Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: photoreceptors • protein structure/function • signal transduction