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 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.