Physiological role of phosducins in the retina

磷酸酯在视网膜中的生理作用

• 批准号: 6889872
• 负责人: BARRY M WILLARDSON
• 金额: $ 22.5万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-03 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889872
• 关键词: G protein; affinity chromatography; genetically modified animals; immunoprecipitation; intracellular transport; kinase inhibitor; laboratory mouse; mass spectrometry; mitogen activated protein kinase; nerve /myelin protein; nuclear proteins; phosphoproteins; phosphorylation; protein binding; protein degradation; protein localization; protein protein interaction; protein sequence; protein signal sequence; protein structure function; protein transport; retina; site directed mutagenesis; small interfering RNA; tissue /cell culture; visual phototransduction

DESCRIPTION (provided by applicant): Eukaryotic cells rely on G protein-mediated signaling pathways to detect and respond to a wide variety of extracellular stimuli. Based on their G protein beta/gamma subunit binding properties, phosducin (Pdc) and phosducin-like protein (PhLP) are believed to regulate G protein signaling. However, recent evidence suggests other roles as well. The long term objective of this research is to define the physiological function of Pdc and PhLP in the photoreceptor cells of the retina and other cell types. Recent studies have demonstrated light-regulated nuclear localization of Pdc in photoreceptor cells, suggesting a role for Pdc in the nucleus. In Specific Aims 1-3, potential mechanisms of regulation of nuclear localization are investigated and potential nuclear binding partners are examined. In Aim 4, the possible role of Pdc in light dependent translocation of G proteins in the photoreceptor is addressed. In Aim 5, the role of PhLP in regulating proteasome-mediated degradation of Gbeta/gamma initiated by extracellular signal-regulated kinase (ERK) activation is investigated. The methods used to achieve these specific aims are multi-disciplinary. They include immunolocalization studies in cultured cells, retinal tissues and transgenic mice using phosphorylation site-specific antibodies; functional proteomics techniques to identify nuclear binding partners; immunoblot-based assays to explore ubiquitin/proteasome-targeted degradation; siRNA-mediated gene silencing; site-directed mutagenesis to identify phosphorylation sites that trigger degradation; and signaling pathway crosstalk assays designed to assess the effect of ERK-mediated degradation of Gbeta/gamma on G protein signaling. A basic understanding of the function of Pdc and PhLP has been very elusive. The experiments proposed herein will give physiologically relevant answers concerning the role of these important proteins in cellular signaling processes.

描述(申请人提供)：真核细胞依靠G蛋白介导的信号通路来检测和响应各种细胞外刺激。根据G蛋白的β/γ亚基结合特性，光导蛋白(PDC)和光导蛋白样蛋白(PhLP)被认为调节G蛋白信号转导。然而，最近的证据表明，它还扮演着其他角色。这项研究的长期目标是确定PDC和PhLP在视网膜感光细胞和其他细胞类型中的生理功能。 最近的研究表明，PDC在光感受器细胞中的核定位是受光调控的，这表明PDC在核中发挥了作用。在具体目标1-3中，研究了核局部化的潜在调节机制，并检查了潜在的核结合伙伴。在目标4中，PDC在光感受器中G蛋白的光依赖性转位中可能起到作用。在目的5中，研究了PhLP在调节蛋白酶体介导的由细胞外信号调节激酶(ERK)激活启动的Gbeta/Gamma降解中的作用。 实现这些具体目标所使用的方法是多学科的。这些方法包括：使用磷酸化位点特异性抗体在培养细胞、视网膜组织和转基因小鼠中的免疫定位研究；确定核结合伙伴的功能蛋白质组学技术；基于免疫印迹的分析以探索泛素/蛋白酶体靶向降解；siRNA介导的基因沉默；定点突变以确定引发降解的磷酸化位点；以及信号通路串扰分析，旨在评估ERK介导的Gbeta/Gamma降解对G蛋白信号的影响。 对PDC和PhLP的功能的基本了解一直非常难以捉摸。这里提出的实验将给出关于这些重要蛋白质在细胞信号过程中的作用的生理学相关的答案。