Physiological role of phosducins in the retina

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DESCRIPTION (provided by applicant): Eukaryotic cells rely on G protein-mediated signaling pathways to detect and respond to a wide variety of extracellular stimuli. Based on their G protein beta/gamma subunit binding properties, phosducin (Pdc) and phosducin-like protein (PhLP) are believed to regulate G protein signaling. However, recent evidence suggests other roles as well. The long term objective of this research is to define the physiological function of Pdc and PhLP in the photoreceptor cells of the retina and other cell types. Recent studies have demonstrated light-regulated nuclear localization of Pdc in photoreceptor cells, suggesting a role for Pdc in the nucleus. In Specific Aims 1-3, potential mechanisms of regulation of nuclear localization are investigated and potential nuclear binding partners are examined. In Aim 4, the possible role of Pdc in light dependent translocation of G proteins in the photoreceptor is addressed. In Aim 5, the role of PhLP in regulating proteasome-mediated degradation of Gbeta/gamma initiated by extracellular signal-regulated kinase (ERK) activation is investigated. The methods used to achieve these specific aims are multi-disciplinary. They include immunolocalization studies in cultured cells, retinal tissues and transgenic mice using phosphorylation site-specific antibodies; functional proteomics techniques to identify nuclear binding partners; immunoblot-based assays to explore ubiquitin/proteasome-targeted degradation; siRNA-mediated gene silencing; site-directed mutagenesis to identify phosphorylation sites that trigger degradation; and signaling pathway crosstalk assays designed to assess the effect of ERK-mediated degradation of Gbeta/gamma on G protein signaling. A basic understanding of the function of Pdc and PhLP has been very elusive. The experiments proposed herein will give physiologically relevant answers concerning the role of these important proteins in cellular signaling processes.