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，并且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。虽然参与视觉信号传导的关键蛋白质的晶体结构现在是已知的，但这些蛋白质在其激活和衰减期间经历的大多数关键结构变化在很大程度上仍然是推测的问题。特别是，我们缺乏甚至基本的信息，涉及衰减视紫红质信号，即，通过该机制，视网膜是从视蛋白结合口袋释放，以及如何视网膜的结合和释放影响抑制蛋白的结合和释放的动态事件。了解这些过程对于视觉研究至关重要-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，尽管对arrestin与视紫红质结合的机制和动力学了解很多，但对结合后是什么使arrestin释放以及这种释放如何与视网膜发色团的状态相关知之甚少。在这个建议的目的我，我们将确定视紫红质如何控制其视网膜席夫碱键的水解。在目标II中，我们将研究视网膜吸收和释放如何发生在视紫红质中，使用视蛋白的最新结构来指导我们的研究。最后，在Aim III中，我们将使用我们的新方法来跟进我们在上一个资助期间的发现-arrestin可以与MIII视紫红质结合，从而捕获和防止视网膜释放。了解arrestin如何调节视网膜释放对健康至关重要，因为arrestin可以在明亮的光线条件下限制游离视网膜的释放，从而有助于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。类似地，了解是什么使arrestin在结合视紫红质后“放手”也是至关重要的-稳定的视紫红质-arrestin复合物被认为是细胞凋亡和常染色体显性视网膜色素变性（ADRP）的促成因素。 公共卫生相关性：拟议的研究将确定参与视网膜结合和释放的分子事件。我们将研究视网膜如何进入和离开视紫红质中的结合口袋，是什么使它留在那里（即，希夫碱键如何形成和水解），以及蛋白质抑制蛋白如何影响这些过程。阐明这些基本问题将有助于阐明视觉中更普遍的问题，例如为什么视杆细胞和视锥细胞视紫红质之间的视网膜结合和释放速率差异如此之大，并有助于阐明几种视网膜疾病的潜在机制。

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 they are the target of most pharmaceutical drugs. We focus our studies primarily on the GPCR rhodopsin and its affiliate proteins. Although crystal structures of key proteins involved in visual signaling are now known, most of the critical structural changes these proteins undergo during their activation and attenuation remain largely a matter of speculation. In particular, we lack even rudimentary information about the dynamic events involved in attenuating rhodopsin signaling, namely, the mechanisms through which retinal is released from the opsin-binding pocket, and how retinal binding and release affects arrestin binding and release. Understanding these processes is of fundamental importance for vision research - the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, although much is known about the mechanism and kinetics of arrestin binding to rhodopsin, little is known about what makes arrestin release after binding, and how this release is related to the status of the retinal chromophore. In Aim I of this proposal we will determine how rhodopsin controls the hydrolysis of its retinal Schiff base linkage. In Aim II we will examine how retinal uptake and release occurs in rhodopsin, using the recent structure of opsin to guide our studies. Finally, in Aim III, we will use our novel methods to follow up on a discovery we made during the last funding period - that arrestin can bind to MIII rhodopsin, thus trapping and preventing retinal release. Understanding how arrestin regulates retinal release is fundamentally important to health, as arrestin may 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). Similarly, understanding what makes arrestin "let go" after binding rhodopsin is also crucial - stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP). PUBLIC HEALTH RELEVANCE: The proposed research will define the molecular events involved in the binding and release of retinal. We will investigate how retinal gets into and out of the binding pocket in rhodopsin, what makes it stay there (i.e., how the Schiff-base linkage forms and hydrolyzes), and how the protein arrestin affects these processes. Answering these fundamental questions will help shed light on more general questions in vision, such as why rates of retinal binding and release vary so greatly between Rod and Cone rhodopsins, and help elucidate underlying mechanisms for several retinal diseases.