Phototransduction in vertebrate rods and cones consists of a series of precisely timed events that are necessary for photoreceptors to function in an environment of continually changing light conditions. We have determined that the retina-specific G protein-coupled receptor kinases, GRK1 and GRK7, which play critical roles in recovery and adaptation in rods and cones, are both substrates for cAMP-dependent protein kinase (PKA) in vitro and in vivo. Phosphorylation by PKA reduces the ability of these kinases to phosphorylate their substrates, the opsins, in vitro. Human cones express both GRK1 and GRK7, unlike mice, which lack the gene for Grk7 and express only Grk1 in cones. Both kinases have been implicated in human retinopathies, such as Oguchi disease, a stationary night blindness syndrome, and Enhanced S Cone Syndrome. Therefore, understanding their regulation in vivo will contribute to the understanding of pathological conditions in the retina. Since mice are not an appropriate model for studying the role of these two kinases in regards to human vision, we propose to use zebrafish larvae as a model to define the functional consequences of Grk1 and Grk7 phosphorylation by PKA. The zebrafish retina is functionally an 'all cone'retina at 4-7 days post fertilization (dpf) and expresses both Grk1 and Grk7 in cones. To evaluate the influence of phosphorylation by PKA, transgenic fish will be generated expressing mutants in which the phosphorylation sites have been eliminated (Ser to Ala) and mutants in which phosphorylation is mimicked by a negatively charged amino acid (Ser to Glu). The wild-type proteins will be suppressed by morpholinos in these fish and the effects of the mutants measured by electroretinogram (ERG) analysis. Our group has recently shown that levels of phosphorylation are affected in null for the adenylyl cyclase 1 (Adcy1) gene and are unaffected in transducin (Tr?) knockout mice. To evaluate the regulation of PKA-mediated phosphorylation in the cone-enriched larval zebrafish, we will examine nof zebrafish that lack cone transducin (Tc?) and wildtype zebrafish in which endogenous Adcy1 has been suppressed with morpholinos. Levels of Grk1 and Grk7 phosphorylation will be determined by immunoblot and immunocytochemical analysis, and visual function measured by ERG analysis. These studies will provide a foundation for understanding the novel role of phosphorylation by PKA on recovery and adaptation in vertebrate cones.
Cones differ from rods in the sensitivity and kinetics of the light response, as well as their susceptibility to genetically- and environmentally-induced disease processes. We have determined that GRK1 and GRK7 are phosphorylated by cAMP-dependent protein kinase. As cAMP is regulated by light in the vertebrate retina, it may regulate the light response of cones via mechanisms not previously described. It is anticipated that these studies will lead to a better understanding of cone function and advance our ability to prevent cone-related diseases.
|Chrispell, Jared D; Rebrik, Tatiana I; Weiss, Ellen R (2015) Electroretinogram analysis of the visual response in zebrafish larvae. J Vis Exp :|