Conformational regulation of arrestin-mediated signaling

描述（由申请人提供）：细胞对持续刺激的反应性降低，通常称为脱敏，是一种广泛存在的生物学现象。G蛋白偶联受体（gpcr）的信号通过两步机制减弱：受体被特定激酶磷酸化，然后抑制蛋白与活性磷受体结合。阻滞蛋白结合终止G蛋白介导的信号，标记gpcr内化，并通过非受体结合伙伴（c-Src和MAP激酶，泛素连接酶等）将信号重定向到其他途径。阻滞蛋白也通过参与受体结合的相同界面与微管相互作用，并动员几个信号分子到细胞骨架上，产生不同的功能后果。本提案的主要目的是阐明作为细胞中多蛋白信号复合物组织者的抑制蛋白功能的结构基础。我们假设阻滞蛋白分子的信号传导能力是由其构象决定的。我们建议确定参与受体和微管结合的抑制因子，以及受体和微管诱导的两种非视觉抑制因子的构象变化的性质，这些变化调节它们与非受体伙伴的相互作用：激酶c-Src、ERK2、JNK3、泛素连接酶Mdm2等。为此，我们建议在活细胞中使用定点诱变、直接结合实验、定点自旋标记阻滞蛋白和EPR光谱以及功能分析。我们还建议使用双自旋标记的阻滞蛋白和自旋标记的阻滞蛋白与自旋标记的模型受体视紫红质，通过常规EPR和双电子-电子共振（DEER）测量阻滞蛋白-受体复合物的自旋间距离，以获得复合物的工作结构模型。这一信息将为设计基于抑制蛋白的分子工具奠定基础，用于有针对性地操纵细胞信号传导，可用于实验和治疗目的。公共卫生相关性：停搏蛋白是一种多功能接头，可调动各种信号分子到G蛋白偶联受体和微管，具有不同的功能后果。本提案的目标是阐明受体结合和微管结合的阻滞蛋白的构象，以了解阻滞蛋白的构象如何影响其与信号蛋白的相互作用及其结合的后果。这一信息将为设计基于抑制蛋白的分子工具奠定基础，用于有针对性地操纵细胞信号传导，可用于实验和治疗目的。

DESCRIPTION (provided by applicant): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. The signaling by G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase followed by arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and redirects signaling to other pathways via non-receptor binding partners (c-Src and MAP kinases, ubiquitin ligases, etc). Arrestins also interact with microtubules via the same interface that is involved in receptor binding and mobilize several signaling molecules to the cytoskeleton with different functional consequences. The main objective of this proposal is to elucidate the structural basis of arrestin function as an organizer of multi-protein signaling complexes in the cell. We hypothesize that the signaling capability of arrestin molecule is determined by its conformation. We propose to identify arrestin elements involved in receptor and microtubule binding and the nature of receptor- and microtubule-induced conformational changes in both non- visual arrestins that regulate their interactions with non-receptor partners: kinases c-Src, ERK2, JNK3, ubiquitin ligase Mdm2, etc. To this end, we propose to use site-directed mutagenesis, direct binding assay, site-directed spin labeling of arrestins and EPR spectroscopy, as well as functional assays in living cells. We also propose to use double spin-labeled arrestins and spin-labeled arrestins with spin-labeled model receptor, rhodopsin, to measure inter-spin distances in arrestin-receptor complex by conventional EPR and double electron-electron resonance (DEER) to obtain a working structural model of the complex. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes. PUBLIC HEALTH RELEVANCE: Arrestins are multi-functional adaptors that mobilize various signaling molecules to G protein-coupled receptors and microtubules with different functional consequences. The goal of this proposal is to elucidate the conformations of receptor-bound and microtubule-bound arrestins to understand how arrestin conformation affects its interactions with signaling proteins and the consequences of their binding. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes.