REGULATION OF INTRACELLULAR TRANSDUCIN HOMEOSTASIS BY PHOSDUCIN IN VERTEBRATE RO

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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.