Abstract
Purpose: :
Phototransduction signaling is a prototypical G-protein coupled receptor signaling pathway. However, accumulating evidence suggests that G-protein coupled receptors can activate more than one signaling pathway. To identify alternative, non-canonical rhodopsin light-activated pathways, we screened for light-induced phosphorylative changes in retinas from transgenic mice deficient for transducin function. This permits the identification of non-transducin mediated signaling pathways.
Methods: :
We performed Western blots, immunohistochemistry, immunoprecipitation (IP), silver staining and mass spectrometry (MS) to characterize differentially phosphorylated proteins in dark- and light-adapted retinas.
Results: :
We have demonstrated light-induced protein kinase D phosphorylation (pPKD) by several different methods. Immunohistochemical methods showed pPKD localization in rod outer segments (ROS) of light-adapted, but not dark-adapted retinas. Western blot analysis confirmed this observation. PKD phosphorylation was specifically mediated through activation of rhodopsin since transgenic Rpe65 mutant mice with light insensitive opsin did not reveal pPKD in ROS. PKD phosphorylation in ROS was rapid, occurring in under five minutes. Dephosphorylation upon return of animals to darkness was also rapid, occurring in less than five minutes. We have identified several possible pPKD binding partners that have yet to be confirmed by additional biochemical methods.
Conclusions: :
These results provide indirect evidence for an alternative rhodopsin-mediated signaling pathway involving PKD that does not require transducin activity.
Keywords: signal transduction • phosphorylation • photoreceptors