Arrestin interactions with non-receptor binding partners

抑制蛋白与非受体结合伙伴的相互作用

• 批准号: 7765525
• 负责人: VSEVOLOD V. GUREVICH
• 金额: $ 27.35万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7765525
• 关键词: Affect; Affinity; Alzheimer' s Disease; Apoptotic; Arrestins; Binding; Binding Sites; Biological Assay; Cell Proliferation; Cell physiology; Cells; Cessation of life; Complex; Crystallization; Disabled Persons; Disease; Drug Delivery Systems; Electron Spin Resonance Spectroscopy; Electronics; Elements; Exclusion; Face; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Human; Individual; Label; Life; Link; MAP2K1 gene; MAPK1 gene; MAPK10 gene; Malignant Neoplasms; Measures; Mediating; Molecular; Molecular Conformation; Mutagenesis; Names; Neurodegenerative Disorders; Nuclear; Parkinson Disease; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Physiological; Play; Protein Binding; Protein Kinase; Proteins; Receptor Signaling; Research Personnel; Role; SRC gene; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Site-Directed Mutagenesis; Spectrum Analysis; Spin Labels; Staging; Surface; Testing; Therapeutic; Therapeutic Intervention; base; coated pit; design; improved; mutant; non-visual arrestins; novel; programs; protein protein interaction; receptor; receptor binding; receptor internalization; reconstitution; small molecule; src-Family Kinases; tool; trafficking; ubiquitin ligase; upstream kinase

DESCRIPTION (provided by applicant): The signaling by G protein-coupled receptors (GPCRs) is regulated by receptor phosphorylation and arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and often redirects the signaling to G-protein-independent pathways via c-Src and ERK1/2 and JNK3 activation cascades. Both receptor-bound and free forms of arrestin regulate the function and affect the intracellular localization of multiple signaling molecules. 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 propose to identify arrestin elements involved in the interactions of both non-visual arrestins in their free and receptor-bound state with non-receptor signaling molecules c-Src, ERK2, JNK3, upstream kinases, and ubiquitin ligase Mdm2. 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 trafficking assays in living cells. We also propose to reconstruct arrestin-containing "signalosomes", i.e., the arrestin-receptor complexes with protein kinases, from purified components in order to elucidate which proteins bind arrestin directly and what are the functional consequences of the interaction of Src, ERK2, and JNK3 with arrestin-receptor complex. Based on the size of the arrestin molecule and the number of its non-receptor interaction partners, we hypothesize that some (if not all) of these kinases will compete with each other for arrestin binding. We propose to test this hypothesis and measure the affinity of Src, ERK2, and JNK3 for free and receptor-bound arrestin. Identification of arrestin binding sites for non-receptor partners will allow us to construct arrestin mutants with selectively enhanced or disabled interaction sites. Using these mutants for targeted experimental manipulation we propose to study the physiological significance of these interactions in the cell. This information will set the stage for designing peptide and small molecule mimics of the interaction surfaces that can be used as experimental and therapeutic tools.

描述（由申请人提供）：G蛋白偶联受体（GPCR）的信号传导受受体磷酸化和抑制蛋白与活性磷酸受体结合的调节。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，并且通常通过c-Src和ERK 1/2和JNK 3激活级联将信号传导重定向到G蛋白非依赖性途径。受体结合和游离形式的arrestin调节多种信号分子的功能并影响其细胞内定位。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。我们建议确定arrestin元素参与的相互作用的非视觉arrestins在其自由和受体结合状态与非受体信号分子c-Src，ERK 2，JNK 3，上游激酶，和泛素连接酶Mdm 2。为此，我们建议使用定点诱变，直接结合测定，定点自旋标记的arrestins和EPR光谱，以及在活细胞中的贩运测定。我们还建议重建含有抑制蛋白的“信号体”，即，抑制蛋白-受体复合物与蛋白激酶，从纯化的组分，以阐明哪些蛋白质结合抑制蛋白直接和什么是Src，ERK 2，和JNK 3与抑制蛋白-受体复合物的相互作用的功能后果。基于arrestin分子的大小及其非受体相互作用伴侣的数量，我们假设这些激酶中的一些（如果不是全部）将相互竞争arrestin结合。我们建议测试这一假设，并测量Src，ERK 2和JNK 3的亲和力为自由和受体结合的抑制蛋白。识别非受体伴侣的抑制蛋白结合位点将使我们能够构建具有选择性增强或禁用相互作用位点的抑制蛋白突变体。使用这些突变体的有针对性的实验操作，我们建议研究这些相互作用在细胞中的生理意义。这些信息将为设计可用作实验和治疗工具的相互作用表面的肽和小分子模拟物奠定基础。