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点。我们的研究主要集中在GPCR视紫红质及其附属蛋白。虽然通过最近的晶体学研究，参与视觉信号的蛋白质的知识已经有了很大的进步，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推测问题。特别是，我们缺乏甚至是最基本的信息，在视紫红质信号衰减过程中发生的动态事件，即，用于释放视网膜视蛋白结合口袋的机制，以及这些过程如何影响arrestin的结合和释放。了解这些过程对于视觉研究至关重要，因为视网膜连接的稳定性在不同的视蛋白中变化很大，并且是某些视觉疾病状态的一个因素。此外，关于抑制蛋白从活化视紫红质释放的机制和动力学知之甚少。 在本提案的目的一，我们将确定通过视紫红质控制其视网膜希夫碱键水解的机制。在目标II中，我们将利用我们的专业知识，在现场定向标记方法，以检查动态和结构变化发生在细胞外环区的视紫红质，因为它结合和释放视网膜。最后，在目标III中，我们将利用我们已经开发的一种新的测定来研究从活化的视紫红质释放抑制蛋白。了解是什么使arrestin结合视紫红质后“放手”是非常重要的，因为稳定的视紫红质-arrestin复合物已被认为是细胞凋亡和常染色体显性视网膜色素变性（ADRP）的一个促成因素。此外，使用我们的新检测方法，我们发现有趣的证据表明，抑制蛋白结合并捕获后Meta II光衰变产物，可能是Meta III。因此，抑制蛋白还可以用于限制在明亮的光条件下游离视网膜的释放，从而帮助限制可能导致萎缩性年龄相关性黄斑变性（AMD）等疾病的氧化视网膜加合物的形成。

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).