rho和AT 1 R激活期间的节段耦合，由闪光照明和压力跳变触发， 分别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，其在GPCR中是保守的或受体特异性的，从而表明药物的靶位点。 设计实现这一建议的目标将揭示分子药理学的新维度， 有可能改善数百万急性或慢性疾病患者的生活。

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.