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The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase–activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

Missense mutations in the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) are associated with autosomal dominant cone/cone-rod (CORD) dystrophies. The nature of the inheritance pattern implies that a pool of normal GCAP proteins is present in photoreceptors together with the mutated variant. To assess whether human GCAP1 and GCAP2 may similarly regulate the activity of the retinal membrane guanylate cyclase GC-1 (GC-E) in the presence of the recently discovered E111V-GCAP1 CORD-variant, we combined biochemical and in silico assays. Surprisingly, human GCAP2 does not activate GC1 over the physiological range of Ca2+ whereas wild-type GCAP1 significantly attenuates the dysregulation of GC1 induced by E111V-GCAP1. Simulation of the phototransduction cascade in a well-characterized murine system, where GCAP2 is able to activate the GC1, suggests that both GCAPs can act in a synergic manner to mitigate the effects of the CORD-mutation. We propose the existence of a species-dependent compensatory mechanism. In murine photoreceptors, slight increases of wild-type GCAPs levels may significantly attenuate the increase in intracellular Ca2+ and cGMP induced by E111V-GCAP1 in heterozygous conditions. In humans, however, the excess of wild-type GCAP1 may only partly attenuate the mutant-induced dysregulation of cGMP signaling due to the lack of GC1-regulation by GCAP2.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase–activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone–rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone–rod dystrophy 6 pathology.

Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.

Copyright: This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca2+. To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+ concentration accelerates the flash response recovery and increases the basal PDE6 activity (βdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs-/- recoverin-/- mice. Our modeling shows that a similar elevation in βdark can fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+ in GCAPs-/- recoverin-/- rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G &gt, T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in M&uuml, ller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

Loss-of-function mutations in the retinal degeneration 3 (RD3) gene cause inherited retinopathy with impaired rod and cone function and fast retinal degeneration in patients and in the natural strain of rd3 mice. The underlying physiopathology mechanisms are not well understood. We previously proposed that guanylate cyclase-activating proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in 6 weeks in rd3 mice, it takes 8 months to halve in rd3/rd3 GCAPs−/− mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free-phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segment in their Ca2+-free, guanylate cyclase activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum (ER) stress, mitochondrial swelling, and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of Leber’s congenital amaurosis 12 (LCA12), this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision.

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone–rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3–4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5–2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude inR838S+rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+phototransduction feedback and abnormally high free-Ca2+concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice inGCAP1,2+/+orGCAP1,2+/−backgrounds, was prevented inGCAP1,2−/−mice lacking Ca2+feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a “phototransduction disease,” which means it is associated with increased free-cGMP and Ca2+levels in photoreceptors.SIGNIFICANCE STATEMENTIn a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1,GUCY2D), a mutation associated with early progressing congenital blindness, cone–rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect ofGUCY2DCORD6-linked mutation on photoreceptor physiologyin vivo. It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones, documents that photoreceptor ROS-GC1 is modulated by gaseous CO(2). Mechanistically, CO(2) is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1 and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca(2+)−modulated phototransduction, the CO(2) pathway is Ca(2+)−independent, yet is linked with it and synergizes it. It, through R(787)C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca(2+) sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

Purpose: The aim of this study was to investigate the genetic basis and pathogenic mechanism of variable maculopathies, ranging from mild photoreceptor degeneration to central areolar choroidal dystrophy, in a five-generation family. Methods: Clinical characterizations, whole-exome sequencing, and genome-wide linkage analysis were carried out on the family. Zebrafish models were used to investigate the pathogenesis of GUCA1A mutations. Results: A novel mutation, GUCA1A p.R120L, was identified in the family and predicted to alter the tertiary structure of guanylyl cyclase-activating protein 1, a photoreceptor-expressed protein encoded by the GUCA1A gene. The mutation was shown in zebrafish to cause significant disruptions in photoreceptors and retinal pigment epithelium, together with atrophies of retinal vessels and choriocapillaris. Those phenotypes could not be fully rescued by exogenous wild-type GUCA1A, suggesting a likely gain-of-function mechanism for p.R120L. GUCA1A p.D100E, another mutation previously implicated in cone dystrophy, also impaired the retinal pigment epithelium and photoreceptors in zebrafish, but probably via a dominant negative effect. Conclusion: We conclude that GUCA1A mutations could cause significant variability in maculopathies, including central areolar choroidal dystrophy, which represents a severe pattern of maculopathy. The diverse pathogenic modes of GUCA1A mutations may explain the phenotypic diversities. Genet Med advance online publication 26 January 2017

Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca(2+) concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs(−/−) hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs(−/−) photoreceptors survived past 6 months, in stark contrast to

Retinal membrane guanylyl cyclase (RetGC) in photoreceptor rod and cone cells is regulated by a family of guanylyl cyclase activating proteins (GCAP1-7). GCAP5 is expressed in zebrafish photoreceptors and promotes Ca(2+)-dependent regulation of RetGC enzymatic activity that regulates visual phototransduction. We report NMR chemical shift assignments of the Ca(2+)-free activator form of GCAP5 (BMRB no. 27705).

Signal transduction processes that are under control of changes in cytoplasmic Ca

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

Ethanol is a widely used beverage and abused drug. Alcoholism causes severe damage to human health and creates serious social problems. Understanding the mechanisms underlying ethanol actions is important for the development of effective therapies. Alcohol has a wide spectrum of effects on physiological activities and behaviours, from sensitization to sedation and even intoxication with increasing concentrations. Animals develop tolerance to ethanol. However, the underlying mechanisms are not well understood. In Caenorhabditis elegans, NPR-1 negatively regulates the development of acute tolerance to ethanol. Here, using in vivo Ca2+ imaging, behavioural tests and chemogenetic manipulation, we show that the soluble guanylate cyclase complex GCY-35/GCY-36—TAX-2/TAX-4 signalling pathway in O2 sensory neurons positively regulates acute functional tolerance in npr-1 worms.

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two non-conserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn2+ and Fe2+). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analysis that demonstrate the binding of one Fe2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe2+, suggesting that Fe2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in both the Fe2+-free and Fe2+-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming a [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Fe2+ binding to GCAP5 decreases its ability to activate photoreceptor human GC-E by decreasing GC-activity more than 10-fold. Our results indicate a strong Fe2+-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

Aim To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB. Methods Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas. Results GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice. Conclusion GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.

Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)-activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca(2+) with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca(2+). The latter mutant also shows a significantly high affinity for Mg(2+), which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca(2+), so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca(2+)-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highest-affinity Ca(2+) binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca(2+). We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca(2+) and cyclic GMP homeostasis and result in retinal degeneration.

One-fifth of all cases of Leber congenital amaurosis are type 1 (LCA1). LCA1 is a severe form of retinal dystrophy caused by loss-of-function mutations in guanylate cyclase 1 (GC1), a key member of the phototransduction cascade involved in modulating the photocurrents. Although GC1 has been studied for some time, the mechanisms responsible for its regulation and membrane targeting are not fully understood. We reported earlier that retinal degeneration 3 (RD3) protein interacts with GC1 and promotes its targeting to the photoreceptor outer segments (POS). Here, we extend our studies to show a direct association between RD3 and guanylate cyclase activating protein 1 (GCAP1). Furthermore, we demonstrate that this functional interaction is important for GC1 targeting to POS. We also show that most LCA1-causing mutations in GC1 result in lost GC1 interaction with RD3 or GC1 being targeted to the plasma membrane. Our data suggest that GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to POS. Interruption of this assembly is likely the underlying mechanism for a subset of LCA1. This study offers insights for the development of therapeutic strategies to treat this severe form of blindness. Background: Defects in the function of guanylate cyclase 1 (GC1) cause Leber congenital amaurosis (LCA) type 1. Results: GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to outer segments. Conclusion: A subset of LCA1 is caused by impaired formation of the RD3-GC1-GCAP1 complex. Significance: Understanding the molecular interaction of RD3 with GC1 and GCAP1 has potential therapeutic benefits for LCA1.

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a ‘proof of concept’ for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Zebrafish express in the retina a large variety of three different membrane-bound guanylate cyclases and six different guanylate cyclase-activating proteins (zGCAPs) belonging to the family of neuronal calcium sensor proteins. Although these proteins are predominantly localized in rod and cone photoreceptor cells of the retina, they differ in their spatial-temporal expression profiles. Further, each zGCAP has a different affinity for Ca(2+) and displays different Ca(2+)-sensitivities of guanylate cyclase activation. Thus, zGCAPs operate as cytoplasmic Ca(2+)-sensors that sense incremental changes of cytoplasmic Ca(2+)-concentration in rod and cone cells and control the activity of their target guanylate cyclases in a Ca(2+)-relay mode fashion.

SummaryRod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R∗), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from rods with and without feedback reveal that the consequences of trial-to-trial fluctuations in R∗ lifetime in normal rods are also dampened by feedback regulation of cGMP synthesis. Thus, calcium feedback trumps the mechanisms of R∗ deactivation in determining the SPR amplitude, attenuating responses arising from longer R∗ lifetimes to a greater extent than those arising from shorter ones. As a result, rod SPRs achieve a more stereotyped amplitude, a characteristic considered important for reliable transmission through the visual system.

Two calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) activate cGMP synthesis in photoreceptor by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) to expedite recovery, but calcium-insensitive constitutively active GCAP1 mutants cause photoreceptor degeneration in human patients and transgenic mice. Although GCAP1 and GCAP2 can both activate RetGC1 and RetGC2in vitro, we find that GCAP1 selectively regulates RetGC1in vivo. Furthermore, elimination of RetGC1 but not RetGC2 isozyme reverses abnormal calcium sensitivity of cGMP synthesis and rescues mouse rods in transgenic mice expressing GCAP1 mutants causing photoreceptor disease. Rods expressing mutant GCAP1 not only survive in the absence of RetGC1 but also remain functional, albeit with reduced electroretinography (ERG) amplitudes typical ofRetGC1−/−genotype. The rod ERG recovery from a strong flash, only slightly affected in bothRetGC1−/−andRetGC2−/−mice, becomes very slow inRetGC1−/−but notRetGC2−/−mice when GCAP2 is not available to provide Ca2+feedback to the remaining RetGC isozyme. The intrinsic biochemical properties of RetGC and GCAP determinedin vitrodo not explain the observed phenomena. Instead, our results argue that there must be a cellular mechanism that limits GCAP1 access to RetGC2 and makes RetGC1 isozyme a preferential target for the disease-causing GCAP1 mutants. A more general conclusion from our findings is that nondiscriminatory interactions between homologous effector enzymes and their regulatory proteins permitted by their intrinsic biochemical properties can be effectively restricted in a living photoreceptor.

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ∼2 μm.

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

RNA interference (RNAi) gene silencing is a potential therapeutic strategy for dominant retinal degeneration disorders. We used self-complementary (sc) AAV2/8 vector to develop an RNAi-based gene therapy in a dominant retinal degeneration mouse model expressing bovine GCAP1(Y99C). We established an in vitro shRNA screening assay based on EGFP-tagged bovine GCAP1, and identified a shRNA that effectively silenced the bovine GCAP1 transgene with ∼80% efficiency. Subretinal injection of scAAV2/8 carrying shRNA expression cassette showed robust expression as early as 1 wk after injection. The gene silencing significantly improved photoreceptor survival, delayed disease onset, and increased visual function. Our results provide a promising strategy toward effective RNAi-based gene therapy by scAAV2/8 delivery for dominant retinal diseases.

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.

Mouse photoreceptor function and survival critically depend on Ca(2+)-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5-28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 μM cGMP s(-1) or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca(2+) in the presence of GCAP1 (EC(50Ca) ∼132-139 nM) than GCAP2 (EC(50Ca) ∼50-59 nM), thus arguing that Ca(2+) sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1, and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca(2+)-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates--C14:2- and C14:1-coenyzme A to C14:0-coenzyme A--were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.

Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approaches that GCAP2 specifically interacts with RIBEYE in photoreceptor synapses. We show that the flexible hinge 2 linker region of RIBEYE(B) domain that connects the nicotinamide adenine dinucleotide (NADH)-binding subdomain with the substrate-binding subdomain (SBD) binds to the C terminus of GCAP2. We demonstrate that the RIBEYE–GCAP2 interaction is induced by the binding of NADH to RIBEYE. RIBEYE–GCAP2 interaction is modulated by the SBD. GCAP2 is strongly expressed in synaptic terminals of light-adapted photoreceptors where GCAP2 is found close to synaptic ribbons as judged by confocal microscopy and proximity ligation assays. Virus-mediated overexpression of GCAP2 in photoreceptor synaptic terminals leads to a reduction in the number of synaptic ribbons. Therefore, GCAP2 is a prime candidate for mediating Ca2+-dependent dynamic changes of synaptic ribbons in photoreceptor synapses.

Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca(2+) sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca(2+)-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR. (13)C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala(9) interact with the membrane surface. Order parameters for Calpha and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static (2)H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from (2)H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.