Structure-function studies of visual arrestin

视觉抑制蛋白的结构功能研究

基本信息

批准号：
10394867
负责人：
VSEVOLOD V. GUREVICH
金额：
$ 58.19万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-04-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10394867
关键词：
Affinity Arrestins Binding Biological Biological Testing Cartilage Diseases Cells Characteristics Collaborations Complex Crystallization Data Defect Disease Drosophila genus Electrons Engineering Ensure Financial compensation G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GRK1 gene GTP-Binding Proteins Goals Human Hyperthyroidism Lead Light Malignant Neoplasms Measurement Mechanics Modeling Molecular Molecular Conformation Mus Mutation Night Blindness Patients Phosphorylation Phosphorylation Site Photoreceptors Physiologic pulse Physiology Positioning Attribute Proteins Recovery Regulation Reproducibility Research Retinal Degeneration Rhodopsin Rod Rodent Role Shapes Signal Transduction Site Spin Labels Structure Techniques Testing Therapeutic Transducin Transgenic Mice Visual arrestin 1 base biophysical techniques clinically relevant cytotoxic cytotoxicity design experimental study gain of function gene therapy improved in vivo in vivo Model monomer mutant novel protein protein interaction retinal rods success

项目摘要

PROJECT SUMMARY We propose to elucidate the molecular mechanisms of arrestin-1 interactions with rhodopsin, which significantly contribute to exquisitely regulated and precisely timed function of photoreceptor cells, using the combination of novel cutting-edge biophysical methods and in vivo experiments. We will compare the shape of the complex of arrestin-1 with rhodopsin using long-range distance measurements between selected points in arrestin-1 and rhodopsin with pulse EPR technique DEER. We will explore the conformation of arrestin-bound rhodopsin and compare it to the conformation of free light-activated rhodopsin and rhodopsin in complex with transducing to determine whether particular conformation states of rhodopsin (a model GPCR) predispose it to interact with G protein and arrestin. This is hypothesized in the field to be the basis of biased GPCR signaling. We will determine the biological role of arrestin-1 self-association in photoreceptors in vivo, which is currently unknown. Finally, we will test novel compensational approach to gene therapy of gain-of-function rhodopsin mutations using engineered enhanced arrestin-1 mutants capable of shutting off rhodopsin signaling independently of its phosphorylation. Proposed studies will significantly improve our understanding of photoreceptor physiology and advance our progress towards gene therapy of gain-of-function rhodopsin mutations. Due to high conservation of mechanics of GPCR regulation, mechanistic studies of arrestin-1 binding to rhodopsin will have broader implications, improving our understanding of the molecular basis of the function of other arrestin subtypes. Proposed studies will shed new light on many aspects of cell signaling. Our strategic goal is to gain sufficient understanding of protein-protein interactions that govern cell signaling to construct mutants with desired functional characteristics for research and therapeutic purposes.

项目摘要我们建议阐明与视紫红质相互作用的分子机制，该机制使用光感受器细胞的精心调节和精确的时机功能显着贡献新型尖端生物物理方法和体内实验的结合。我们将比较使用Rhodopsin逮捕蛋白1的复合蛋白使用长距离距离测量用脉冲EPR技术鹿逮捕蛋白-1和视紫红质。我们将探索限制逮捕的构象 Rhodopsin并将其与复合物中的游离光激活Rhodopsin和Rhodopsin的构象转换以确定视紫红质（GPCR模型）的特定构象是否使其倾向于与G蛋白和逮捕蛋白相互作用。在现场中假设这是偏见的GPCR信号传导的基础。我们将确定抑制素-1自我关联在体内的生物学作用，目前是未知。最后，我们将测试新的补偿方法来实现功能获得的视紫红质的基因治疗使用工程增强的逮捕蛋白-1突变体的突变，能够关闭视紫红质信号传导独立于其磷酸化。拟议的研究将大大提高我们对光感受器的生理并提高了我们朝着功能收益视紫红蛋白获得基因治疗的进步突变。由于GPCR调节力学的高度保存，抑制蛋白1的机械研究与视紫红质的结合将具有更广泛的影响，从而提高了我们对分子基础的理解其他逮捕的亚型的功能。拟议的研究将为细胞信号的许多方面提供新的启示。我们的战略目标是对控制细胞信号传导的蛋白质 - 蛋白质相互作用有足够的了解构建具有所需功能特征的突变体，以进行研究和治疗目的。