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H. Song, M. Belcastro, A. Jivotovskaya, M. Sokolov; Phosducin Regulates Trafficking of Transducin in Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5445.
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Phosducin (Pdc) is a regulator of heterotrimeric G proteins that forms a complex with the Gβγ dimer and controls its association with the G subunit. Since the association of the G and Gβγ subunits of rod transducin is required for their translocation to the outer segment membranes, we hypothesized that Pdc regulates this process. We tested this hypothesis by comparing the in vivo rates of transducin trafficking to the rod outer segment in transgenic mice expressing normal Pdc, Pdc lacking two phosphorylation sites that control its interaction with Gβγ, and Pdc knockout mice.
Pdc cDNA was cloned from the mouse retina and FLAG-tagged. The double phosphorylation mutant Pdc was created by site-directed mutagenesis of this Pdc cDNA so that it would code for Ala instead of Ser at positions 54 and 71. Normal and mutant Pdc were expressed in mice under the control of rhodopsin promoter. Lines with the highest levels of transgene expression were back-crossed into a Pdc-null background. Photoreceptor viability and function were assessed using light microscopy and ERG, and protein levels in the retina were quantified using Western blotting. To study the translocation of signaling proteins in rods, mice were subjected to controlled illumination, sacrificed, and localization of photoreceptor proteins was determined using both Western blot analysis of serial tangential sections of the retina and immunofluorescence.
We have generated several transgenic lines expressing normal and double phosphorylation mutant FLAG-tagged Pdc at high levels. Analysis of their retinas revealed that this genetic manipulation exerted no detrimental effects on photoreceptor function and viability. Furthermore, transgenic Pdc appeared to be functional, as it restored normal levels of the transducin β subunit, otherwise significantly reduced in Pdc-null rods. Consistent with previous observations, we were able to detect robust light-driven translocation of transducin and arrestin between the outer segments and the rest of the rods. However, only translocation of transducin was found to be Pdc-dependent. The quantitative analysis of our findings will be presented.
Our data indicate that Pdc plays an important role in regulating trafficking of transducin in rod photoreceptors.
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