Spatial Organization and Molecular Interactions of Visual Opsins

视觉视蛋白的空间组织和分子相互作用

• 批准号: 8958257
• 负责人: Adam W. Smith
• 金额: $ 44.14万
• 依托单位: UNIVERSITY OF AKRON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8958257
• 关键词: Adrenergic Receptor; Atomic Force Microscopy; Binding; Brain; Cattle; Cell membrane; Cells; Chemicals; Complex; Cues; Data; Dimerization; Disease; Dopamine Receptor; Electron Microscopy; Environment; Equilibrium; Event; Fluorescence; G-Protein-Coupled Receptors; Heterogeneity; Heterotrimeric GTP-Binding Proteins; Homo; Hormones; Immune system; In Situ; Integral Membrane Protein; Life; Light; Literature; Measures; Mediating; Membrane; Membrane Proteins; Methods; Molecular; Mutation; Neurotransmitters; Opsin; Photoreceptors; Phototransduction; Physiologic pulse; Physiological; Play; Prevalence; Property; Protein Family; Proteinase-Activated Receptors; Proteins; Receptor Activation; Receptors, Adrenergic, beta-3; Regulation; Research; Resolution; Retina; Retinal; Rhodopsin; Rod Outer Segments; Role; Series; Signal Transduction; Signal Transduction Pathway; Spectrum Analysis; Structure; Testing; Thermodynamics; Time; Vertebrate Photoreceptors; Vision; Vision Disorders; Visual; Work; base; cellular imaging; density; dimer; drug market; imaging modality; in vitro Assay; in vivo; insight; melanopsin; molecular scale; monomer; programs; public health relevance; quantitative imaging; receptor; reconstitution; response; retinal rods; time use; tool

 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.

 描述（由申请人提供）：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、脑功能和免疫系统调节中起着至关重要的作用，是上市药物靶向的最大蛋白质家族。视紫红质是一种典型的A类GPCR，以高密度存在于视网膜视杆细胞的外段。视杆细胞中的感光蛋白在视觉中起着核心作用。视紫红质突变与广泛的视觉障碍有关，其中许多是不可逆和无法治疗的。虽然分离的视紫红质作为单体起作用，但有压倒性的证据表明它在视杆外节（ROS）膜内二聚化。视紫红质二聚体的功能相关性已通过纯化视紫红质的体外测定得到证实，但由于缺乏能够在生理条件下解析分子尺度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，因此尚未建立天然膜中二聚体与生理功能之间的结构联系。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），以确定视蛋白寡聚化和动态活细胞质膜。我们的假设是，视蛋白二聚化是普遍存在于细胞膜，结构选择性，并积极的信号。为了验证这一假设，本研究的具体目标是（1）研究视蛋白的流行性和动态稳定性（2）测量几种原型A类GPCR（包括多巴胺受体、蛋白酶激活受体和β-肾上腺素能受体）的二聚化平衡，以及（3）开发一个平台来研究光激活的视紫红质和黑视蛋白的二聚化并测量二聚化对信号转导的影响。支持视紫红质二聚体的存在是丰富的，但从目前的文学作品中缺少的是视蛋白二聚化的热力学和二聚化在细胞信号事件中起积极作用的方式的充分表征。这是重要的，因为它将推进该领域从辩论视紫红质二聚体的存在，以全面了解的化学平衡，支配二聚和性质的视紫红质复合物。通过确定视蛋白寡聚化的分子水平细节，我们将深入了解驱动GPCR寡聚化的机制。成功完成这一提案的目标将对GPCR寡聚化的日益增长的领域产生直接影响，并将促进我们对这一类重要的膜受体的理解。

项目成果

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

• DOI: 10.1007/978-1-4939-2330-4_14
• 发表时间: 2015
• 期刊: Methods in molecular biology
• 作者: Adam W. Smith