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: 7381126
• 负责人: MAXIM SOKOLOV
• 金额: $ 24.89万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381126
• 关键词: REGULATION; INTRACELLULAR; TRANSDUCIN; HOMEOSTASIS; PHOSDUCIN

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 utilizing G protein mediated signaling to capture light and transduce it into a neural signal. Individual rods maintain high, invariable amounts of heterotrimeric G protein transducin optimal for the detection of visual stimuli, throughout their lives. Importantly, drastic change of transducin intracellular amounts often puts photoreceptor on the path of elimination resulting in retina degeneration and loss of vision. The mechanisms underlying rod transducin homeostasis are in the focus of this research proposal. The long-term goal of our laboratory is to explore the role of G protein mediated signaling in function of retinal photoreceptors. 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 through a cycle of light-dependent phosphorylation. This hypothesis is based on our observation that rods of the phosducin knockout mice accumulates significantly reduced amount of transducin, together with the in vitro properties of phosducin, such as its interaction with transcription factor Crx, which regulates expression of several photoreceptor genes including transducin, and phosducin?s ability to protect transducin beta-gamma subunit from the proteasome-assisted proteolysis. In Aims 1 and 2 we will elucidate the role of phosducin in transducin transcriptional and proteolytic control. For that we will compare transducin beta? mRNA levels, as well as values of transducin beta 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 using a multi-tiered experimental approach, including intracellular localization of the phosducin phospho-states within the rod, transgenic expression of phosducin phosphorylation mutant on the phosducin knockout background, and identification of protein partners of phosducin in preparations of rod major subcellular compartments using immunoprecipitation coupled to mass spectrometric protein identification. The proposed studies are set to elucidate the role of phosducin in regulation of transducin homeostasis in photoreceptors, which is important for our understanding of molecular mechanisms of photoreceptor degeneration and for developing of strategies to counteract retinal degeneration. We expect that our results will also have a significant impact on our understanding of the principals of cellular regulation of G protein mediated signaling.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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蛋白介导的信号来捕捉光线并将其转化为神经信号。在它们的一生中，单个视杆维持着高的、恒定量的异三聚体G蛋白转导蛋白，这是检测视觉刺激的最佳选择。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，导致视网膜退化和视力丧失。杆转导蛋白动态平衡的机制是这项研究计划的重点。本实验室的长期目标是探索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。这一假说是基于我们的观察结果，即光导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，以及光导蛋白的体外性质，如它与调节包括转导蛋白在内的几个光感受器基因表达的转录因子CRx的相互作用，以及光导蛋白S保护转导蛋白β-伽马亚单位免受蛋白酶体辅助蛋白降解的能力。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，我们将比较转导蛋白β？转基因小鼠和野生型小鼠视网膜中转导蛋白β半衰期的值。目的3将致力于利用多层次的实验方法研究光依赖的光敏光导蛋白磷酸化，包括光导蛋白磷酸化状态在杆状细胞内的定位，在光导蛋白敲除背景下的转基因表达，以及利用免疫沉淀和质谱蛋白质鉴定相结合的方法在杆状主要亚细胞间隔的制备中鉴定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行性变的分子机制和发展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。