Rhodopsin Kinase Research Articles

Modulating signalling lifetime to optimise a prototypical animal opsin for optogenetic applications

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.

Rhodopsin, light-sensor of vision.

The light sensor of vertebrate scotopic (low-light) vision, rhodopsin, is a G-protein-coupled receptor comprising a polypeptide chain with bound chromophore, 11-cis-retinal, that exhibits remarkable physicochemical properties. This photopigment is extremely stable in the dark, yet its chromophore isomerises upon photon absorption with 70% efficiency, enabling the activation of its G-protein, transducin, with high efficiency. Rhodopsin's photochemical and biochemical activities occur over very different time-scales: the energy of retinaldehyde's excited state is stored in <1 ps in retinal-protein interactions, but it takes milliseconds for the catalytically active state to form, and many tens of minutes for the resting state to be restored. In this review, we describe the properties of rhodopsin and its role in rod phototransduction. We first introduce rhodopsin's gross structural features, its evolution, and the basic mechanisms of its activation. We then discuss light absorption and spectral sensitivity, photoreceptor electrical responses that result from the activity of individual rhodopsin molecules, and recovery of rhodopsin and the visual system from intense bleaching exposures. We then provide a detailed examination of rhodopsin's molecular structure and function, first in its dark state, and then in the active Meta states that govern its interactions with transducin, rhodopsin kinase and arrestin. While it is clear that rhodopsin's molecular properties are exquisitely honed for phototransduction, from starlight to dawn/dusk intensity levels, our understanding of how its molecular interactions determine the properties of scotopic vision remains incomplete. We describe potential future directions of research, and outline several major problems that remain to be solved.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

Genetic analysis and clinical features of three Chinese patients with Oguchi disease.

Oguchi disease is a rare autosomal recessive form of congenital stationary night blindness caused by disease-causing variants in the rhodopsin kinase gene (GRK1) or the arrestin gene (SAG). Our study aims to describe the clinical features and identify the genetic defects for three Chinese patients with Oguchi disease. We conducted detailed ophthalmologic examinations for three patients from three unrelated non-consanguineous Chinese families. Targeted next-generation sequencing (targeted NGS) and copy number variations (CNVs) analysis were applied to screen pathogenic variants. Sanger sequencing validation, quantitative real-time PCR (qPCR), and segregation analysis were further performed for confirmation. Subsequently, a combined genetic and structural biology approach was used to infer the likely functional consequences of novel variants. All three patients presented with typical clinical features of Oguchi disease, including night blindness, characteristic fundus appearance (Mizuo-Nakamura phenomenon), attenuated rod responses, and negative ERG waveforms. Their visual acuity and visual field were normal. Genetic analysis revealed two pathogenic variants in SAG and four pathogenic variants in GRK1. Patient 1 was identified to harbor compound heterozygous SAG variants c.874C > T (p.R292*) and exon2 deletion. Compound heterozygous GRK1 variants c.55C > T (p.R19*) and c.1412delC (p.P471Lfs*52) were found in patient 2. In patient 3, compound heterozygous GRK1 variants c.946C > A (p.R316S) and c.1388T > C (p. L463P) were detected. We reported the first two Chinese Oguchi patients with novel GRK1 pathogenic variants (P471Lfs*52, R316S, L463P) and one Oguchi case with SAG, indicating both GRK1 and SAG are important causative genes in Chinese Oguchi patients.

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin.

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.

Non-Invasive Diagnostics of Renal Cell Carcinoma Using Ultrasensitive Immunodetection of Cancer-Retina Antigens.

Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.

Dydrogesterone and levonorgestrel at environmentally relevant concentrations have antagonist effects with rhythmic oscillation in brain and eyes of zebrafish.

Synthetic progestins levonorgestrel (LNG) and dydrogesterone (DDG) are frequency detected in surface water. Combined effects of LNG and DDG on gonad differentiation are similar to LNG single exposure in juvenile zebrafish. However, LNG and DDG mixtures have stronger effects on spermatogenesis in testes of adult zebrafish, which show variable at different life stage. Effects of LNG and DDG mixtures on eyes and brain remain unknown. Here we investigated effects of LNG, DDG and their mixtures on eyes and brain. Zebrafish were exposed to LNG, DDG and their mixtures from 2 hpf to 144 dpf. Rhythm and vision related biological processes were enriched in eyes and brain in LNG and DDG treatments, which indicated rhythmic oscillation in eyes and brain. The qPCR data revealed that both LNG and DDG decreased transcription of arntl2 and clocka, while increased transcription of per1a, per1b, rpe65a and tefa in eyes and brain. However, DDG and LNG mixtures had slight effect on transcription of genes related to rhythm and vision. In addition, LNG and DDG reduced the thickness of inner nuclear layer in the eyes. Bliss independent model revealed that LNG and DDG had antagonist effects on transcription and histology in eyes and brain. Moreover, LNG and DDG formed the same hydrogen bonds with green-sensitive opsin-4 and rhodopsin kinase GRK7a. Taken together, LNG and DDG competed with each other for the same binding residues resulting in antagonist effect in their mixtures treatments, and have significant ecological implications to assess combined effects of progestins mixtures on fish in different organs.

Tropism of AAV Vectors in Photoreceptor-Like Cells of Human iPSC-Derived Retinal Organoids.

PurposeTo expand the use of human retinal organoids from induced pluripotent stem cells (iPSCs) as an in vitro model of the retina for assessing gene therapy treatments, it is essential to establish efficient transduction. To date, targeted transduction of the photoreceptor-like cells of retinal organoids with adeno-associated virus (AAV) vectors has had varied degrees of success, which we have looked to improve in this study.MethodsRetinal organoids were differentiated from iPSCs of healthy donors and transduced with reporter AAV containing a CAG.GFP, CAG.RFP, GRK1.GFP, or EFS.GFP transgene. Capsid variants assessed were AAV5, AAV2 7m8, AAV2 quad mutant, AAV2 Y444F, and AAV8 Y733F. At 27 days post-transduction, retinal organoids were assessed for reporter expression and viability.ResultsThe short intron-less elongation factor 1 alpha (EFS) promoter provided minimal reporter expression, whereas vectors containing the CAG promoter enabled transduction in 1% to 37% of cells depending on the AAV serotype; the AAV2 quad mutant (average 19.4%) and AAV2 7m8 (16.4%) outperformed AAV5 (12%) and AAV8 Y733F (2.1%). Reporter expression from rhodopsin kinase (GRK1) promoter transgenes occurred in ∼5% of cells regardless of the serotype. Positive co-localization with recoverin-expressing cells was achieved from all GRK1 vectors and the CAG AAV2 quad mutant variant. Treatment with the AAV vectors did not influence retinal organoid viability.ConclusionsReliable transduction of the photoreceptor-like cells of retinal organoids can be readily achieved. When using a CAG-driven transgene, transduction of a broad range of cell types is observed, and GRK1 transgenes provide a more restricted expression profile locating to the outer layer of photoreceptor-like cells of retinal organoids.Translational RelevanceThis study expands the AAV capsid and transgene options for preclinical testing of gene therapy in iPSC-derived human retinal organoids.

Farnesylation and lipid unsaturation are critical for the membrane binding of the C-terminal segment of G-Protein Receptor Kinase 1

Many proteins are modified by the covalent addition of different types of lipids, such as myristoylation, palmitoylation and prenylation. Lipidation is expected to promote membrane association of proteins. Visual phototransduction involves many lipid-modified proteins. The G-Protein-coupled receptor of rod photoreceptors, rhodopsin, is inactivated by G-Protein-coupled Receptor Kinase 1 (GRK1). The C-terminus of GRK1 is farnesylated and its truncation has been shown to result in a very high decrease of its enzymatic activity, most likely because of the loss of its membrane localization. Little information is available on the membrane binding of GRK1 as well as of most prenylated proteins. Measurements of the membrane binding of the non-farnesylated and farnesylated C-terminal segment of GRK1 were thus performed using lipids typical of those found in rod outer segment disk membranes. Their random coil secondary structure was determined using circular dichroism and infrared spectroscopy. The non-farnesylated C-terminal segment of GRK1 has no surface activity. In contrast, the farnesylated C-terminal segment of GRK1 shows a particularly strong binding to lipid monolayers bearing at least one unsaturated fatty acyl chain. No binding is observed in the presence of monolayers of saturated phospholipids, in agreement with the low affinity of farnesylated Ras proteins for lipids in the liquid-ordered state. Altogether, these data demonstrate that the farnesyl group of the C-terminal segment of GRK1 is mandatory for its membrane binding, which is favored by particular lipids or lipid mixtures. This information will also be useful for the understanding of the membrane binding of other prenylated proteins.

Circadian regulation of vertebrate cone photoreceptor function.

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm-dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. Functional rhythmicity persists in continuous darkness, and it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.

Identification and Characterization of a Rhodopsin Kinase Gene in the Suckers of Octopus vulgaris: Looking around Using Arms?

Simple SummaryOctopus arms are a fascinating and evolutionarily unique sensory organ, with hundreds of motile suckers, each with thousands of sensory cells, lining eight highly flexible arms. Scientifically, there are many open questions regarding the sensory capabilities of the arms and specifically the highly innervated suckers. In our present work, we used a multidisciplinary approach to fully characterize the light-sensing molecule, Ov-GRK1, in the suckers, skin and retina of Octopus vulgaris. We sequenced the O. vulgaris GRK1 gene, defining a phylogenetic tree and performing a 3D structure model prediction. We found differences in the relative expression of mRNA in different sucker types at several locations along the arm, which might indicate a functional difference. Using labeling methods, we localized the expression to the highly sensitive sucker rim. Our findings indicate that octopus suckers, in specific areas of the arm, might have the ability for light sensing. We therefore suggest that suckers are tactile, chemical and light sensors.In their foraging behavior octopuses rely on arm search movements outside the visual field of the eyes. In these movements the environment is explored primarily by the suckers that line the entire length of the octopus arm. In this study, for the first time, we report the complete characterization of a light-sensing molecule, Ov-GRK1, in the suckers, skin and retina of Octopus vulgaris. We sequenced the O. vulgaris GRK1 gene, defining a phylogenetic tree and performing a 3D structure model prediction. Furthermore, we found differences in relative mRNA expression in different sucker types at several arm levels, and localized it through in situ hybridization. Our findings suggest that the suckers in octopus arms are much more multimodal than was previously shown, adding the potential for light sensing to the already known mechanical and chemical sensing abilities.

Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1.

G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization1. Although it is unclear how GRKs recognize these receptors2-4, a conserved region at the GRK N terminus is essential for this process5-8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.

Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics

Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.

Abstract P-17: Photophysical Properties of Freely Diffusing and Immobilized Fluorescent Conjugate Based on Calcium-Binding Protein Recoverin and Alexa647 Dye

Background: Recoverin is a calcium sensor membrane-associated protein that inhibits rhodopsin kinase thereby participating in the regulation of visual transduction. Here we examined calcium-induced conformational changes in recoverin conjugated with fluorescent dye Alexa647. Methods: Photophysical properties of immobilized and freely diffusing recoverin were investigated using fluorescence lifetime imaging microscopy and fluorescence emission spectroscopy. In solution, the formation and dissociation of the Ca2+-recoverin complex manifested as changes in Alexa647 spectra and the lifetime. In contrast, immobilization of recoverin on the microscopy glass via biotin–NeutrAvidin–biotinylated polyethylene glycol (PEG) tether inhibited changes in fluorescent signal. That can be provided by PEG as it prevented the calcium-induced changes in spectrum and lifetime of recoverin-bound Alexa647 in solution. The use of another immobilization facilitator, bovine serum albumin (BSA), did not affect calcium-induced changes in fluorescence of the conjugate in solution but produced the matrix, which was ineffective in recoverin immobilization. Results: Microscale thermophoresis demonstrated that biotinylated recoverin interacted with NeutrAvidin in solution indicating that immobilization affinity depended mainly on the geometry of the glass coating surface. Conclusion: Our results highlight the challenge of specific protein immobilization that does not affect protein functionality. By the example of recoverin, we showed that the employment of two common immobilization facilitators, PEG and BSA, yielded surfaces with different space geometry, which differently affect NeutrAvidin-based immobilization affinity as well as Ca2+-dependent conformational changes of the biotinylated protein.

Structure of the rhodopsin–rhodopsin kinase complex defines the rules of engagement between G protein‐coupled receptors (GPCRs) and GPCR kinases

Structure of the rhodopsin–rhodopsin kinase complex defines the rules of engagement between G protein‐coupled receptors (GPCRs) and GPCR kinases

Functional identification of an opsin kinase underlying inactivation of the pineal bistable opsin parapinopsin in zebrafish

In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency.

Label-free Quantification of Direct Protein-protein Interactions with Backscattering Interferometry.

The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.