Structural Dynamics of Retinal Binding and Release

描述（由申请人提供）：我们研究的长期目标是了解g蛋白偶联受体（gpcr）被激活和减弱的分子机制。这些受体代表了人类基因组中最大的家族，最重要的是，它们是大多数药物的靶标。我们的研究主要集中在GPCR视紫红质及其附属蛋白上。尽管通过最近的晶体学研究，对参与视觉信号的蛋白质的了解已经取得了巨大的进展，但它们在激活和衰减过程中所经历的关键结构变化在很大程度上仍然是一个猜测问题。特别是，我们甚至缺乏关于视紫质信号衰减过程中发生的动态事件的最基本信息，即用于从视蛋白结合口袋中释放视网膜的机制，以及这些过程如何影响捕集蛋白的结合和释放。了解这些过程对视觉研究至关重要，因为视网膜连接的稳定性在不同视蛋白之间差异很大，并且是一些视觉疾病状态的一个因素。此外，关于从活化的视紫红质释放阻滞素的机制和动力学知之甚少。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. These receptors represent the largest family in the human genome, and most importantly, are the target of most pharmaceutical drugs. Our studies focus primarily on the GPCR rhodopsin and its affiliate proteins. Although knowledge of the proteins involved in visual signaling has been enormously advanced through recent crystallographic studies, the critical structural changes they undergo during activation and attenuation remain largely a matter of speculation. In particular, we lack even the most rudimentary information about the dynamic events that occur during the attenuation of rhodopsin signaling, namely, the mechanisms used to release retinal from the opsin-binding pocket, and how these processes affect arrestin binding and release. Understanding these processes is of fundamental importance for vision research, as the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, little is known regarding the mechanism and kinetics of arrestin release from activated rhodopsin. In Aim I of this proposal we will determine the mechanisms through which rhodopsin controls the hydrolysis of its retinal Schiff base linkage. In Aim II we will use our expertise in site-directed labeling methods to examine dynamic and structural changes occurring in the extracellular loop region of rhodopsin as it binds and releases retinal. Finally, in Aim III, we will utilize a new assay we have developed to study arrestin release from activated rhodopsin. Understanding what makes arrestin "let go" after binding rhodopsin is fundamentally important, since stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP). Furthermore, using our new assay, we find intriguing evidence that arrestin binds to and traps a post-Meta II photodecay product, possibly Meta III. Thus, arrestin may also serve to limit the release of free retinal under bright light conditions, and thus help limit the formation of oxidative retinal adducts that can contribute to diseases like atrophic age-related macular degeneration (AMD).