REGULATION OF INTRACELLULAR TRANSDUCIN HOMEOSTASIS BY PHOSDUCIN IN VERTEBRATE RO

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 视网膜光感受器（视杆细胞和视锥细胞）是高度特化的感觉神经元，其利用G蛋白介导的信号传导来获取视觉信息。视杆细胞在其整个生命过程中维持高水平的、恒定量的异源三聚体G蛋白转导素，这是检测视觉刺激所必需的。重要的是，细胞内transducin数量的急剧变化往往使光感受器处于消除的路径上，这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蛋白介导的信号在视网膜感光功能中的作用。本申请的目的是阐明杆中转导蛋白稳态的分子机制。本申请的中心假设是，杆中的转导蛋白积累受G蛋白介导的信号传导的调节因子--这一假设是基于所报道的转导蛋白的体外性质和我们的观察结果，即转导蛋白敲除小鼠的视杆细胞积累的转导蛋白的量显著减少。在目的1和2中，我们将阐明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蛋白在视网膜中的半衰期的值的敲除和野生型小鼠。目标3将致力于光依赖性的视杆细胞蛋白磷酸化的研究，其中我们将确定视杆细胞内视杆细胞蛋白磷酸化状态的细胞内定位，使用视杆细胞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，并使用视网膜微生物技术鉴定各种视杆细胞亚细胞区室中光敏导管蛋白的蛋白伴侣解剖和免疫沉淀结合质谱蛋白质鉴定。 这些研究旨在阐明Ducin在调节光感受器中转导素稳态中的作用，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。我们希望我们的研究结果也将对我们理解G蛋白介导的信号传导的细胞调节原理产生重大影响。

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.