REGULATION OF INTRACELLULAR TRANSDUCIN HOMEOSTASIS BY PHOSDUCIN IN VERTEBRATE RO

磷酸酯蛋白对脊椎动物 RO 细胞内转导蛋白稳态的调节

• 批准号: 7719933
• 负责人: MAXIM SOKOLOV
• 金额: $ 16.84万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7719933
• 关键词: Address; Afferent Neurons; Blindness; Computer Retrieval of Information on Scientific Projects Database; Coupled; Detection; Dissection; Funding; GTP-Binding Proteins; Goals; Grant; Half-Life; Heterotrimeric GTP-Binding Proteins; Homeostasis; Immunoprecipitation; In Vitro; Institution; Knock-out; Knockout Mice; Laboratories; Life; Mediating; Messenger RNA; Molecular; Phosphorylation; Photoreceptors; Property; Proteins; Regulation; Reporting; Research; Research Design; Research Personnel; Resources; Retina; Retinal; Retinal Degeneration; Retinal Photoreceptors; Role; Signal Transduction; Source; Transducin; Transgenic Organisms; United States National Institutes of Health; Vertebrate Photoreceptors; Wild Type Mouse; base; beta Subunit Transducin; cell growth regulation; in vivo; mutant; phosducin; photoreceptor degeneration; retinal rods; visual information; visual stimulus

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Retinal photoreceptors, rods and cones, are highly specialized sensory neurons that utilize G protein-mediated signaling for acquisition of visual information. Rods maintain high, invariable amounts of heterotrimeric G protein transducin required for the detection of visual stimuli, throughout their lives. Importantly, drastic change of transducin intracellular amounts often puts photoreceptors on the path to elimination, which causes retina degeneration and loss of vision. The long-term goal of our laboratory is to understand the role of G protein-mediated signaling in retinal photoreceptor function. The objective of this application is to elucidate molecular mechanisms underlying transducin homeostasis in rods. The central hypothesis of this application is that transducin accumulation in rods is regulated by phosducin, a regulator of G protein mediated signaling, which undergoes a cycle of light-dependent phosphorylation. This hypothesis is based on the reported in vitro properties of phosducin and our observation that rods of the phosducin knockout mouse accumulate significantly reduced amount of transducin. In Aims 1 and 2 we will elucidate the role of phosducin in transducin transcriptional and proteolytic control. For that we will compare the transducin beta mRNA levels, as well as values of transducin beta protein half-life in the retinas of the phosducin knockout and wild type mice. Aim 3 will be dedicated to studies of light-dependent phosducin phosphorylation, where we will determine intracellular localization of the phosducin phospho-states within the rod, address the in vivo function of this regulation using transgenic expression of phosducin phosphorylation mutants, and identify protein partners of phosducin in various rod subcellular compartments using retinal micro-dissection and immunoprecipitation coupled to mass spectrometric protein identification. The proposed studies are designed to elucidate the role of phosducin in regulation of transducin homeostasis in photoreceptors, which is important for our understanding of the molecular mechanisms of photoreceptor degeneration and for developing strategies to counteract retinal degeneration. We expect that our results will also have a significant impact on our understanding of the principles of cellular regulation of G protein-mediated signaling.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 视网膜感光细胞、视杆细胞和视锥细胞是高度专业化的感觉神经元，利用 G 蛋白介导的信号传导来获取视觉信息。视杆细胞在其一生中都保持着检测视觉刺激所需的大量、恒定的异源三聚体 G 蛋白转导蛋白。重要的是，细胞内转导蛋白数量的急剧变化通常会使光感受器走上消除之路，从而导致视网膜变性和视力丧失。 我们实验室的长期目标是了解 G 蛋白介导的信号传导在视网膜感光功能中的作用。本申请的目的是阐明视杆细胞中转导蛋白稳态的分子机制。该应用的中心假设是视杆细胞中转导蛋白的积累受到磷酸蛋白的调节，磷酸蛋白是 G 蛋白介导的信号传导的调节剂，其经历光依赖性磷酸化循环。这一假设是基于报道的光导蛋白的体外特性以及我们观察到光导蛋白敲除小鼠的视杆细胞积累的转导蛋白数量显着减少。在目标 1 和 2 中，我们将阐明磷导蛋白在转导蛋白转录和蛋白水解控制中的作用。为此，我们将比较转导蛋白 β mRNA 水平，以及转导蛋白 β 蛋白在磷酸蛋白敲除小鼠和野生型小鼠视网膜中的半衰期值。目标 3 将致力于光依赖性磷酸化的研究，其中我们将确定视杆细胞内磷酸化蛋白磷酸化状态的细胞内定位，使用磷酸化蛋白磷酸化突变体的转基因表达来解决这种调节的体内功能，并使用视网膜显微解剖和免疫沉淀来鉴定各种视杆细胞亚细胞区室中磷酸化蛋白的蛋白质伴侣 与质谱蛋白质鉴定相结合。 拟议的研究旨在阐明磷导蛋白在光感受器转导蛋白稳态调节中的作用，这对于我们理解光感受器变性的分子机制和制定对抗视网膜变性的策略非常重要。我们预计我们的结果也会对我们理解 G 蛋白介导的信号传导的细胞调节原理产生重大影响。