REGULATION OF INTRACELLULAR TRANSDUCIN HOMEOSTASIS BY PHOSDUCIN IN VERTEBRATE RO

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。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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捕获光并将其转化为神经信号。个别杆保持高，不变的量的异源三聚体G蛋白转导最佳的视觉刺激的检测，在他们的生活。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，导致视网膜变性和视力丧失。杆转导蛋白稳态的机制是本研究的重点。 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。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由G蛋白介导的信号传导的调节因子--这一假设是基于我们的观察，即杆的β球蛋白基因敲除小鼠积累显着减少量的transformin，连同β球蛋白基因的体外特性，如其与转录因子Crx的相互作用，调节表达的几个感光基因，包括transformin，和β球蛋白？的能力，以保护transformin β-γ亚基从蛋白酶体辅助的蛋白水解。 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半衰期值在视网膜中的β-葡糖蛋白敲除小鼠和野生型小鼠。目的3将致力于研究光依赖性的p53-ducin磷酸化，使用多层次的实验方法，包括p53-ducin磷酸化状态的细胞内定位在杆，p53-ducin磷酸化突变体的转基因表达p53-ducin敲除背景下，和鉴定p53-ducin的蛋白伴侣在杆的主要亚细胞区室的制剂，使用免疫沉淀结合质谱蛋白鉴定。 本研究旨在阐明光感受器转导蛋白在调节光感受器内转导蛋白稳态中的作用，这对于我们理解光感受器变性的分子机制和开发对抗视网膜变性的策略具有重要意义。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 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.