Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors in the dark and mediates recovery of the cone photoresponse in response to forskolin

Abstract

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. In vitro analysis demonstrates that GRK1 and GRK7 are phosphorylated by PKA and that phosphorylation is associated with a decreased capacity to phosphorylate rhodopsin, while in vivo observations show that GRK phosphorylation is cAMP-dependent. In many vertebrates, including humans, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently reported that a mutation deleting the phosphorylation site, Ser21, in GRK1 is associated with a delay in dark adaptation in mouse rods but not in cones, suggesting that GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, we present immunochemical and electrophysiological studies using zebrafish as a model. Since zebrafish display a retinal GRK expression profile similar to humans, these studies will allow us to further evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery that may be relevant to human physiology. ERG analyses of wildtype and Grk-knockout zebrafish larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgenic zebrafish, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses in rod grk1a and cone grk1b knockout zebrafish, as well as the all cone Nrl-/- mouse, show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation in the context of cone adaptation and recovery.