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.

Timely 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.

Broad spectrum metabolomics for detection of abnormal metabolic pathways in a mouse model for retinitis pigmentosa.

Retinitis pigmentosa (RP) is a degenerative disease of the retina that affects approximately 1 million people worldwide. There are multiple genetic causes of this disease, for which, at present, there are no effective therapeutic strategies. In the present report, we utilized broad spectrum metabolomics to identify perturbations in the metabolism of the rd10 mouse, a genetic model for RP that contains a mutation in Pde6ß. These data provide novel insights into mechanisms that are potentially critical for retinal degeneration. C57BL/6J and rd10 mice were raised in cyclic light followed by either light or dark adaptation at postnatal day (P) 18, an early stage in the degeneration process. Mice raised entirely in the dark until P18 were also evaluated. After euthanasia, retinas were removed and extracted for analysis by ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-QTOF-MS). Compared to wild type mice, rd10 mice raised in cyclic light or in complete darkness demonstrate significant alterations in retinal pyrimidine and purine nucleotide metabolism, potentially disrupting deoxynucleotide pools necessary for mitochondrial DNA replication. Other metabolites that demonstrate significant increases are the Coenzyme A intermediate, 4'-phosphopantothenate, and acylcarnitines. The changes in these metabolites, identified for the first time in a model of RP, are highly likely to disrupt normal energy metabolism. High levels of nitrosoproline were also detected in rd10 retinas relative to those from wild type mice. These results suggest that nitrosative stress may be involved in retinal degeneration in this mouse model.

Reduced phosphoCREB in Müller glia during retinal degeneration in rd10 mice.

PURPOSE: The mechanisms that trigger retinal degeneration are not well understood, despite the availability of several animal models with different mutations. In the present report, the rd10 mouse, a model for retinitis pigmentosa (RP) that contains a mutation in the gene for PDE6ß (Pde6b), is used to evaluate gliosis, as a marker for retinal stress, and cyclic AMP response element binding protein (CREB) phosphorylation, which may be important early in retinal degeneration. METHODS: Wild-type C57Bl6J and rd10 mice raised under cyclic light were examined for changes in gliosis and CREB phosphorylation for approximately 3 weeks beginning at P14 to P17 using immunocytochemistry. Mice raised under normal cyclic light and in complete darkness were also compared for changes in CREB phosphorylation. RESULTS: Gliosis in rd10 mice raised under cyclic light was apparent at P17, before extensive degeneration of the photoreceptor layer is visible, and increased over time. Phosphorylation of CREB at Ser133 (pCREB) was detected in Müller glia (MG) in the wild-type and rd10 mice. However, at all phases of photoreceptor degeneration, the pCREB levels were lower in the rd10 mice. We also observed extensive migration of MG cell bodies to the outer nuclear layer (ONL) during degeneration. In contrast to the mice raised under cyclic light, the rd10 mice raised in the dark exhibited slower rates of degeneration. When the dark-reared mice were exposed to cyclic light, the photoreceptor layer degenerated within 4 days to approximately one to two rows of nuclei. Interestingly, the pCREB levels in the MG also decreased during this 4-day cyclic light exposure compared to the levels in the rd10 mice raised continuously in the dark. CONCLUSIONS: The results of these studies suggest that photoreceptors communicate directly or indirectly with MG at early stages, inducing gliosis before extensive retinal degeneration is apparent in rd10 mice. Surprisingly, phosphorylation of CREB is downregulated in the MG. These results raise the interesting possibility that Müller glia undergo CREB-mediated transcriptional changes that influence photoreceptor degeneration either positively or negatively. Unlike canine models of RP, no increase in pCREB was observed in photoreceptor cells during this period suggesting possible mechanistic differences in the role of CREB in photoreceptors between these species.

Phosphorylation of G protein-coupled receptor kinase 1 (GRK1) is regulated by light but independent of phototransduction in rod photoreceptors.

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 α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.

Phosphorylation of GRK7 by PKA in cone photoreceptor cells is regulated by light.

The retina-specific G protein-coupled receptor kinases, GRK1 and GRK7, have been implicated in the shutoff of the photoresponse and adaptation to changing light conditions via rod and cone opsin phosphorylation. Recently, we have defined sites of phosphorylation by cAMP-dependent protein kinase (PKA) in the amino termini of both GRK1 and GRK7 in vitro. To determine the conditions under which GRK7 is phosphorylated in vivo, we have generated an antibody that recognizes GRK7 phosphorylated on Ser36, the PKA phosphorylation site. Using this phospho-specific antibody, we have shown that GRK7 is phosphorylated in vivo and is located in the cone inner and outer segments of mammalian, amphibian and fish retinas. Using Xenopus laevis as a model, GRK7 is phosphorylated under dark-adapted conditions, but becomes dephosphorylated when the animals are exposed to light. The conservation of phosphorylation at Ser36 in GRK7 in these different species (which span a 400 million-year evolutionary period), and its light-dependent regulation, indicates that phosphorylation plays an important role in the function of GRK7. Our work demonstrates for the first time that cAMP can regulate proteins involved in the photoresponse in cones and introduces a novel mode of regulation for the retinal GRKs by PKA.

Grk1b and Grk7a Both Contribute to the Recovery of the Isolated Cone Photoresponse in Larval Zebrafish.

Purpose: To define the functional roles of Grk1 and Grk7 in zebrafish cones in vivo. Methods: Genome editing was used to generate grk7a and grk1b knockout zebrafish. Electroretinogram (ERG) analyses of the isolated cone mass receptor potential and the b-wave were performed in dark-adapted zebrafish using a paired flash paradigm to determine recovery of cone photoreceptors and the inner retina after an initial flash. In addition, psychophysical visual response was measured using the optokinetic response (OKR). Results: ERG analysis demonstrated that deletion of either Grk1b or Grk7a in zebrafish larvae resulted in modestly lower rates of recovery of the isolated cone mass receptor potential from an initial flash compared to wildtype larvae. On the other hand, grk1b-/- and grk7a-/- larvae exhibited a b-wave recovery that was similar to wildtype larvae. We evaluated the OKR and found that deletion of either Grk1b or Grk7a leads to a small decrease in temporal contrast sensitivity and alterations in visual acuity. Conclusions: For the first time, we demonstrate that Grk1b and Grk7a both contribute to visual function in larval zebrafish cones. Since the difference between wildtype and each knockout fish is modest, it appears that either GRK is sufficient for adequate cone visual function.

Cone deactivation kinetics and GRK1/GRK7 expression in enhanced S cone syndrome caused by mutations in NR2E3.

PURPOSE: To determine the relationship between cone deactivation kinetics in patients with the enhanced S cone syndrome (ESCS) caused by mutations in NR2E3 and the immunoreactivity to G-protein-coupled receptor kinase 1 (GRK1) and GRK7. METHODS: Electroretinogram (ERG) photoresponses were used to investigate activation kinetics of cones with a model of cone phototransduction. Deactivation kinetics of cones after bright flashes was quantified with a paired-flash ERG paradigm. Immunocytochemistry was performed with antibodies against cone opsins and kinases GRK1 and GRK7 in postmortem normal and ESCS retinal tissue. RESULTS: Activation kinetics of long/middle-wavelength-sensitive (L/M) cone-mediated responses in patients with ESCS were similar to those of normal L/M cones. Activation kinetics of ESCS short-wavelength-sensitive (S) cones, when compared with normal L/M cone responses evoked by the same stimulus, were slower by an amount consistent with the expected differences in spectral sensitivities. After bright flashes chosen to evoke identical activation kinetics, ESCS S cones deactivated much more slowly than ESCS or normal L/M cones. Normal human retina revealed strongly labeled cone outer segments with anti-GRK1 and anti-GRK7. In an ESCS retina, outer segments positive for L/M opsin were strongly labeled with anti-GRK1, whereas outer segments positive for S opsin showed no detectable GRK1 reactivity. GRK7 labeling was absent in all photoreceptors of the ESCS retina. CONCLUSIONS: The cone-dominant human retina resulting from NR2E3 mutations affords greater understanding of the physiological roles of GRK1 and GRK7 in human cone photoreceptors. Normal deactivation kinetics in human L/M cones can occur without GRK7 when GRK1 is present in ESCS, but does not occur when GRK7 is present but GRK1 is deficient in Oguchi disease. Lack of both GRK1 and GRK7 in S cones of patients with ESCS results in a more pronounced abnormality in deactivation kinetics and suggests the existence of partial compensation by either GRK when the other is deficient.

Direct binding of visual arrestin to a rhodopsin carboxyl terminal synthetic phosphopeptide.

PURPOSE: The phosphorylated carboxyl terminus of rhodopsin is required for the stable binding of visual arrestin to the full length rhodopsin molecule. Phosphorylation of the carboxyl terminus has been shown to induce conformational changes in arrestin, which promote its binding to the cytoplasmic loops of rhodopsin. However, it has not been determined whether phosphorylation is also responsible for the direct binding of the rhodopsin carboxyl terminus to arrestin. To further investigate the role of rhodopsin phosphorylation on arrestin binding, surface plasmon resonance was used to measure the interaction between a synthetic phosphopeptide corresponding to the carboxyl terminus of rhodopsin and visual arrestin in real time. METHODS: Synthetic peptides were generated that correspond to the phosphorylated and nonphosphorylated carboxyl terminus of bovine rhodopsin. These peptides were immobilized on a biosensor chip and their interaction with purified visual arrestin was monitored by surface plasmon resonance on a BIAcore 2000 or 3000. RESULTS: A synthetic peptide phosphorylated on residues corresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovine rhodopsin was sufficient for direct binding to visual arrestin. In contrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342 and a nonphosphorylated synthetic peptide were not able to bind arrestin. A peptide fully substituted at all serine and threonine residues with glutamic acid was unable to substitute for phosphorylation. CONCLUSIONS: Surface plasmon resonance is a sensitive method for detecting small differences in affinity. We were successful in using this technique to detect differences in the affinity of phosphorylated and nonphosphorylated rhodopsin peptides for visual arrestin. The data suggest that these are low-affinity interactions and indicate that phosphorylation is responsible for the direct binding of the rhodopsin carboxyl terminus to visual arrestin. Four phosphorylated residues are sufficient for this interaction. Because the affinity of the synthetic phosphopeptide for arrestin is substantially lower than the full length rhodopsin molecule, the cytoplasmic loops and rhodopsin carboxyl terminus appear to interact in a cooperative manner to stably bind arrestin.

Mouse cone arrestin expression pattern: light induced translocation in cone photoreceptors.

PURPOSE: Arrestins are a superfamily of regulatory proteins that down-regulate activated and phosphorylated G-protein coupled receptors (GPCRs). Cone arrestin (CAR) is expressed in cone photoreceptors and pinealocytes and may contribute to the shutoff mechanisms associtated with high acuity color vision. To initiate a study of CAR's function in cone phototransduction, the mouse CAR (mCAR) transcript and protein expression patterns are examined and in vitro binding assays are also presented. METHODS: Tissue distribution of mCAR was determined by Northern and immunoblot analyses and its cellular localization identified by In situ hybridization and immunohistochemistry. The protein expression pattern of mCAR in the postnatal developmental and adult mouse retina was analyzed by immunoblotting in normal C57 and rd/rd mouse retinas. In vitro binding assays with in vitro translated arrestins were used to study the interaction of mCAR and mouse S-antigen (mSAG) with embryonic chicken outer segment (OS) membranes containing both rod and cone opsins. RESULTS: MCAR has a high level of amino acid sequence identity with orthologous sequences reported for other species except the C-terminal region, which is highly conserved between mouse and rat but divergent in other species. MCAR is expressed exclusively in the retina and the pineal gland, and unique isoforms are expressed during postnatal development of the retina and the pineal gland. The postnatal developmental expression pattern of mCAR and mSAG in the rd/rd mouse retina parallels the generation and degeneration of the cone and rod photoreceptors in these mice. In situ and immunohistochemistry both reveal cone-specific expression of mCAR in the retina. Immunofluorescent staining of retinal sections from dark-adapted or light-exposed mice suggests a light-dependent translocation of mCAR immunoreactivity from the cone inner segments (CIS) and other parts of the cell body to the cone outer segments (COS), similar to but not as dramatic as rod arrestin. In vitro binding assays show a small yet significant increase in binding of the full-length mCAR (mCARFL) to embryonic chicken OS membranes following light activation and phosphorylation of the opsins in the membranes. CONCLUSIONS: MCAR is expressed in retinal cone photoreceptors and the pineal gland. The light-dependent translocation of mCAR immunoreactivity and the increase of mCAR binding to light-activated, phosphorylated embryonic chicken OS membranes, compared to its binding to dark, unphosphorylated membranes, suggest the possibility that mCAR is involved in shutting off the phototransduction cascade in cone photoreceptors as rod arrestin does in rod photoreceptors. However, prominent differences exist between rod arrestin and CAR, suggesting other functions for CAR.

M opsin phosphorylation in intact mammalian retinas.

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.

Phosphorylation of GRK1 and GRK7 by cAMP-dependent protein kinase attenuates their enzymatic activities.

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.

The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding.

The binding of arrestin to rhodopsin is initiated by the interaction of arrestin with the phosphorylated rhodopsin C-terminus and/or the cytoplasmic loops, followed by conformational changes that expose an additional high-affinity site on arrestin. Here we use an arrestin mutant (R175E) that binds similarly to phosphorylated and unphosphorylated, wild-type rhodopsin to identify rhodopsin elements other than C-terminus important for arrestin interaction. R175E-arrestin demonstrated greatly reduced binding to unphosphorylated cytoplasmic loop mutants L72A, N73A, P142A and M143A, suggesting that these residues are crucial for high-affinity binding. Interestingly, when these rhodopsin mutants are phosphorylated, R175E-arrestin binding is less severely affected. This effect of phosphorylation on R175E-arrestin binding highlights the co-operative nature of the multi-site interaction between arrestin and the cytoplasmic loops and C-terminus of rhodopsin. However, a combination of any two mutations disrupts the ability of phosphorylation to enhance binding of R175E-arrestin. N73A, P142A and M143A exhibited accelerated rates of dissociation from wild-type arrestin. Using sensitivity to calpain II as an assay, these cytoplasmic loop mutants also demonstrated reduced ability to induce conformational changes in arrestin that correlated with their reduced ability to bind arrestin. These results suggest that arrestin bound to rhodopsin is in a distinct conformation that is co-ordinately regulated by association with the cytoplasmic loops and the C-terminus of rhodopsin.