Biophysical approaches to investigate the biological significance of GPCR dimers

 描述(申请人提供)：近年来积累的大量实验证据表明，G蛋白偶联受体(GPCRs)在质膜上相互结合，形成二聚体/寡聚体。然而，关于受体亚型之间的可变性、受体-受体相互作用的特异性以及最重要的是GPCR寡聚对信号的影响，大多数研究都没有定论。这些方面需要解决，以阐明GPCR寡聚体的功能作用，并最终通过对GPCR信号的特定影响来设计新的治疗策略。这项研究计划的目的是提供突破性的机制洞察，了解细胞膜上主要阿片受体(OR)亚型作为原型GPCRs的时空组织，并最终了解其与功能的关系。为此，我们建议使用一种协同的、跨学科的策略，集成最先进的分子动力学(MD)模拟、单分子显微镜和Förster共振能量转移(FRET)显微镜分析GPCR信号。这一整合是及时的，并通过GPCR领域的前沿理论和实验进展而取得突破性进展，包括新的高分辨率晶体结构、活细胞中单个GPCR原体单粒子跟踪的最新技术发展、GPCR信号的高效FRET分析的实施，以及由高效并行化代码和多尺度系统表示补充的高性能计算能力。虽然我们将使用ORS作为模型系统，但我们提出的策略可以应用于任何GPCRs或其他膜蛋白的复合体。具体地说，我们建议使用单分子全反射荧光显微镜(TIR-FM)和GPCR体之间的FRET/BRET信号来研究高效、共价标记的OR亚型的双/低聚物的形成，以表征它们可能独特的二/低聚模式。基于迭代的计算-实验方法，我们将预测、产生和研究突变体，以探索形成不同OR二聚体的界面。最后，我们将通过评估使用经典信号分析和新开发的实时FRET传感器测量的二聚体稳定化或去稳定化突变对信号的影响，来研究二聚体形成和信号之间的可能关系。我们将把这些研究从简单的转基因细胞系转移到原代细胞，未来也将致力于从转基因细胞转移到原代细胞 用我们独立合作开发的荧光标记配体研究内源性受体的荧光标记受体。因此，我们的研究最终将有助于解开GPCR二聚体/寡聚化的一般机制及其对信号的影响，并旨在为GPCR化治疗寻找新的靶点。

 DESCRIPTION (provided by applicant): A wealth of experimental evidence accumulated over recent years suggests that G protein-coupled receptors (GPCRs) associate with each other in the plasma membrane, forming di-/oligomers. However, most studies have been inconclusive with regard to the variability across receptor subtypes, specificity of receptor-receptor interactions and, most importantly, the impact of GPCR oligomerization on signaling. These aspects need to be addressed to clarify the functional role of GPCR oligomers and, ultimately, devise new therapeutic strategies via specific effects on GPCR signaling. The objective of this research proposal is to provide breakthrough mechanistic insights into the spatio-temporal organization of the major opioid receptor (OR) subtypes, serving as prototypic GPCRs, in the cell membrane, and ultimately, its relation to function. To this end, we propose to use a synergistic, inter-disciplinary strategy integrating state-of-the-art molecular dynamics (MD) simulations, single molecule microscopy, and Förster resonance energy transfer (FRET) microscopy assays of GPCR signaling. This integration is timely and made ground-breaking by cutting-edge theoretical and experimental advances in the GPCR field, including new high- resolution crystal structures, recent technological developments for single-particle tracking of individual GPCR protomers in a living cell, implementation of efficient FRET assays for GPCR signaling, and high performance computational capabilities complemented by efficiently parallelized codes and multiscale system representations. Although we will use ORs as model systems, our proposed strategy can be applied to any complex of GPCRs or other membrane proteins. Specifically, we propose to study di-/oligomer formation of high- efficiency, covalently labeled OR subtypes with fluorescent dyes using single molecule total internal reflection fluorescence microscopy (TIR-FM) and FRET/BRET signals between GPCR protomers to characterize their possibly distinctive di-/oligomerization patterns. Based on an iterative computational-experimental approach, we will predict and then generate and investigate mutants to explore the interfaces forming the different OR dimers. Finally, we will investigate a possible relationship between dimer formation and signaling by assessing the effects of dimer-stabilizing or de-stabilizing mutations on signaling as measured with classic signaling assays and with newly developed real-time FRET sensors. We will move these studies from simple transfected cell lines to primary cells, and will also aim to move, in the future, from transfected fluorescently labeled receptors to endogenous receptors studied with fluorescently labeled ligands that we are developing in an independent collaboration. Thus, our studies will ultimately help to unravel general mechanisms of GPCR di/oligomerization and their effects on signaling and aim to identify new targets for GPCR-directed therapies.