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.

项目摘要阻止蛋白是G蛋白偶联受体（GPCR）信号的主要调节剂，并以两种方式起作用。首先，逮捕蛋白通过物理阻断G蛋白终止G蛋白与同源受体的偶联耦合网站。其次，逮捕蛋白可以支持G蛋白独立的信号传导。最好的Studiod逮捕介导的信号通路包括有丝分裂原活化蛋白（MAP）激酶的激活。最近我们已经确定了激活的停滞蛋白3的结构，该结构显示了预期的域间扭曲。我们也是确定的局部，以前未被认可的会议变化激活逮捕素。结构分析与功能测量相结合，我们提出了一个新的范式用于通过这些地点逮捕效果的脚手架，在该地点过渡素至主动构象可以“打开”效应子结合。在功能上类似于G蛋白的“开关区域”，这些激活 - 效应子结合区域的依赖性会议变化距接收器位点的远端为30-40Å（或非受体激活剂）结合，与激活相关的会议变化不同文献中描述。我们利用这一新发现的三个目标来探讨激活和效应子在三个目标中的结合。在AIM 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（购物中心）和亲和力测量值，双电子共振（鹿）作为一个单分子和细胞中功能测量的替代方法）研究在稳定受体非依赖性阻滞蛋白激活中的寡聚。在AIM 2中，我们结合了诱变和功能分析，以揭示抑制磷酸抑制和抑制素抑制蛋白激活位点的蛋白质相互作用在变构中传播到效应子结合位点，即远端是30 - 40Å。在AIM 3中，我们使用新方法稳定抑制素3的主动形式，并使用X射线晶体学到确定逮捕蛋白效应相互作用的细节。