Conformational regulation of arrestin-mediated signaling

抑制蛋白介导的信号传导的构象调节

• 批准号: 7464846
• 负责人: VSEVOLOD V. GUREVICH
• 金额: $ 30.58万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464846
• 关键词: Affect; Arrestin; Arrestins; Attenuated; Binding; Binding Sites; Biochemical; Biological Assay; Biological Phenomena; Biological Process; Cells; Cessation of life; Characteristics; Chimera organism; Classification; Complex; Cytoskeleton; Electron Spin Resonance Spectroscopy; Electronics; Electrons; Elements; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Individual; Life; Light; Localized; MAPK1 gene; MAPK10 gene; Measures; Mediating; Methods; Microtubules; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Conformation; Names; Nature; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Pliability; Process; Property; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Public Health; Purpose; Range; Regulation; Retinal Cone; Rhodopsin; Role; SRC gene; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Site-Directed Mutagenesis; Specificity; Spectrum Analysis; Spin Labels; Staging; Stimulus; Structural Models; Structure; Testing; Therapeutic; Visual; Work; arrestin3; base; desensitization; design; functional outcomes; improved; inhibitor/antagonist; mdm2 protein; mutant; preference; protein-tyrosine kinase c-src; receptor; receptor binding; receptor internalization; retinal rods; tool; ubiquitin ligase

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.

描述(由申请人提供)：细胞对持续刺激的反应性降低，通常被称为脱敏，是一种普遍的生物学现象。G蛋白偶联受体(GPCRs)的信号通过两步机制减弱：受体被特定的激酶磷酸化，然后arrestin与活性的磷受体结合。Arrestin结合终止G蛋白介导的信号转导，标记GPCRs进行内化，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。抑制素还通过参与受体结合的相同界面与微管相互作用，并将几个信号分子动员到细胞骨架上，产生不同的功能后果。这一建议的主要目的是阐明arrestin作为细胞内多蛋白信号复合体的组织者的结构基础。我们假设arrestin分子的信号传递能力是由其构象决定的。我们建议鉴定与受体和微管结合有关的arrestin元件，以及受体和微管诱导的调控它们与非受体伙伴相互作用的非视觉抑制蛋白的构象变化的性质：激酶c-Src、ERK2、JNK3、泛素连接酶MDM2等。为此，我们建议使用定点突变、直接结合试验、阻止蛋白的定点自旋标记和EPR光谱，以及活细胞中的功能分析。我们还建议使用双自旋标记的拦阻蛋白和自旋标记的拦阻蛋白与自旋标记的模型受体视紫红质，通过常规的EPR和双重电子-电子共振(DER)来测量arrestin-受体复合体的自旋间距离，以获得该复合体的工作结构模型。这些信息将为设计基于arrestin的分子工具奠定基础，这些工具用于靶向操纵细胞信号，可用于实验和治疗目的。与公共健康相关：阻滞素是一种多功能适配器，可以将各种信号分子动员到G蛋白偶联受体和微管上，产生不同的功能后果。这个建议的目的是阐明受体结合和微管结合的拦阻蛋白的构象，以了解arrestin的构象如何影响其与信号蛋白的相互作用以及它们结合的后果。这些信息将为设计基于arrestin的分子工具奠定基础，这些工具用于靶向操纵细胞信号，可用于实验和治疗目的。