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

Abstract

phosphodiesterase 6; PP2A, protein phosphatase 2A; pGRK1, GRK1 phosphorylated on Serine 21; RPE, retinal pigment epithelium; SEM, standard error of the mean. AbstractTimely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors. K E Y W O R D ScAMP, photoreceptor, phototransduction, protein phosphorylation, vision 2678 |

Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors and mediates cone photoresponse recovery to elevated cAMP

“…These observations are consistent with our previous report showing that mice with a mutation in Grk1 that converts the phosphorylation site, Ser21, to alanine have delayed dark adaptation ( 30 ) in rods but not cones and led us to examine the phosphorylation of Grk1 in the ‘all-cone’ retina of Nrl−/− mice. Mice deficient for the Nrl transcription factor possess retinas lacking rods and instead consist mostly of modified S-cones ( 37 ).…”

“…Interestingly, while PKA⍺ phosphorylated both GRK1 and GRK7 in vitro , our observations of Grk1 phosphorylation in retinas of grk1a−/− and grk1b−/− zebrafish as well as in the ‘all cone’ retina of the Nrl−/− mouse strongly suggest that Grk1 in vertebrate cones is not phosphorylated in a cAMP-dependent manner. This observation agrees with both our previous report of GRK1-S21A mice displaying a significant delay in dark adaptation in rods but not in cones ( 30 ), as well the results of this report showing that Grk7a but not Grk1b modulates recovery of the cone photoresponse in forskolin-treated zebrafish larvae.…”

“…We previously reported that in zebrafish, which possess a retinal GRK expression pattern similar to humans, both Grk1b and Grk7a contribute to recovery of the cone photoresponse, confirming the participation of both kinases in the return of cone photoreceptors to the dark-adapted state ( 29 ). We also observed that dark adaptation is significantly decreased in vivo in the rods of mice expressing GRK1 with the mutation Ser21Ala, which blocks phosphorylation ( 30 ). This suggests a role for cAMP-dependent phosphorylation of GRK1 in modulating the lifetime of activated rhodopsin.…”

Rhodopsin-mediated light-off-induced protein kinase A activation in mouse rod photoreceptor cells

“…Taken together, these findings suggest that PKA promotes the dark adaptation of rods. In support of this notion, recent studies by Kolesnikov and colleagues reported a delayed rod dark adaptation in mutant mice whose GRK1 is mutated to block its cAMP-dependent phosphorylation (65). PKA phosphorylates GRK1 to reduce its rhodopsin phosphorylation ability (19), which, in turn, extends the active lifetime of rhodopsin to increase the photosensitivity.…”

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

“…We previously reported that both Grk1b and Grk7a contribute to recovery of the cone photoresponse in zebrafish, which possess a retinal GRK expression pattern similar to humans, and confirm the participation of both kinases in the return of cone photoreceptors to the dark-adapted state (27). We recently reported that dark adaptation was significantly decreased in vivo in the rods of mice expressing GRK1 with the mutation Ser21Ala, which blocks phosphorylation (28). This suggests a role for cAMP-dependent phosphorylation of GRK1 in modulating the lifetime of activated rhodopsin.…”