Arestin Interaction with Endocytic Components

描述（由申请人提供）：抑制蛋白在调节G蛋白偶联受体（GPCR）的信号传导和运输中发挥重要作用。视觉抑制蛋白（抑制蛋白-1和4）与视蛋白特异性相互作用，而非视觉抑制蛋白（抑制蛋白-2和3）与多种GPCR相互作用，并结合磷酸肌醇、内吞机制的组分（网格蛋白和β 2-适配蛋白）和各种信号分子（Src家族成员和iMAP激酶）。晶体学分析表明，arrestin-1和2具有相似的基本结构，包含由极性核心连接的N-末端和C-末端结构域。极性核心的破坏被认为发生在受体结合时，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、内吞组分和信号分子相互作用的结构基础在很大程度上仍然未知。在本申请中，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arrestin-2激活的结构和分子基础以及与内吞机制的组分的相互作用。具体目标是：1.对抑制蛋白与磷酸肌醇、网格蛋白和β 2-适应蛋白复合物进行晶体学分析。虽然结构/功能分析提供了重要的洞察域，介导抑制蛋白与磷酸肌醇和内吞成分的相互作用，这些组件的结合界面的详细图片是缺乏的。我们建议结晶和解决野生型和激活的arrestin-2与肌醇六磷酸，网格蛋白，和β 2-适应蛋白复合物的结构。这些复合物的高分辨率晶体学分析应提供以下结构方面的见解：1）arrestin-2与磷酸肌醇、网格蛋白和β 2-适应蛋白的结合界面; 2）arrestin-2激活的机制;和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β 2-适应蛋白相互作用的生物学特性。抑制蛋白与网格蛋白包被的小凹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。我们将使用我们的结构分析的见解，以进一步确定抑制蛋白介导的调节GPCR内吞作用的机制基础。这些研究将利用各种生物化学，分子和细胞生物学策略来探测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，这些研究应该提供结构上的差异，在基础和激活形式的arrestin-2，并提供了一个机制的基础上，如何非视觉arrestins作为GPCR调控的支架蛋白的功能。

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.