Targeted Engineering of Designer Arrestins to Regulate Cell Signaling

设计抑制蛋白的靶向工程以调节细胞信号传导

• 批准号: 9275751
• 负责人: VSEVOLOD V. GUREVICH
• 金额: $ 34.14万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275751
• 关键词: Apoptotic; Arrestins; Binding; Biochemistry; Biological Assay; Biophysics; Cell Death; Cell Proliferation; Cell Survival; Cells; Cessation of life; Complex; Crystallization; Data; Development; Disease; Dopamine D1 Receptor; Elements; Engineering; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Human; Levodopa; MAPK3 gene; MAPK8 gene; Malignant Neoplasms; Mediating; Mitogen-Activated Protein Kinases; Molecular; Molecular Conformation; Mus; Neurodegenerative Disorders; Parkinson Disease; Pathway interactions; Peptides; Phosphotransferases; Play; Role; Signal Transduction; Specificity; Structure; Testing; Therapeutic; X-Ray Crystallography; arrestin3; base; behavioral sensitization; experimental study; mouse model; mutant; non-visual arrestins; novel; preference; receptor; receptor binding; scaffold; tool

PROJECT SUMMARY Arrestins were discovered as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. For example, free and receptor-bound arrestin-3 scaffolds ASK1-MKK4/7-JNK1/2/3 cascades, promoting the activation of JNK family kinases. The two non-visual arrestins interact with ~800 different GPCRs in humans. We propose to identify arrestin elements responsible for receptor specificity, and elucidate the structural basis of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestins. Our new crystal structure of arrestin-3 in the presence of an abundant cytoplasmic molecule IP6 revealed receptor-bound-like (active) conformation of arrestin-3. The ability of arrestin-3 to assume this conformation in the absence of GPCRs likely explains receptor-independent scaffolding activity of arrestin-3. We identified arrestin-3 elements critical for JNK and ERK activation, as well as conformational requirements of distinct branches of arrestin-mediated signaling. We constructed arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation and identified short arrestin-3 peptides that enhance or suppress JNK activity in cells. We will test the potential of signaling-biased arrestins and arrestin-derived molecular tools to facilitate cell death or survival. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). Using arrestin-3 mutant specific for dopamine D1 receptor we showed that arrestin-mediated signaling from D1 plays a role in the behavioral sensitization to L-DOPA and development of L-DOPA-induced diskinesia in mouse model of Parkinson’s disease, but other GPCRs also contribute to these phenomena. We believe that signaling-biased arrestins and arrestinbased molecular tools with specific functional capability will help elucidate the intricacies of cellular signaling and yield novel potent therapeutic tools.

项目摘要 逮捕蛋白被发现是通过G的G蛋白偶联受体（GPCR）信号的负调节剂 蛋白质。新数据表明，免费和接收器的逮捕蛋白通过MAP启动信号 激酶调节细胞死亡，生存和增殖。例如，免费和接收器结合 逮捕素-3脚手架ask1-mkk4/7-jnk1/2/3级联，促进了JNK家族激酶的激活。 这两种非视觉停滞蛋白与人类中约800种不同的GPCR相互作用。我们建议确定 负责接收器特异性的逮捕蛋白元素，并阐明组装的结构基础 由抑制蛋白组织的多蛋白信号传导复合物（信号体）。我们的新晶体结构 在存在丰富的细胞质分子IP6的情况下，逮捕蛋白3揭示了受体结合的（活性） 逮捕素3的构象。在没有GPCR的情况下，逮捕蛋白3假设这种构象的能力 可能解释了与接收器无关的脚手架活性。我们确定了逮捕素-3元素 对于JNK和ERK激活至关重要，以及不同分支的构象要求 逮捕蛋白介导的信号传导。我们构建了绑定ask1，mkk和jnk的逮捕蛋白-3突变体，但要这样做 没有促进JNK激活，并确定了增强或抑制JNK活性的简短逮捕蛋白-3辣椒 在细胞中。我们将测试信号偏见的逮捕蛋白和逮捕蛋白衍生的分子工具的潜力 促进细胞死亡或生存。专门增加或阻止促凋亡信号的分子工具 具有与过量细胞增殖（例如癌症）或死亡相关的疾病中的热潜力 （例如，神经退行性疾病）。使用抑制素-3突变体特异性多巴胺D1受体WE 表明，D1的抑制素介导的信号在行为敏感性中起作用 以及帕金森氏病小鼠模型中L-DOPA引起的疾病的发展，但其他 GPCR也有助于这些现象。我们认为发出信号偏见的逮捕蛋白和 具有特定功能功能的基于逮捕的分子工具将有助于阐明 细胞信号传导并产生新型潜在的治疗工具。