Structural Dynamics in Rhodopsin Activation and Attenuation

项目摘要 我们研究的一个长期目标是了解G蛋白偶联的分子机制 受体(GPCRs)被激活和减弱。GPCR是人类基因组中最大的家族，而 大多数药物的靶标。一个例外是视紫红质--尽管第一个GPCR 尽管已发现，但到目前为止，它对直接的药物治疗一直难以奏效。 在这里，Kriger和Farrens实验室联手定义了 人类视紫红质和视锥细胞光敏蛋白的光激活，决定视紫红质如何与其配体相互作用， 视网膜，确定其功能如何随着导致视网膜疾病的突变而变化，并确定如何 这些相互作用使其能够与其附属蛋白Arrestin相互作用，并受其相互作用的调节。尽管 视网膜、视紫红质和芳香素的结构现在是已知的，使它们能够 相互之间的互动是不存在的。因此，我们在这里提出的研究类型是必要的。 特定目标1将确定人类红色和绿色锥体色素的光活化动力学，确定 人类视紫红质的激活是如何被与adrp相关的突变短路的，并测试如何 这些动力学受用于治疗和稳定错误折叠的视蛋白的小分子伴侣的影响。特定的 目标2将确定新的受体构象在视网膜摄取和释放过程中发挥什么作用， 测试以前未知的受体构象是否能够结合11-顺式视网膜(11CR)，并继续扩展 我们的发现是，视蛋白在释放全反式视网膜(ATR)后可以短暂地停留在类活性状态。 最后，特定的目标3将确定arrestin结合是否使ATR能够结合光漂白的视紫红质 平衡，并定义arrestin与视紫红质二聚体的结合对这一现象有什么影响。 了解是什么调节视紫红质的光激活、视网膜摄取和释放的过程，以及 从健康的角度来看，arrestin如何规范这些行为是至关重要的。视网膜必须 适应这些事件的巨大变化，因为它适应非常不同的光条件，但像差 随着时间的推移，这一过程被认为会导致视网膜氧化加合物的形成，从而促进疾病 如萎缩性老年性黄斑变性(AMD)。因此，看起来阿雷斯汀必须谨慎行事。 一方面控制在不同光照条件下释放的视网膜游离量，另一方面 适时从受体释放视网膜及其自身，避免形成稳定的视紫红质芳香素 可导致细胞凋亡和某些形式的视网膜色素变性的复合体。 这里的工作补充了我们最近的发现，即ATR可以与一些视紫红质进行平衡交换 照片产品，以及其他人最近发现的非视网膜配体，可以结合和稳定错误折叠的视蛋白。 这些发现极大地增加了开发药物以与之竞争或 增强视网膜结合，从而为用药理药物治疗这个关键的光感受器打开了大门。

Project Summary A long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. GPCRs are the largest family in the human genome, and the target of most pharmaceutical drugs. One exception has been rhodopsin – although the first GPCR discovered, it has so far been refractory to direct pharmacological treatments. Here the Kliger and Farrens lab join forces to define the dynamic events and mechanisms involved in the photo-activation of human rhodopsin and cone photopsins, determine how rhodopsin interacts with its ligand, retinal, determine how its function changes with mutations responsible for retinal diseases and determine how these interactions enable, and are modulated by, interactions with its affiliate protein arrestin. Although the structures of retinal, rhodopsin, and arrestin are now known, the dynamic processes that enable them to interact with each other are not. Thus, the types of studies we propose here are required. Specific Aim 1 will determine the photoactivation kinetics of human red and green cone pigments, determine how the activation of human rhodopsin is short-circuited by mutations associated with ADRP, and test how these kinetics are effected by small molecule chaperones used to treat and stabilize misfolded opsins. Specific Aim 2 will determine what role novel receptor conformations play in the process of retinal uptake and release, test if a previously unidentified receptor conformation enables binding of 11-cis retinal (11CR), and expand on our discovery that opsin can transiently linger in an active-like state after releasing all-trans retinal (ATR). Finally, Specific Aim 3 will determine if arrestin binding enables ATR to bind photobleached rhodopsin in equilibrium, and define what effect arrestin binding to rhodopsin dimers has on this phenomenon. Understanding what regulates the process of rhodopsin photoactivation, and retinal uptake and release, and how arrestin regulates these actions is critically important from a health perspective. The retina must accommodate huge variations in these events as it adapts to widely different light conditions, yet aberrations in this process over time are thought to result in the formation of oxidative retinal adducts that promote diseases like atrophic age-related macular degeneration (AMD). Thus, it appears that arrestin must walk a fine line – on the one hand controlling the amount of free retinal released under varying light conditions, and on the other releasing retinal and itself from the receptor at the appropriate time to avoid forming stable rhodopsin-arrestin complexes that can contribute to apoptosis and some forms of retinitis pigmentosa. The work here complements our recent discovery that ATR can exchange in equilibrium with some rhodopsin photoproducts, and recent discoveries by others of non-retinal ligands that bind and stabilize misfolded opsins. These findings dramatically increase the possibility that drugs can be developed to either compete with or enhance retinal binding, thus opening the door for treating this key photoreceptor with pharmacological agents.