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: 7609756
• 负责人: MAXIM SOKOLOV
• 金额: $ 27.83万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7609756
• 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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 视网膜光感受器，视杆和视锥，是高度专门化的感觉神经元，利用G蛋白介导的信号来获取视觉信息。视杆细胞在其一生中维持着检测视觉刺激所需的高量、恒定量的异三聚体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往往会使光感受器走上消除的道路，这会导致视网膜退化和视力丧失。 我们实验室的长期目标是了解G蛋白介导的信号在视网膜光感受器功能中的作用。这项应用的目的是阐明杆状体内转导蛋白稳态的分子机制。这一应用的中心假设是，转导蛋白在杆状细胞中的积累受G蛋白介导的信号调节因子--磷酸脱氢蛋白的调节，该蛋白经历一个光依赖的磷酸化周期。这一假说是基于已报道的硫代蛋白的体外性质，以及我们观察到硫代蛋白基因敲除小鼠的杆状体内积累的转导蛋白数量显著减少。在目标1和2中，我们将阐明光导蛋白在转导蛋白转录和蛋白降解调控中的作用。为此，我们将比较posducin基因敲除小鼠和野生型小鼠视网膜中转导蛋白βmRNA水平以及转导蛋白β蛋白半衰期的值。目的3将致力于光依赖的光导蛋白磷酸化的研究，其中我们将确定光导蛋白磷酸化状态在视杆中的细胞内定位，通过转基因表达光导蛋白磷酸化突变体来解决这一调控的体内功能，并使用视网膜显微解剖和免疫沉淀结合质谱学蛋白质鉴定来确定不同视杆亚细胞间隔中的光导蛋白的蛋白质伙伴。 这些研究旨在阐明光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退变的分子机制和开发对抗视网膜退行性变的策略具有重要意义。我们预计，我们的结果也将对我们理解G蛋白介导的信号的细胞调控原理产生重大影响。