Structural Dynamics of Retinal Binding and Release

描述(申请人提供)：我们研究的长期目标是了解G蛋白偶联受体(GPCRs)被激活和减弱的分子机制。这些受体代表着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视紫质及其附属蛋白上。虽然通过最近的结晶学研究，对参与视觉信号的蛋白质的了解已经有了很大的进步，但它们在激活和衰减过程中经历的关键结构变化在很大程度上仍然是一个猜测的问题。特别是，我们甚至缺乏关于视紫红质信号衰减过程中发生的动态事件的最基本信息，即用于从视黄素结合袋中释放视网膜的机制，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是至关重要的，因为视网膜连接的稳定性在不同的视蛋白中差异很大，并且是某些视觉疾病状态的一个因素。此外，关于从激活的视紫红质中释放arrestin的机制和动力学还知之甚少。 在这项提议的目标I中，我们将确定视紫红质控制其视网膜Schiff碱基连接的水解性的机制。在AIM II中，我们将使用我们在定点标记方法方面的专业知识来检查视紫红质结合和释放视网膜时细胞外环区域发生的动态和结构变化。最后，在目标III中，我们将利用我们开发的一种新的方法来研究激活的视紫红质释放arrestin。由于稳定的视紫红质-arrestin复合体被认为是细胞凋亡和常染色体显性遗传性视网膜色素变性(Adrp)的促成因素，因此了解是什么使arrestin在与视紫红质结合后“松开”是非常重要的。此外，使用我们的新检测方法，我们发现了有趣的证据，即arrestin与Meta II后的光衰产物结合并捕获，可能是Meta III。因此，arrestin可能还有助于限制在强光条件下自由视网膜的释放，从而有助于限制导致萎缩性老年性黄斑变性(AMD)等疾病的视网膜氧化加合物的形成。

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).