Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes

Hiromi, Yamaoka; Tachibanaki, Shuji; Kawamura, Sadao

doi:10.1074/jbc.m115.674101

Cited by 7 publications

(7 citation statements)

References 33 publications

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“…However, cones could also potentially express a higher compensatory amount of total PP2A and/or other opsin phosphatases than rods. Notably, it has been demonstrated previously that the dephosphorylation of cone opsin in vertebrates proceeds significantly faster than that of rhodopsin . Furthermore, a very rapid thermal decay of photoactivated cone pigment would quickly reduce the efficiency of its subsequent interactions with GRK1 within the first several seconds of dark adaptation and thus minimize the overall effect of kinase activity on the following cone opsin regeneration with 11‐ cis ‐retinal.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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 |

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Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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“…However, despite the remarkable progress in understanding G-protein signaling in general and phototransduction in particular, the mammalian rod enzyme(s) responsible for catalyzing this reaction in vivo has remained unknown (16). Furthermore, although in vertebrate cones both the decay of photoactivated pigment and its subsequent regeneration with 11-cis-retinal (55,56), as well as their opsin dephosphorylation (40,57), proceed significantly faster than in rods, the identity of the cone pigment phosphatase is also unknown.…”

Section: Discussionmentioning

confidence: 99%

Dephosphorylation by protein phosphatase 2A regulates visual pigment regeneration and the dark adaptation of mammalian photoreceptors

Kolesnikov

Orban

Jin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone photoreceptors arguably represent the best-understood example of such GPCR signaling. Their visual pigments (opsins) are activated by light, transduce the signal, and are then inactivated by a GPCR kinase and arrestin. Although pigment inactivation by phosphorylation is well understood, the enzyme(s) responsible for pigment dephosphorylation and the functional significance of this reaction remain unknown. Here, we show that protein phosphatase 2A (PP2A) acts as opsin phosphatase in both rods and cones. Elimination of PP2A substantially slows pigment dephosphorylation, visual chromophore recycling, and ultimately photoreceptor dark adaptation. These findings demonstrate that visual pigment dephosphorylation regulates the dark adaptation of photoreceptors and provide insights into the role of this reaction in GPCR signaling.

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“…Note that this contamination is the percentage of the total CIS proteins, and therefore, the actual contamination of CIS proteins is dependent on the total CIS proteins and total COS proteins. Our previous estimation of the volumes of COS and CIS in carp showed that the IS volume is ~6 times higher than the OS volume [ 10 ]. Based on this volume ratio, we estimated the actual contamination of CIS proteins in COS-rich fraction.…”

Section: Resultsmentioning

confidence: 99%

“…In other words, the emPAI value of cone transducin α-subunit could be much higher when all of them were analyzed. In the above process, ROS and COS volume differences [ 10 ] were taken into account to compare the emPAI values in the equal volume. Proteins in Table 2 are listed in descending order of COS/(COS+CIS).…”

Section: Resultsmentioning

confidence: 99%

“…To evaluate the purity of membranes separated with sucrose density gradient centrifugation described above, OS- or IS-specific marker proteins were quantified in each separated membrane fraction. A marker of OS membranes, visual pigments, was quantified spectrophotometrically as described [ 4 , 9 , 10 ]. As the markers of membranes in the IS, F1 ATPase β subunit was used for mitochondrial inner membranes, TOM20 for mitochondrial outer membranes, calnexin for endoplasmic reticulum (ER) membranes, and Na + /K + ATPase α subunit for IS plasma membranes.…”

Section: Methodsmentioning

confidence: 99%

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Purification of cone outer segment for proteomic analysis on its membrane proteins in carp retina

et al. 2017

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Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.

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Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes

Cited by 7 publications

References 33 publications

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

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

Dephosphorylation by protein phosphatase 2A regulates visual pigment regeneration and the dark adaptation of mammalian photoreceptors

Purification of cone outer segment for proteomic analysis on its membrane proteins in carp retina

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