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

项目摘要/摘要 人类有800多种不同的G蛋白偶联受体(GPCRs)，它们形成了 最大的细胞表面受体家族，约占所有治疗药物靶点的30%。 在被激动剂激活后，GPCRs采用不同的激活状态，使它们能够偶联 或者到G蛋白，以启动G蛋白介导的信号转导，或者到GPCR激酶(GRKs)，后者 磷酸化GPCRs使其与拦阻蛋白相互作用终止G蛋白 激活并启动arrestin介导的信号转导。而生理激动剂启动了两种G 蛋白质和arrestin介导的信号传递，通常只有两条途径中的一条具有治疗作用 有益的，而另一种途径则负责产生不想要的副作用。给定 GPCRs作为药物靶标的重要性，人们对开发“有偏见的”GPCRs有巨大的兴趣 主要通过两条途径中的一条发出信号的激动剂。然而，关键的是 合理开发偏向激动剂的障碍是关于GRK如何相互作用的有限信息 具有GPCRs，并且完全没有任何高分辨率的GPCRK结构。结晶体 G蛋白复合体中4个GPCRs和复合体中1个GPCRs的冷冻电子显微镜结构 提供了对受体构象的新的理解 G蛋白和Arrestin偶联所必需的，但对GRK如何结合有机械理解 由于这种高度瞬变的性质，磷酸化的GPCR仍然难以捉摸 互动。这项建议的目标是通过以下方式弥合这一关键的知识差距 利用视紫红质和GRK1之间的相互作用作为范式来确定 GPCRs和GRK之间有针对性的相互作用的机制和结构基础。 意义：拟议目标的完成将揭示 GRK/GPCR识别并揭示GPCR/GRK构象，使GRK结合并 受体磷酸化。这些信息对于合理地设计 治疗偏向激动剂，用于治疗广泛的疾病。

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.