Arestin Interaction with Endocytic Components

Arestin 与内吞成分的相互作用

• 批准号: 6767838
• 负责人: Jeffrey L Benovic
• 金额: $ 31.71万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767838
• 关键词: X ray crystallography; arrestins; binding proteins; biological signal transduction; cell biology; cell surface receptors; clathrin; electron microscopy; endocytosis; fluorescence microscopy; immunofluorescence technique; immunoprecipitation; phosphatidylinositols; protein localization; protein protein interaction; protein purification; protein transport; surface plasmon resonance; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Arrestins play an essential role in regulating signaling and trafficking of G protein-coupled receptors (GPCRs). Visual arrestins (arrestin-1 and 4) specifically interact with opsins while non-visual arrestins (arrestin-2 and 3) interact with multiple GPCRs and also bind phosphoinositides, components of the endocytic machinery (clathrin and beta2-adaptin), and various signaling molecules (Src family members and iMAP kinases). Crystallographic analysis reveals that arrestin-1 and 2 have a similar basal structure containing N-terminal and C-terminal domains joined by a polar core. Disruption of the polar core is thought to occur upon receptor binding and result in an "active" conformation that regulates arrestin interaction with various proteins. The structural basis for arrestin activation as well as for arrestin interaction with GPCRs, phosphoinositides, endocytic components, and signaling molecules remains largely unknown. In this application, we propose to use X-ray crystallography and various biochemical and cell biological strategies to analyze the structural and molecular basis for arrestin-2 activation and interaction with components of the endocytic machinery. The specific objectives are to: 1. Perform crystallographicanalysis of arrestin complexed with phosphoinositides, clathrin, and beta2-adaptin. Although structure/function analysis has provided important insight into the domains that mediate arrestin interaction with phosphoinositides and endocytic components, a detailed picture of the binding interfaces of these components is lacking. We propose to crystallize and solve structures of wild-type and activated arrestin-2 in complex with inositol hexakisphosphate, clathrin, and beta2-adaptin. High-resolution crystallographic analysis of these complexes should provide structural insight into: 1) the binding interfaces of arrestin-2 with phosphoinositides, clathrin, and beta2-adaptin; 2) the mechanism of arrestin-2 activation; and 3) the mechanism of assembly of a multi-protein endocytic complex. 2. Characterize the biology of arrestin interaction with phosphoinositides, clathrin, and beta2-adaptin. The dynamics of arrestin interaction with clathrin-coated pits is complex and involves multiple protein-protein and protein-lipid interactions. We will use insight from our structural analysis to further define the mechanistic basis for arrestin-mediated regulation of GPCR endocytosis. These studies will utilize various biochemical, molecular, and cell biological strategies to probe the interaction of arrestins with endocytic components and elucidate the function of such interactions in GPCR trafficking. Overall, these studies should provide structural insight into differences in the basal and activated forms of arrestin-2 and provide a mechanistic basis for how non-visual arrestins function as GPCR-regulated scaffolding proteins.

描述（由申请人提供）：抑制素在调节 G 蛋白偶联受体 (GPCR) 的信号传导和运输中发挥重要作用。视觉视紫红质抑制蛋白（arrestin-1 和 4）与视蛋白特异性相互作用，而非视觉视紫红质抑制蛋白（arrestin-2 和 3）与多个 GPCR 相互作用，还结合磷酸肌醇、内吞机制组件（网格蛋白和 β2-适应素）以及各种信号传导分子（Src 家族成员和 iMAP 激酶）。晶体学分析表明，arrestin-1 和 2 具有相似的基础结构，包含由极性核心连接的 N 端和 C 端结构域。极性核心的破坏被认为是在受体结合时发生的，并导致调节抑制蛋白与各种蛋白质相互作用的“活性”构象。抑制蛋白激活以及抑制蛋白与 GPCR、磷酸肌醇、内吞成分和信号分子相互作用的结构基础仍然很大程度上未知。在此应用中，我们建议使用 X 射线晶体学和各种生化和细胞生物学策略来分析抑制蛋白-2 激活以及与内吞机制组件相互作用的结构和分子基础。具体目标是： 1. 对与磷酸肌醇、网格蛋白和 β2-适应蛋白复合的视紫红质抑制蛋白进行晶体分析。尽管结构/功能分析为介导视紫红质抑制蛋白与磷酸肌醇和内吞成分相互作用的结构域提供了重要的见解，但缺乏这些成分的结合界面的详细图片。我们建议结晶并解析野生型和活化的抑制蛋白-2与肌醇六磷酸、网格蛋白和β2-适应蛋白的复合物的结构。这些复合物的高分辨率晶体分析应提供以下结构方面的见解： 1) 抑制蛋白-2 与磷酸肌醇、网格蛋白和 β2-适应蛋白的结合界面； 2) 抑制蛋白-2激活机制； 3）多蛋白内吞复合物的组装机制。 2. 表征抑制蛋白与磷酸肌醇、网格蛋白和 β2-适应蛋白相互作用的生物学特性。视紫红质抑制蛋白与网格蛋白包被的凹坑相互作用的动力学是复杂的，涉及多种蛋白质-蛋白质和蛋白质-脂质相互作用。我们将利用结构分析的见解来进一步定义抑制蛋白介导的 GPCR 内吞作用调节的机制基础。这些研究将利用各种生化、分子和细胞生物学策略来探测视紫红质抑制蛋白与内吞成分的相互作用，并阐明这种相互作用在 GPCR 运输中的功能。总的来说，这些研究应该提供对arrestin-2基础形式和激活形式差异的结构洞察，并为非视觉视紫红质抑制蛋白如何作为GPCR调节的支架蛋白发挥作用提供机制基础。