Physiological role of phosducins in the retina

磷酸酯在视网膜中的生理作用

• 批准号: 7226620
• 负责人: BARRY M WILLARDSON
• 金额: $ 21.85万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226620
• 关键词: Physiological; role; phosducins; retina

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（由申请人提供）：真核细胞依赖于G蛋白介导的信号通路来检测和响应各种细胞外刺激。基于其G蛋白β/伽马亚基结合特性，硫糖素（PDC）和磷素样蛋白（PHLP）被认为可以调节G蛋白信号传导。但是，最近的证据也表明了其他角色。这项研究的长期目标是定义视网膜和其他细胞类型的感光细胞中PDC和PHLP的生理功能。 最近的研究表明，PDC在感光细胞中的光调节核定位，表明PDC在细胞核中的作用。在特定的目标1-3中，研究了核定位调节的潜在机制，并检查了潜在的核结合伙伴。在AIM 4中，解决了PDC在光感受器中G蛋白的光依赖性转运中的可能作用。在AIM 5中，研究了PHLP在调节细胞外信号调节激酶（ERK）激活蛋白酶体介导的GBETA/GAMMA降解中的作用。 用于实现这些特定目标的方法是多学科的。它们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进行的免疫定位研究。识别核结合伴侣的功能蛋白质组学技术；基于免疫印迹的测定法以探索泛素/蛋白酶体靶向降解； siRNA介导的基因沉默；定向诱变以鉴定触发降解的磷酸化位点；以及旨在评估ERK介导的GBETA/GAMMA降解对G蛋白信号传导的降解的影响的信号通路串扰测定。 对PDC和PHLP功能的基本理解非常难以捉摸。本文提出的实验将给出有关这些重要蛋白在细胞信号传导过程中的作用的生理相关答案。