Phosphorylation of G Protein-coupled Receptor Kinase 1 (GRK1) Is Regulated by Light but Independent of Phototransduction in Rod Photoreceptors

Abstract

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser 21 in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark-and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser 21 . Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin ␣-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.

Phosphorylation at Serine 21 in G protein‐coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors

“…These data were originally published in the Journal of Biological Chemistry ©The American Society for Biochemistry and Molecular Biology (Ref. 25). B, Western blot analysis of retinas from 4‐ to 5‐month‐old control and GRK1‐S21A mice.…”

“…Retinas from light exposed mice were removed from eye cups in the light using a conventional dissection microscope. Alternatively, retinas from mice maintained in the dark were removed under dim red light using an infrared dissection microscope . For homogenization, each retina was placed in 100 μL of HEPES‐Ringer buffer, followed by the addition of 150 μL of buffer consisting of 125 mM Tris‐HCl, pH 6.8, 5% SDS, 20% glycerol, and 100 mM NaF preheated to 95°C.…”

G protein-coupled receptor kinases: More than just kinases and not only for GPCRs

“…Using the Xenopus laevis retina as a model, it was shown that the level of the PKA-mediated phosphorylation of grk7 was diminished by light exposure (Osawa et al, 2008). Similarly, the level of GRK1 phosphorylation was higher in the dark-adapted mouse retina as compared to the light-adapted animals (Osawa et al, 2011). These data suggest that PKA phosphorylation of GRKs 1 and 7 serve as a feedback mechanism: dephosphorylation increases kinase activity upon light exposure when rapid opsin deactivation is required.…”

“…These data suggest that PKA phosphorylation of GRKs 1 and 7 serve as a feedback mechanism: dephosphorylation increases kinase activity upon light exposure when rapid opsin deactivation is required. Interestingly, phototransduction does not seem to be required for the light-induced dephosphorylation of GRK1, since it is preserved in mice lacking transducin α-subunit (Osawa et al, 2011). …”

RGS Protein Regulation of Phototransduction

“…68 While the stoichiometry of phosphorylation was not quantitatively measured, introducing phosphomimetic mutations into recombinant RGS9-1 reduced its GAP activity, suggesting that this may be a modulation mechanism for phototransduction recovery, akin to what was more recently shown for GRK1 by PKA. 69,70 While reversible protein phosphorylation is a common way of modulating enzyme activity in biochemistry, the extent to which it is used to modulate phototransduction was not understood to its full extent. This is another fertile area of discovery.…”