Abstract
Purpose :
In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases is essential for sustained visual function. GRK1 and GRK7 are phosphorylated in a cAMP-dependent manner in vivo in the retinas of dark-adapted vertebrates. We have shown that a phosphomimetic of GRK1 affects the rate of dark adaptation in mouse rods but not in cones, suggesting a role in the disparate dark adaptation rates in these two cell types. The present work evaluates phosphorylation of GRKs in rods and cones of mice and zebrafish.
Methods :
Phosphorylation was analyzed by western blot analysis of retinal tissue (mice and adult zebrafish) and isolated eyes (larval zebrafish) using anti-phosphoGrk antibodies. Grk1a was targeted for knockout in zebrafish using CRISPR gene editing. Recovery of the cone photoresponse in zebrafish was evaluated by ERG using a white light dual flash with increasing interstimulus intervals. To evaluate the effect of elevated cAMP on the rate of cone recovery in vivo, zebrafish larvae were incubated with forskolin.
Results :
GRK1 was not observed to be phosphorylated in dark-adapted Nrl-/- mice, which only have cones. Phosphorylation of Grk1 is detected in dark-adapted grk1b-/- zebrafish lacking the cone-specific paralog, suggesting that phosphorylated Grk1 is Grk1a in rods and that cone Grk1b is not phosphorylated. To evaluate this hypothesis, fish lacking the grk1a paralog are being generated. F0 mosaic larvae exhibit significant deletion of Grk1a and an absence of Grk1 phosphorylation in the dark. Wildtype larvae treated with forskolin display a significant decrease in the rate of cone recovery compared to vehicle. This decrease is also observed in grk1b-/- larvae, which express only grk7a in cones. In contrast, forskolin has no effect on the rate of cone recovery in grk7a-/- larvae, consistent with our observation that only Grk7 is phosphorylated in zebrafish cones.
Conclusions :
These data are consistent with our report of a GRK1 phosphomimetic in mice suggesting a role for GRK1 phosphorylation in rod adaptation. Grk7, but not Grk1, phosphorylation seems to be important in cone adaptation in zebrafish. Further experiments using complete Grk1a knockouts and dark adaptation analyses will be used to determine the roles of GRK phosphorylation in vertebrates that, like humans, express both GRKs in cones.
This is a 2021 ARVO Annual Meeting abstract.