Spatial Organization and Molecular Interactions of Visual Opsins

 描述(申请人提供)：G蛋白偶联受体(GPCR)是参与多种生理功能的七螺旋跨膜蛋白。它们在视觉、大脑功能和免疫系统调节中起着至关重要的作用，是市场上销售的药物所针对的最大蛋白质家族。视紫红质是一种典型的A类GPCR，在视网膜视杆细胞外段高密度分布。它在视觉中起着中心作用，视杆细胞中的光感受器蛋白。视紫红质突变与广泛的视觉障碍有关，其中许多是不可逆转和不可治疗的。虽然分离的视紫红质是一种单体，但有压倒性的证据表明，它在视杆细胞外段(ROS)膜内发生二聚化。视紫红质二聚体的功能相关性已通过纯化的视紫红质的体外分析得到证实，但由于缺乏能够在生理条件下解决分子尺度的相互作用并具有足够的时间分辨率来测量实时受体激活事件的实验工具，二聚体与天然膜上的生理功能之间的结构联系尚未建立。为了研究这些相互作用，我们将使用定量成像方法，包括脉冲交错激发荧光互相关光谱(PIE-FCCS)，来确定活细胞质膜中视蛋白的齐聚和动力学。我们的假设是视蛋白二聚化在细胞膜中普遍存在，具有结构选择性，并且在信号转导中起活跃作用。为了验证这一假说，这项建议特别旨在(1)调查视蛋白(视蛋白(视杆、视锥和黑素))蛋白二聚体在活细胞膜中的发生率和动态稳定性，(2)测量几种典型的A类GPCRs(包括多巴胺受体、蛋白水解酶激活受体和β-肾上腺素能受体)的二聚化平衡，以及(3)建立一个研究光激活视紫红质和黑色素蛋白二聚化的平台，并测量二聚化对信号转导的影响。对视紫红质二聚体存在的支持很多，但目前的文献缺乏对视红质二聚体的热力学以及二聚体在细胞信号事件中发挥积极作用的方式的全面描述。这一点意义重大，因为它将推动该领域从关于视紫红质二聚体是否存在的辩论，到对支配二聚作用的化学平衡和视紫红质复合体性质的全面理解。通过确定视蛋白寡聚的分子水平细节，我们将深入了解驱动GPCR寡聚的机制。这项提议的目标的成功完成将直接影响GPCR齐聚这一日益增长的领域，并将促进我们对这一重要类别的膜受体的理解。

 DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCR) are seven-helix transmembrane proteins that participate in numerous physiological functions. They play an essential role in vision, brain function, and immune-system regulation, and are the largest protein family targeted by marketed drugs. Rhodopsin is a prototypical class A GPCR found in high densities at the outer segment of rod cells in the retina. It plays a central role in vision a the photoreceptor protein in rod cells. Rhodopsin mutations are implicated in a wide range of visual disorders, many of which are irreversible and untreatable. While isolated rhodopsin is functional as a monomer, there is overwhelming evidence that it dimerizes within rod outer segment (ROS) membranes. The functional relevance of rhodopsin dimers has been demonstrated through in vitro assays of purified rhodopsin, but the structural connection between dimerization and physiological function in native membranes has not been established due to the lack of experimental tools that can resolve molecular-scale interactions under physiological conditions and with sufficient time resolution to measure real-time receptor activation events. To investigate these interactions, we will use a quantitative imaging approach, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), to determine opsin oligomerization and dynamics in the live-cell plasma membrane. Our hypothesis is that opsin dimerization is prevalent in cell membranes, structurally selective, and active in signaling. To test that hypothesis, this proposal specifically aims to (1) investigate th prevalence and dynamic stability of opsin (rod, cone and melanopsin) protein dimers in live-cell membranes, (2) measure the dimerization equilibrium of several prototypical class A GPCRs including dopamine receptors, protease-activated receptors, and beta-adrenergic receptors, and (3) develop a platform to investigate dimerization in light-activated rhodopsin and melanopsin and measure the effect of dimerization on signal transduction. Support for the existence of rhodopsin dimers is abundant, but missing from the current body of literature is a full characterization of the thermodynamics of opsin dimerization and the way in which dimerization plays an active role in cell signaling events. This is significant because it will advance the fiel from a debate about the existence of rhodopsin dimers to a comprehensive understanding of the chemical equilibria that govern dimerization and the properties of the rhodopsin complexes. By determining the molecular-level details of opsin oligomerization, we will obtain insight into th mechanisms that drive GPCR oligomerization. Successful completion of the aims of this proposal will have a direct impact on the growing field of GPCR oligomerization and will advance our understanding of this important class of membrane receptors.