Molecular basis for arrestin-mediated signaling

抑制蛋白介导的信号传导的分子基础

• 批准号: 9324338
• 负责人: T M Iverson
• 金额: $ 31.01万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324338
• 关键词: Abbreviations; Affinity; Apoptosis; Apoptotic; Arrestins; Binding; Binding Sites; Biological Assay; Biological Models; Cells; Coupling; Crystallography; Cytoplasm; Data; Distal; Drug Targeting; Electrons; Eukaryotic Cell; Fingers; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; In Vitro; Lasers; Literature; MAPK10 gene; MAPK8 gene; Malignant Neoplasms; Measurement; Mediating; Methods; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Conformation; Mutagenesis; Names; Nature; Neurodegenerative Disorders; Phosphotransferases; Phytic Acid; Play; Property; Protein Isoforms; Proteins; Role; SRC gene; Signal Pathway; Signal Transduction; Site; Structure; Surface; Testing; Time; Work; X-Ray Crystallography; arrestin 2; arrestin3; biophysical techniques; experimental study; inorganic phosphate; insight; light scattering; non-visual arrestins; novel; protein activation; protein-tyrosine kinase c-src; receptor; receptor binding; sarcoma; scaffold; single molecule; src-Family Kinases

PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking the G protein coupling site. Second, arrestins can support G protein independent signaling. The best studied arrestin- mediated signaling pathways include the activation of mitogen activated protein (MAP) kinases. Recently we have determined the structure of activated arrestin-3, which shows the expected inter-domain twist. We also identified localized, previously unrecognized conformational changes in the effector binding regions of activated arrestin. Combining structural analysis with functional measurements, we propose a new paradigm for arrestin scaffolding of effectors via these sites, where the transition of arrestin into the active conformation can “turn on” effector binding. Functionally analogous to `switch regions' of G proteins, these activation- dependent conformational changes in effector binding regions are 30-40 Å distal from the site of receptor (or non-receptor activator) binding and are distinct from activation-associated conformational changes previously described in the literature. We leverage this new finding with three aims that explore the connection between activation and effector binding in three aims. In Aim 1, we combine in vitro structural and biophysical techniques (X-ray crystallography, multi-angle laser light scattering (MALLS), and affinity measurements, with double electron electron resonance (DEER) as an alternative approach) with single molecule and functional measurements in cells to investigate the role of oligomerization in stabilizing receptor-independent arrestin activation. In Aim 2, we combine mutagenesis and functional analysis to reveal how arrestin-phosphate and arrestin- protein interactions at the activation sites of arrestin is allosterically transmitted to the effector binding sites, which are 30 – 40 Å distal. In Aim 3, we use new methods to stabilize the active form of arrestin-3 and use X-ray crystallography to identify the details of arrestin-effector interactions.

项目总结 Arrestin蛋白是G蛋白偶联受体(GPCR)信号的主要调节者，有两种作用方式。 首先，阻滞剂通过物理阻断G蛋白来终止G蛋白与同源受体的偶联 联结点。第二，阻滞素可以支持G蛋白非依赖性信号转导。研究得最好的芳香素- 介导的信号通路包括丝裂原活化蛋白(MAP)激酶的激活。最近我们 确定了活化的arrestin-3的结构，显示了预期的结构域间扭曲。我们也 效应器结合区中已识别的先前未识别的局部性构象变化 激活了arrestin。将结构分析与功能测量相结合，提出了一种新的范式 对于通过这些位置的效应器的arrestin支架，其中arrestin到活性构象的转变 可以“打开”效应器绑定。在功能上类似于G蛋白的“开关区域”，这些激活- 效应器结合区的依赖构象变化是在受体(或 非受体激活剂)结合，不同于先前激活相关的构象变化 文献中所描述的。我们利用这一新发现有三个目的来探索 激活和效应器绑定在三个目的。 在目标1中，我们结合了体外结构和生物物理技术(X射线结晶学、多角度激光 光散射(MALL)和亲和力测量，以双电子电子共振(DER)为基础 另一种方法)在细胞中进行单分子和功能测量，以研究 齐聚在稳定受体非依赖性arrestin激活中的作用。 在目标2中，我们结合突变和功能分析来揭示arrestin-磷酸和arrestin是如何- Arrestin活化部位的蛋白质相互作用以变构方式传递到效应器结合部位， 它们是30-40？远端的。 在目标3中，我们使用新的方法来稳定arrestin-3的活性形式，并使用X射线结晶学来 确定arrestin-效应器相互作用的细节。