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，是一种普遍的生物学现象。通过G蛋白偶联受体（GPCRs）的信号传导通过两步机制减弱：通过特异性激酶的受体磷酸化，然后抑制蛋白结合活性磷酸化受体。抑制蛋白结合终止G蛋白介导的信号传导，标记GPCR用于内化，并通过非受体结合配偶体（c-Src和MAP激酶、泛素连接酶等）将信号传导重定向至其他途径。抑制蛋白也通过参与受体结合的相同界面与微管相互作用，并将几种信号分子动员到具有不同功能后果的细胞骨架。该提案的主要目的是阐明arrestin作为细胞中多蛋白信号复合物的组织者功能的结构基础。我们推测arrestin分子的信号传导能力是由其构象决定的。我们提出鉴定参与受体和微管结合的抑制蛋白元件，以及调节它们与非受体伴侣相互作用的两种非视觉抑制蛋白中受体和微管诱导的构象变化的性质：激酶c-Src、ERK 2、JNK 3、泛素连接酶Mdm 2等。为此，我们建议使用定点诱变，直接结合测定，抑制蛋白的定点自旋标记和EPR光谱，以及活细胞中的功能测定。我们还建议使用双自旋标记的arrestins和自旋标记的arrestins与自旋标记的模型受体，视紫红质，测量自旋间距arrestins受体复合物的常规EPR和双电子-电子共振（DEER），以获得一个工作的结构模型的复合物。这些信息将为设计基于抑制蛋白的分子工具奠定基础，这些分子工具可用于实验和治疗目的，用于靶向操纵细胞信号传导。公共卫生相关性：Arrestins是一种多功能的衔接子，可以将各种信号分子动员到G蛋白偶联受体和微管上，具有不同的功能后果。该提案的目标是阐明受体结合和微管结合arrestins的构象，以了解arrestin构象如何影响其与信号蛋白的相互作用及其结合的后果。这些信息将为设计基于抑制蛋白的分子工具奠定基础，这些分子工具可用于实验和治疗目的，用于靶向操纵细胞信号传导。

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.