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H. Song, M. Belcastro, A. Jivotovskaya, M. Sokolov; Regulation of Transducin Homeostasis by Phosducin in Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1675.
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© ARVO (1962-2015); The Authors (2016-present)
Homeostasis of heterotrimeric G protein, transducin, is essential for normal function and even survival of rod photoreceptor cells. We previously demonstrated that the deletion of phosducin (Pdc), a major protein partner of free transducin βγ subunits in photoreceptors, caused significant down-regulation of transducin βγ expression. Intriguingly, expression of Pdc itself was reported to be modulated by transducin α subunit. We have explored molecular mechanisms of this mutual co-regulation of Pdc and transducin expression.
Expression levels of transducin α, β and γ subunits and Pdc were compared in the retinas of normal, Pdc knockout, and transducin α knockout mice, using quantitative RT-PCR and Western blotting. The rates of protein turnover were determined using metabolic labeling and pulse-chase assay. Similar analyses of transient co-expression of these proteins were conducted in cultured HEK 293 cells.
We confirmed that the knockout of Pdc reduced protein content of transducin βγ subunits in the retina by up to two fold, while the knockout of transducin α increased protein levels of Pdc by two fold. However no significant differences in the Ct values of transducin β, γ and Pdc were observed, indicating no change in the corresponding mRNA levels due to the Pdc or transducin α gene deletion. In the cell-culture, co-transfection of Pdc and transducin β subunit was required for the overexpression of transducin β, while the overexpression of transducin α appeared to be Pdc-independent. The studies of transducin βγ folding and assembly will be presented.
Our data provide evidence that Pdc and two functional parts of transducin, α and βγ subunits, co-regulate each other’s protein levels. This regulation occurs on a post-translational, rather than a transcriptional level, and is based on a mutual stabilization of transducin βγ and Pdc. The model describing mechanisms of mutual co-regulation of transducin and Pdc expression will be presented.
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