Molecular basis for arrestin-mediated signaling

抑制蛋白介导的信号传导的分子基础

基本信息

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
项目类别：
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

项目摘要

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.

项目概要抑制蛋白是 G 蛋白偶联受体 (GPCR) 信号传导的主要调节因子，并以两种方式发挥作用。首先，视紫红质抑制蛋白通过物理阻断 G 蛋白来终止 G 蛋白与同源受体的偶联其次，抑制蛋白可以支持 G 蛋白独立信号传导。介导的信号通路包括丝裂原激活蛋白（MAP）激酶的激活。我们还确定了激活的抑制蛋白-3 的结构，这显示了预期的域间扭曲。确定了效应器结合区域中先前未识别的局部构象变化将结构分析与功能测量相结合，我们提出了一种新的范例。对于通过这些位点的效应子的视紫红质抑制蛋白支架，视紫红质抑制蛋白转变为活性构象可以“打开”效应器结合，其功能类似于 G 蛋白的“开关区域”，这些激活- 效应器结合的依赖性区域构象变化距离受体位点（或非受体激活剂）结合，与之前激活相关的构象变化不同我们利用这一新发现来实现三个目的，探索之间的联系。激活和效应器结合三个目标。在目标 1 中，我们结合了体外结构和生物物理技术（X 射线晶体学、多角度激光光散射（MALLS）和亲和力测量，以双电子电子共振（DEER）作为替代方法）通过单分子和细胞中的功能测量来研究寡聚化稳定受体独立的抑制蛋白激活。在目标 2 中，我们结合诱变和功能分析来揭示视紫红质抑制蛋白-磷酸和视紫红质抑制蛋白- 视紫红质抑制蛋白激活位点的蛋白质相互作用以变构方式传递到效应子结合位点，远端 30 – 40 Å。在目标 3 中，我们使用新方法来稳定抑制蛋白-3 的活性形式，并使用 X 射线晶体学来确定抑制蛋白-效应器相互作用的细节。