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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。视网膜感受器，棒和锥体是利用G蛋白介导的信号传导的高度专业的感觉神经元来捕获光并将其转换为神经信号。单个杆在其一生中保持高量的异三聚体G蛋白透明蛋白可检测视觉刺激。重要的是，细胞内量的急剧变化通常会使光感受器处于消除路径，从而导致视网膜变性和视力丧失。杆状稳态稳态的基础机制是该研究建议的重点。 我们实验室的长期目标是探索G蛋白介导的信号传导在视网膜感受器功能中的作用。该应用的目的是阐明杆中跨丁质稳态的基础机制。该应用的中心假设是，杆中的透明蛋白积累受到G蛋白介导的信号传导的调节剂的调节，该信号通过光依赖性磷酸化的循环经历。这一假设是基于我们的观察结果，即磷酸蛋白敲除小鼠的棒可显着降低过ducin素的量，以及磷酸的体外特性，例如它与转录因子CRX的相互作用，这些特性调节了几种光感受器基因的表达，包括多核基因和phosduciss beTamem？蛋白水解。 在AIMS 1和2中，我们将阐明磷酸素在转杜蛋白转录和蛋白水解对照中的作用。为此，我们将比较透明蛋白β？ mRNA水平以及phosducin敲除和野生型小鼠视网膜中跨蛋白β半衰期的值。 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免疫沉淀耦合到质谱蛋白鉴定。 拟议的研究设置为阐明硫糖素在光感受器中跨核稳态调节中的作用，这对于我们理解光感受器变性的分子机制以及制定抵抗视网膜变性的策略很重要。我们期望我们的结果还将对我们对G蛋白介导的信号传导细胞调节原理的理解产生重大影响。