Gene augmentation for autosomal dominant retinitis pigmentosa using rhodopsin genomic loci nanoparticles in the P23H+/- knock-in murine model.

Gene therapy for autosomal dominant retinitis pigmentosa (adRP) is challenged by the dominant inheritance of the mutant genes, which would seemingly require a combination of mutant suppression and wild-type replacement of the appropriate gene. We explore the possibility that delivery of a nanoparticle (NP)-mediated full-length mouse genomic rhodopsin (gRho) or human genomic rhodopsin (gRHO) locus can overcome the dominant negative effects of the mutant rhodopsin in the clinically relevant P23H+/--knock-in heterozygous mouse model. Our results demonstrate that mice in both gRho and gRHO NP-treated groups exhibit significant structural and functional recovery of the rod photoreceptors, which lasted for 3 months post-injection, indicating a promising reduction in photoreceptor degeneration. We performed miRNA transcriptome analysis using next generation sequencing and detected differentially expressed miRNAs as a first step towards identifying miRNAs that could potentially be used as rhodopsin gene expression enhancers or suppressors for sustained photoreceptor rescue. Our results indicate that delivering an intact genomic locus as a transgene has a greater chance of success compared to the use of the cDNA for treatment of this model of adRP, emphasizing the importance of gene augmentation using a gDNA that includes regulatory elements.

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

In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous in vitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. In vivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones in vivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.

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.

Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa.

The use of gene therapy may allow replacement of the defective gene. Minigenes, such as cDNAs, are often used. However, these may not express normal physiological genetic profiles due to lack of crucial endogenous regulatory elements. We constructed DNA nanoparticles (NPs) that contain either the mouse or human full-length rhodopsin genomic locus, including endogenous promoters, all introns, and flanking regulatory sequences of the 15-16 kb genomic rhodopsin DNA inserts. We transduced the NPs into primary retinal cell cultures from the rhodopsin knockout (RKO) mouse in vitro and into the RKO mouse in vivo and compared the effects on different functions to plasmid cDNA NP counterparts that were driven by ubiquitous promoters. Our results demonstrate that genomic DNA vectors resulted in long-term high levels of physiological transgene expression over a period of 5 months. In contrast, the cDNA counterparts exhibited low levels of expression with sensitivity to the endoplasmic reticulum (ER) stress mechanism using the same transgene copy number both in vitro and in vivo. This study demonstrates for the first time the transducing of the rhodopsin genomic locus using compacted DNA NPs.

Low availability of choline in utero disrupts development and function of the retina.

Adequate supply of choline, an essential nutrient, is necessary to support proper brain development. Whether prenatal choline availability plays a role in development of the visual system is currently unknown. In this study, we addressed the role of in utero choline supply for the development and later function of the retina in a mouse model. We lowered choline availability in the maternal diet during pregnancy and assessed proliferative and differentiation properties of retinal progenitor cells (RPCs) in the developing prenatal retina, as well as visual function in adult offspring. We report that low choline availability during retinogenesis leads to persistent retinal cytoarchitectural defects, ranging from focal lesions with displacement of retinal neurons into subretinal space to severe hypocellularity and ultrastructural defects in photoreceptor organization. We further show that low choline availability impairs timely differentiation of retinal neuronal cells, such that the densities of early-born retinal ganglion cells, amacrine and horizontal cells, as well as cone photoreceptor precursors, are reduced in low choline embryonic d 17.5 retinas. Maintenance of higher proportions of RPCs that fail to exit the cell cycle underlies aberrant neuronal differentiation in low choline embryos. Increased RPC cell cycle length, and associated reduction in neurofibromin 2/Merlin protein, an upstream regulator of the Hippo signaling pathway, at least in part, explain aberrant neurogenesis in low choline retinas. Furthermore, we find that animals exposed to low choline diet in utero exhibit a significant degree of intraindividual variation in vision, characterized by marked functional discrepancy between the 2 eyes in individual animals. Together, our findings demonstrate, for the first time, that choline availability plays an essential role in the regulation of temporal progression of retinogenesis and provide evidence for the importance of adequate supply of choline for proper development of the visual system.-Trujillo-Gonzalez, I., Friday, W. B., Munson, C. A., Bachleda, A., Weiss, E. R., Alam, N. M., Sha, W., Zeisel, S. H., Surzenko, N. Low availability of choline in utero disrupts development and function of the retina.

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.

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.

Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model. We have shown improved gene expression and fewer epigenetic modifications compared with the use of a Rho cDNA expression construct. In the current study, we altered our strategy by delivering a codon-optimized genomic form of Rho (co-sgRho) (for gene replacement) in combination with an RNAi-based inactivation of endogenous Rho alleles (gene suppression of both mutant Rho alleles, but mismatched with the co-sgRho) into a homozygous RhoP23H/P23H knock-in (KI) RP mouse model, which has a severe phenotype of adRP. In addition, we have conjugated a cell penetrating TAT peptide sequence to our previously established CK30PEG10 diblock co-polymer. The DNAs were compacted with CK30PEG10-TAT diblock co-polymer to form DNA nanoparticles (NPs). These NPs were injected into the sub-retinal space of the KI mouse eyes. As a proof of concept, we demonstrated the efficiency of this strategy in the partial improvement of visual function in the RhoP23H/P23H KI 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.

AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress.

NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.

Sox2-Deficient Müller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina.

PURPOSE: Müller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Müller glia has yet to be determined. METHODS: We use inducible, MG-specific genetic ablation of Sox2 in vivo at the peak of MG genesis to analyze its function in the maturation of murine MG and effects on other cells in the retina. Histologic and functional analysis of the Sox2-deficient retinas is conducted at key points in postnatal development. RESULTS: Ablation of Sox2 in the postnatal retina results in disorganization of MG processes in the inner plexiform layer and mislocalized cell bodies in the nuclear layers. This disorganization is concurrent with a thinning of the neural retina and disruption of neuronal processes in the inner and outer plexiform layers. Functional analysis by electroretinography reveals a decrease in the b-wave amplitude. Disruption of MG maturation due to Sox2 ablation therefore negatively affected the function of the retina. CONCLUSIONS: These results demonstrate a novel role for SOX2 in glial process outgrowth and adhesion, and provide new insights into the essential role Müller glia play in the development of retinal cytoarchitecture. Prior to this work, SOX2 was known to have a primary role in determining cell fate. Our experiments bypass cell fate conversion to establish a new role for SOX2 in a committed cell lineage.

Electroretinogram analysis of the visual response in zebrafish larvae.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

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.

Mechanistic basis for the failure of cone transducin to translocate: why cones are never blinded by light.

The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.

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.

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.

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.

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.

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.