Structural and Functional Studies of Rhodopsin and G-Protein Coupled Receptor Kinases

项目总结/摘要 人类有超过800种不同的G蛋白偶联受体（GPCR），它们形成了 是细胞表面受体的最大家族，占所有治疗药物靶标的约30%。 在被激动剂激活后，GPCR采取不同的激活状态，使它们能够偶联 或者是G蛋白，启动G蛋白介导的信号传导，或者是GPCR激酶（GRKs）， 使GPCR磷酸化以使其能够与抑制蛋白相互作用以终止G蛋白 激活并启动抑制蛋白介导的信号传导。虽然生理激动剂启动两个G 蛋白质和抑制蛋白介导的信号传导，通常只有一个这两个途径是治疗 有益的，而另一种途径是负责不必要的副作用。鉴于 由于GPCR作为药物靶点的重要性，人们对开发“有偏见的”GPCR产生了巨大的兴趣。 主要通过两种途径之一发出信号的激动剂。然而，关键的 合理开发偏向性激动剂的障碍是关于GRKs如何相互作用的信息有限 具有GPCR并且完全不存在任何高分辨率GPCR/GRK结构。晶体 和与G蛋白复合的四个GPCR和与G蛋白复合的一个GPCR的冷冻-EM结构 提供了一个新兴的理解受体构象 G蛋白和抑制蛋白偶联所需的，但GRKs如何结合的机械理解， 和磷酸化的GPCR由于其高度瞬时的性质而仍然是难以捉摸的。 互动本提案的目的是通过以下方式弥补这一关键的知识差距： 利用视紫红质和GRK 1之间的相互作用作为范例来确定 GPCRS和GRKs之间靶向相互作用的机制和结构基础。 意义：完成所提出的目标将揭示第一个机械基础， GRK/GPCR识别，并揭示GPCR/GRK构象，使GRK结合， 受体磷酸化这些信息对于合理设计 用于治疗广谱疾病的治疗性偏向激动剂。

PROJECT SUMMARY/ABSTRACT Humans have more than 800 different G protein-coupled receptors (GPCRs), which form the largest family of cell-surface receptors and account for ~30% of all therapeutic drug targets. Upon activation by agonists, GPCRs adopt distinct activated states that allow them to couple either to G proteins, to initiate G protein-mediated signaling, or to GPCR kinases (GRKs), which phosphorylate GPCRs to enable their interactions with arrestins to terminate G protein activation and initiate arrestin-mediated signaling. While physiological agonists initiate both G protein- and arrestin-mediated signaling, often only one of the two pathways is therapeutically beneficial, while the other pathway is responsible for unwanted side effects. Given the importance of GPCRs as drug targets, there is huge interest in developing “biased” GPCR agonist that signal predominantly through only one of the two pathways. However, the critical barrier for rationally developing biased agonist is the limited information on how GRKs interact with GPCRs and the complete absence of any high resolution GPCR/GRK structure. Crystal and cryo-EM structures of four GPCRs in complex with G proteins and of one GPCR in complex with an arrestin have provided an emerging understanding of the receptor conformations required for G protein and arrestin coupling, yet a mechanistic understanding of how GRKs bind and phosphorylate GPCRs have remained elusive due to the highly transient nature of this interaction. The objective of this proposal is to bridge this critical gap in knowledge by exploiting the interaction between rhodopsin and GRK1 as a paradigm to determine the mechanistic and structural basis of the targetable interaction between GPCRS and GRKs. SIGNIFICANCE: Completion of the proposed aims will reveal the first mechanistic basis of GRK/GPCR recognition and reveal the GPCR/GRK conformation that enables GRK binding and receptor phosphorylation. This information is of paramount importance for the rational design of therapeutic biased agonists for the treatment of a broad spectrum of diseases.