Conformational regulation of arrestin-mediated signaling

抑制蛋白介导的信号传导的构象调节

基本信息

批准号：
7884252
负责人：
VSEVOLOD V. GUREVICH
金额：
$ 29.25万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
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/7884252
关键词：
Affect Arrestins Attenuated Binding Binding Sites Biochemical Biological Assay Biological Phenomena Biological Process Cells Cessation of life Characteristics Chimera organism 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 MAPK1 gene MAPK10 gene Measures Mediating Methods Microtubules Mitogen-Activated Protein Kinases Modeling Molecular Molecular Conformation Names Nature Pathway interactions Phosphorylation Phosphotransferases Play Process Property Protein Kinase Protein Phosphatase 2A Regulatory Subunit PR53 Protein phosphatase Proteins 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 flexibility functional outcomes improved inhibitor/antagonist mdm2 protein mutant non-visual arrestins preference protein-tyrosine kinase c-src public health relevance receptor receptor binding receptor internalization retinal rods tool ubiquitin ligase

Affect Arrestins Attenuated Binding Binding Sites Biochemical Biological Assay Biological Phenomena Biological Process Cells Cessation of life Characteristics Chimera organism 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 MAPK1 gene MAPK10 gene Measures Mediating Methods Microtubules Mitogen-Activated Protein Kinases Modeling Molecular Molecular Conformation Names Nature Pathway interactions Phosphorylation Phosphotransferases Play Process Property Protein Kinase Protein Phosphatase 2A Regulatory Subunit PR53 Protein phosphatase Proteins 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 flexibility functional outcomes improved inhibitor/antagonist mdm2 protein mutant non-visual arrestins preference protein-tyrosine kinase c-src public health relevance receptor receptor binding receptor internalization retinal rods tool ubiquitin ligase

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项目摘要

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）：通常称为脱敏的细胞对持续刺激的反应性的降低是一种广泛的生物学现象。 G蛋白偶联受体（GPCR）的信号通过两步机制减弱：受体磷酸化是通过特定激酶的受体磷酸化来抑制了与活性磷酸受体的抑制蛋白结合。阻止蛋白结合终止G蛋白介导的信号传导，将GPCR标记为内在化，并通过非受体结合伴侣（C-SRC和MAP激酶，泛素连接酶等）将信号转移到其他途径中。逮捕蛋白还通过与受体结合有关的相同界面与微管相互作用，并动员了具有不同功能后果的细胞骨架的几个信号分子。该提案的主要目的是阐明抑制蛋白功能作为细胞中多蛋白信号复合物的组织者的结构基础。我们假设抑制蛋白分子的信号传导能力取决于其构象。 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,逮捕蛋白和EPR光谱的位置定向自旋标记，以及活细胞中的功能测定。我们还建议使用带自旋标记的模型受体Rhodopsin使用双自旋标记的逮捕蛋白和自旋标记的阻止蛋白，以通过常规EPR和双电子电子共振（DEER）（DEER）测量抗抑制蛋白受阻络合物中的旋转间距离（DEER），以获得该综合体的工作结构模型。该信息将为设计基于逮捕蛋白的分子工具奠定阶段，以针对细胞信号的靶向操纵，该工具可用于实验和治疗目的。公共卫生相关性：逮捕蛋白是多功能适配器，将各种信号分子动员到具有不同功能后果的G蛋白偶联受体和微管。该提案的目的是阐明受体结合和微管结合抑制蛋白的构象，以了解抑制素构象如何影响其与信号蛋白的相互作用及其结合的后果。该信息将为设计基于逮捕蛋白的分子工具奠定阶段，以针对细胞信号的靶向操纵，该工具可用于实验和治疗目的。