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Microtubules form flexible fibers, which are utilized in cell proliferation and differentiation. Although the flexibility of microtubules was shown to be regulated by various microtubule-associated proteins, this regulation is still far from complete understanding. Here, we report a new potential regulator of microtubules in mammals. Gcap14 colocalizes with microtubules in mammalian cells transfected with Gcap14 expression vector. Association of Gcap14 with microtubules was confirmed by biochemical subcellular fractionation. Recombinant Gcap14 protein cosedimented with pure microtubules, indicating a direct binding between the two. Furthermore, recombinant Gcap14 was shown to have the ability of inducing microtubule bundling in vitro.Structured summary of protein interactionsGcap14 physically interacts with Gcap14 by anti tag coimmunoprecipitation (View Interaction: 1, 2)

The expression pattern and property profile of the neuronal Ca(2+) sensor guanylate cyclase-activating protein 3 (zGCAP3) was studied by immunochemical approaches, biophysical methods and enzymatic assays. Using affinity purified antibodies immunoreactivity towards zGCAP3 was weakly detected in the outer and strongly in the inner segments of cone cells as well as in the outer plexiform layer, to a lesser degree also in the inner plexiform and ganglion cell layer of the zebrafish retina. This cellular distribution was independent of a dark/light cycle. Some neuronal Ca(2+) sensors are acylated (mainly myristoylated) at the amino-terminus. Probing larval and adult stages of the developing zebrafish retina indicated that zGCAP3 was first expressed in a non-myristoylated form, but was finally present in the adult retina as a myristoylated protein. While zGCAP3 did not undergo a classical Ca(2+) -myristoyl switch as investigated by surface plasmon resonance spectroscopy, myristoylation had two main other consequences: it enhanced the Ca(2+) -sensitivity of the Ca(2+) -induced conformational change and it stabilized the protein conformation. Differences between myristoylated and non-myristoylated zGCAP3 were also observed in modulating the kinetic and catalytic parameters of the GCAP-target, a membrane bound guanylate cyclase. Thus, the stabilizing effect of the myristoyl group is apparently less important in the larval than in the adult fish.

Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.

The light responses of rod and cone photoreceptors in the vertebrate retina are quantitatively different, yet extremely stable and reproducible because of the extraordinary regulation of the cascade of enzymatic reactions that link photon absorption and visual pigment excitation to the gating of cGMP-gated ion channels in the outer segment plasma membrane. While the molecular scheme of the phototransduction pathway is essentially the same in rods and cones, the enzymes and protein regulators that constitute the pathway are distinct. These enzymes and regulators can differ in the quantitative features of their functions or in concentration if their functions are similar or both can be true. The molecular identity and distinct function of the molecules of the transduction cascade in rods and cones are summarized. The functional significance of these molecular differences is examined with a mathematical model of the signal-transducing enzymatic cascade. Constrained by available electrophysiological, biochemical and biophysical data, the model simulates photocurrents that match well the electrical photoresponses measured in both rods and cones. Using simulation computed with the mathematical model, the time course of light-dependent changes in enzymatic activities and second messenger concentrations in non-mammalian rods and cones are compared side by side.

The spatial and temporal pattern of cone packing during marmoset foveal development was explored to understand the variables involved in creating a high acuity area. Retinal ages were between fetal day (Fd) 125 and 6 years. Cone density was determined in wholemounts using a new hexagonal quantification method. Wholemounts were labeled immunocytochemically with rod markers to identify reliably the foveal center. Cones were counted in small windows and density was expressed as cones × 103/mm2 (K). Two weeks before birth (Fd 125–130), cone density had a flat distribution of 20–30 K across the central retina encompassing the fovea. Density began to rise at postnatal day 1 (Pd 1) around, but not in, the foveal center and reached a parafoveal peak of 45–55 K by Pd 10. Between Pd 10 and 33, there was an inversion such that cone density at the foveal center rose rapidly, reaching 283 K by 3 months and 600 K by 5.4 months. Peak foveal density then diminished to 440 K at 6 months and older. Counts done in sections showed the same pattern of low foveal density up to Pd 1, a rapid rise from Pd 30 to 90, followed by a small decrease into adulthood. Increasing foveal cone density was accompanied by 1) a reduction in the amount of Müller cell cytoplasm surrounding each cone, 2) increased stacking of foveal cone nuclei into a mound 6–10 deep, and 3) a progressive narrowing of the rod-free zone surrounding the fovea. Retaining foveal cones in a monolayer precludes final foveal cone densities above 60 K. However, high foveal adult cone density (300 K) can be achieved by having cone nuclei stack into columns and without reducing their nuclear diameter. Marmosets reach adult peak cone density by 3–6 months postnatal, while macaques and humans take much longer. Early weaning and an arboreal environment may require rapid postnatal maturation of the marmoset fovea.

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1−/− mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1−/− photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1−/− retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

A negative phototransduction feedback in rods and cones is critical for the timely termination of their light responses and for extending their function to a wide range of light intensities. The calcium feedback mechanisms that modulate phototransduction in rods have been studied extensively. However, the corresponding modulation mechanisms that enable cones to terminate rapidly their light responses and to adapt in bright light, properties critical for our daytime vision, are still not understood. In cones, calcium feedback to guanylyl cyclase is potentially a key step in phototransduction modulation. The guanylyl cyclase activity is modulated by the calcium-binding guanylyl cyclase activating proteins (GCAP1 and GCAP2). Here, we used single-cell and transretinal recordings from mouse to determine how GCAPs modulate dark-adapted responses as well as light adaptation in mammalian cones. Deletion of GCAPs increased threefold the amplitude and dramatically prolonged the light responses in dark-adapted mouse cones. It also reduced the operating range of mouse cones in background illumination and severely impaired their light adaptation. Thus, GCAPs exert powerful modulation on the mammalian cone phototransduction cascade and play an important role in setting the functional properties of cones in darkness and during light adaptation. Surprisingly, despite their better adaptation capacity and wider calcium dynamic range, mammalian cones were modulated by GCAPs to a lesser extent than mammalian rods. We conclude that a disparity in the strength of GCAP modulation cannot explain the differences in the dark-adapted properties or in the operating ranges of mammalian rods and cones.

This study documents the identity of an intriguing transduction mechanism of the [Ca(2+)](i) signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca(2+) transmitter switch (Ca(2+)TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is (657)WTAPELL(663). The two Ca(2+) signaling GCAP pathways converge in Ca(2+)TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca(2+)-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.

Guanylate cyclase activating protein-2 (GCAP-2) is a Ca2+-binding protein of the neuronal calcium sensor (NCS) family. Ca2+-free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca2+-dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca2+-free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca2+-myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca2+-independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca2+-bound and Ca2+-free forms, respectively, to gain information on the nature of the Ca2+-dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca2+, the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca2+ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca2+-free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca2+-bound state.

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+)-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+)-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+) around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca(2+)/Mg(2+)-dependent conformational changes and in the Ca(2+)-dependent monomer-dimer equilibrium. Direct Ca(2+)-binding revealed that all zGCAPs bound at least three Ca(2+). The corresponding apparent affinity constants reflect binding of Ca(2+) with high (≤ 100 nM), medium (0.1-5 µM) and/or low (≥ 5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+)-sensor system in zebrafish rod and cone cells supporting a Ca(2+)-relay model of differential zGCAP operation in these cells.

The sensitivity of receptor cells places a fundamental limit upon the sensitivity of sensory systems. For example, the signal-to-noise ratio of sensory receptors has been suggested to limit absolute thresholds in the visual and auditory systems. However, the necessity of optimally processing sensory receptor signals for behaviour to approach this limit has received less attention. We investigated the behavioural consequences of increasing the signal-to-noise ratio of the rod photoreceptor single-photon response in a transgenic mouse, the GCAPs-/- knockout. The loss of fast Ca2+ feedback to cGMP synthesis in phototransduction for GCAPs-/- mice increases the magnitude of the rod single-photon response and dark noise, with the increase in size of the single-photon response outweighing the increase in noise. Surprisingly, despite the increased rod signal-to-noise ratio, behavioural performance for GCAPs-/- mice was diminished near absolute visual threshold. We demonstrate in electrophysiological recordings that the diminished performance compared to wild-type mice is explained by poorly tuned postsynaptic processing of the rod single-photon response at the rod bipolar cell. In particular, the level of postsynaptic saturation in GCAPs-/- rod bipolar cells is not sufficient to eliminate rod noise, and degrades the single-photon response signal-to-noise ratio. Thus, it is critical for retinal processing to be optimally tuned near absolute threshold; otherwise the visual system fails to utilize fully the signals present in the rods.

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca(2+) sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca(2+)-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca(2+) binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca(2+) affinity and thermal stability, whereas all the mutants showed decreased Ca(2+) affinity and significantly lower thermal stability in both apo and Ca(2+)-loaded forms. No apparent cooperativity of Ca(2+) binding was detected for any variant. Finally, the non-myristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca(2+) concentrations. Thus, we conclude that distorted Ca(2+)-sensor properties could lead to cone dysfunction.

Retinal membrane guanylyl cyclase (RetGC) and Ca(2+)/Mg(2+) sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg(2+)-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn(2+). These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg(2+)-bound activator form of GCAP1.

Neuronal Ca(2+) sensors (NCS) are high-affinity Ca(2+)-binding proteins critical for regulating a vast range of physiological processes. Guanylate cyclase-activating proteins (GCAPs) are members of the NCS family responsible for activating retinal guanylate cyclases (GCs) at low Ca(2+) concentrations, triggering synthesis of cGMP and recovery of photoreceptor cells to the dark-adapted state. Here we use amide hydrogen-deuterium exchange and radiolytic labeling, and molecular dynamics simulations to study conformational changes induced by Ca(2+) and modulated by the N-terminal myristoyl group. Our data on the conformational dynamics of GCAP1 in solution suggest that Ca(2+) stabilizes the protein but induces relatively small changes in the domain structure; however, loss of Ca(+2) mediates a significant global relaxation and movement of N- and C-terminal domains. This model and the previously described "calcium-myristoyl switch" proposed for recoverin indicate significant diversity in conformational changes among these highly homologous NCS proteins with distinct functions.

Progressive visual impairment leading to blindness is often associated with inherited retinal dystrophies. These disorders correlate in most cases with mutations in genes that code for proteins of the visual transduction system in rod and cone photoreceptor cells. Recent progress has highlighted the involvement of a neuronal calcium sensor protein that is specifically expressed in rod and cone cells and operates as a guanylate cyclase-activating protein (GCAP). A group of patients suffering from cone or cone-rod dystrophies carry mutations in the GCAP1 gene, and biochemical analysis of GCAP1 function revealed that for most of these mutations GCAP1 exhibits a disturbance in its Ca2+-sensing and its guanylate cyclase-activating properties. Cellular consequences of different GCAP1 mutations are compared and discussed.

We discuss the heterogeneity of autosomal dominant cone and cone-rod dystrophies (adCD, and adCORD, respectively). As one of the best characterized adCD genes, we focus on the GUCA1A gene encoding guanylate cyclase activating protein 1 (GCAP1), a protein carrying three high affinity Ca(2+) binding motifs (EF hands). GCAP1 senses changes in cytoplasmic free [Ca(2+)] and communicates these changes to GC1, by either inhibiting it (at high free [Ca(2+)]), or stimulating it (at low free [Ca(2+)]). A number of missense mutations altering the structure and Ca(2+) affinity of EF hands have been discovered. These mutations are associated with a gain of function, producing dominant cone and cone rod dystrophy phenotypes. In this article we review these mutations and describe the consequences of specific mutations on GCAP1 structure and GC stimulation.We discuss the heterogeneity of autosomal dominant cone and cone-rod dystrophies (adCD, and adCORD, respectively). As one of the best characterized adCD genes, we focus on the GUCA1A gene encoding guanylate cyclase activating protein 1 (GCAP1), a protein carrying three high affinity Ca(2+) binding motifs (EF hands). GCAP1 senses changes in cytoplasmic free [Ca(2+)] and communicates these changes to GC1, by either inhibiting it (at high free [Ca(2+)]), or stimulating it (at low free [Ca(2+)]). A number of missense mutations altering the structure and Ca(2+) affinity of EF hands have been discovered. These mutations are associated with a gain of function, producing dominant cone and cone rod dystrophy phenotypes. In this article we review these mutations and describe the consequences of specific mutations on GCAP1 structure and GC stimulation.

Cones show briefer light responses than rods and do not saturate even under very bright light. Using purified rod and cone homogenates, we measured the activity of guanylate cyclase (GC), an enzyme responsible for cGMP synthesis and therefore recovery of a light response. The basal GC activity was 36 times higher in cones than in rods: It was mainly caused by higher expression levels of GC in cones (GC-C) than in rods (GC-R). With identification and quantification of GC-activating protein (GCAP) subtypes expressed in rods and cones together with determination of kinetic parameters of GC activation in the presence and absence of GCAP, we estimated the in situ GC activity in rods and cones at low and high Ca 2+ concentrations. It was revealed that the GC activity would be >10 times higher in cones than in rods in both the dark-adapted and the light-adapted states. Electrophysiological estimation of the GC activity measured in the truncated preparations of rod and cone outer segments gave consistent results. Our estimation of the in situ GC activity reasonably explained the rapid recovery and nonsaturating behavior of cone light responses.

Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone-rod dystrophy. Cyclase activity is regulated by Ca(2+) which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca(2+)-binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca(2+) levels rise following a light flash.

Light adaptation in vertebrate photoreceptors is mediated by multiple mechanisms, one of which could involve nuclear feedback and changes in gene expression. Therefore, we have investigated light adaptation-associated changes in gene expression using microarrays and real-time PCR in isolated photoreceptors, in cultured isolated retinas and in acutely isolated retinas. In all three preparations after 2 h of an exposure to a bright light, we observed an up-regulation of almost 100% of three genes, Sag, Guca1a and Guca1b, coding for proteins known to play a major role in phototransduction: arrestin, GCAP1 and GCAP2. No detectable up-regulation occurred for light exposures of less than 1 h. Functional in vivo electroretinographic tests show that a partial recovery of the dark current occurred 1-2 h after prolonged illumination with a steady light that initially caused a substantial suppression of the photoresponse. These observations demonstrate that prolonged illumination results in the up-regulation of genes coding for proteins involved in the phototransduction signalling cascade, possibly underlying a novel component of light adaptation occurring 1-2 h after the onset of a steady bright light.

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca(2+) and Mg(2+) binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). GCAP1 binds cooperatively to Ca(2+) at EF3 and EF4 (DeltaH(EF3) = -3.5 kcal/mol, and DeltaH(EF4) = -0.9 kcal/mol) with nanomolar affinity (K(EF3) = 80 nM, and K(EF4) = 200 nM), and a third Ca(2+) binds entropically at EF2 (DeltaH(EF2) = 3.1 kcal/mol, and K(EF2) = 0.9 microM). GCAP1 binds functionally to Mg(2+) at EF2 (DeltaH(EF2) = 4.3 kcal/mol, and K(EF2) = 0.7 mM) required for RetGC activation. Ca(2+) and/or Mg(2+) binding to GCAP1 dramatically alters DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3, and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange-broadened, upfield-shifted, and insensitive to Ca(2+), consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg(2+) or Ca(2+) binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.

OBJECTIVE: Evaluation of the presence of a Ca(2+)-regulated membrane guanylate cyclase signal transudation system in the spermatozoa. DESIGN: Experimental study. SETTING: Research university laboratory. PATIENT(S): Human sperm obtained from healthy donors who met the criteria of the World Health Organization for normozoospermia and bovine semen collected from bulls of proven fertility. INTERVENTION(S): Radioimmunoassay and immunohistochemistry of human and bovine spermatozoa. MAIN OUTCOME MEASURE(S): The membrane guanylate cyclase activity and the presence of membrane guanylate cyclase transduction machinery components in the spermatozoa. RESULT(S): The identity of a Ca(2+)-modulated membrane guanylate cyclase transduction machinery in human and bovine spermatozoa has been documented. The machinery is both inhibited and stimulated within nanomolar to semimicromolar range of free Ca(2+). The transduction component of this machinery is the rod outer segment membrane guanylate cyclase type 1 (ROS-GC1). The enzyme coexists with three Ca(2+)-dependent modulators: guanylate cyclase activating protein type 1 (GCAP1), S100B and neurocalcin delta. ROS-GC1 and its modulators are present in the heads and tails of both species' spermatozoa. CONCLUSION(S): The coexpression of ROS-GC1 and its activators in spermatozoa suggests that the Ca(2+)-modulated ROS-GC1 transduction system may be a part of the fertilization machinery.

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca(2+)-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca(2+)] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca(2+)-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.

Cyclic GMP serves as the second messenger in visual transduction, linking photon absorption by rhodopsin to the activity of ion channels. Synthesis of cGMP in photoreceptors is supported by a pair of retina-specific guanylyl cyclases, retGC1 and -2. Two neuronal calcium sensors, GCAP1 and GCAP2, confer Ca2+ sensitivity to guanylyl cyclase activity, but the importance and the contribution of each GCAP is controversial. To explore this issue, the gene GUCA1B, coding for GCAP2, was disrupted in mice, and the capacity for knock-out rods to regulate retGC and generate photoresponses was tested. The knock-out did not compromise rod viability or alter outer segment ultrastructure. Levels of retGC1, retGC2, and GCAP-1 expression did not undergo compensatory changes, but the absence of GCAP2 affected guanylyl cyclase activity in two ways; (a) the maximal rate of cGMP synthesis at low [Ca2+] dropped 2-fold and (b) the half-maximal rate of cGMP synthesis was attained at a higher than normal [Ca2+]. The addition of an antibody raised against mouse GCAP2 produced similar effects on the guanylyl cyclase activity in wild type retinas. Flash responses of GCAP2 knock-out rods recovered more slowly than normal. Knock-out rods became more sensitive to flashes and to steps of illumination but tended to saturate at lower intensities, as compared with wild type rods. Therefore, GCAP2 regulation of guanylyl cyclase activity quickens the recovery of flash and step responses and adjusts the operating range of rods to higher intensities of ambient illumination.

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca(2+)-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520-570 nM free Ca(2+) concentration. Furthermore, the Ca(2+)-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca(2+)-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca(2+)-myristoyl switch, a feature typical of some but not all neuronal Ca(2+)-sensor proteins. However, tryptophan fluorescence studies and Ca(2+)-dependent differences in protease accessibility revealed Ca(2+)-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca(2+) sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca(2+)-sensitive regulator of guanylate cyclase activity in cone vision.

Detailed biochemical, structural and physiological studies of the role of Ca2(+)-binding proteins in mammalian retinal neurons have yielded new insights into the function of these proteins in normal and pathological states. In phototransduction, a biochemical process that is responsible for the conversion of light into an electrical impulse, guanylate cyclases (GCs) are regulated by GC-activating proteins (GCAPs). These regulatory proteins respond to changes in cytoplasmic Ca2+ concentrations. Disruption of Ca2+ homeostasis in photoreceptor cells by genetic and environmental factors can result ultimately in degeneration of these cells. Pathogenic mutations in GC1 and GCAP1 cause autosomal recessive Leber congenital amaurosis and autosomal dominant cone dystrophy, respectively. This report provides a recent account of the advances, challenges, and possible future prospects of studying this important step in visual transduction that transcends to other neuronal Ca2+ homeostasis processes.

There is increasing interest in developing viral vectors capable of reliably delivering multiple therapeutic genes to targeted cell populations. Currently, bicistronic vectors carrying two transgenes linked by an internal ribosomal entry site (IRES) are the most commonly employed vectors to accomplish this goal. We and others have found that the protein encoded downstream of the IRES in these vectors is not reliably expressed. The purpose of this study was to determine if replacement of the IRES in our self-inactivating, insulated, lentiviral vectors with a second, independent, cell-specific promoter would produce a vector that reliably expressed two proteins in targeted retinal cells in vivo.Five dual promoter lentiviral vectors were constructed using our self-inactivating (SIN), insulated, lentiviral backbone. Each vector carried two independent transgenes encoding a fluorescent protein (GFP or tdTomato) whose expression was driven by three photoreceptor promoters (interphotoreceptor retinoid binding protein-IRPB1783; guanylate cyclase activating protein 1-GCAP292; rhodopsin-mOP500) and one ubiquitously expressed promoter (elongation factor 1alpha-EF1alpha). Constructs were packaged and injected into the optic vesicles of developing chicken embryos. The day before hatching, the retinas were removed and examined as whole mount tissues and as frozen sections using fluorescent microscopy.In our first experiment, we characterized the expression of the three photoreceptor promoters in chicken retina. The activities of GCAP292 and IRBP1783 were restricted to cone cells. GCAP292 was also active in a small sub-group of inner nuclear cells. The activity of mOP500 was restricted to rod cells. In our second experiment, we characterized the activity of three dual promoter vectors: GCAP292-GFP-IRBP1783-tdTomato, IRBP-tdTomato-GCAP292-GFP, and IRBP1783-tdTomato-mOP500-GFP. All three vectors produced easily detectable levels of GFP and tdTomato in transduced retinas, a result that prompted further analyses of the expression characteristics of these vectors. In retinas treated with either of the GCAP292/IRBP1783 dual promoter vectors, GFP and tdTomato were only detected in cone cells. No GFP was detected in the inner retina. In retinas treated with IRBP1783-tdTomato-mOP500-GFP, tdTomato was detected only in cone cells and GFP was detected only in rod cells, a result indicating that these promoters retained their intrinsic expression specificities in this dual promoter vector. In our final experiment, the ubiquitously expressed EF1alpha promoter was paired with either GCAP292 or mOP500 creating EF1alpha-tdTomato-GCAP292-GFP and EF1alpha-tdTomato-mOP-GFP. In retinas treated with EF1alpha-tdTomato-GCAP292-GFP, GFP was only detected in cone cells. In retinas treated with EF1alpha-tdTomato-mOP500-GFP, GFP was detected in rod cells and in several cells within the inner retina.The results of this study show that it is possible to construct dual promoter lentiviral vectors that reliably express two proteins in a cell-specific manner. Among the dual promoter vectors created for this study, we have identified two vectors that specifically target expression of both transgenes to cone cells and one vector that specifically targets expression of one transgene to cone cells and the other transgene to rod cells. The ability to create one lentiviral vector that is capable of targeting expression of multiple genes to single or multiple cells in vivo should prove very useful in the development and delivery of complex, combination therapies to diseased tissues.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca2+sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca2+in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca2+measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca2+sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca2+concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca2+can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.

TEZ6397 Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2007. Kaynakça (s.51-55) var. x, 56 s. : res. ; 29 cm. Retinal guanylate cyclases, retGC-1 and retGC-2, are a subclass of the membrane dependent guanylate cyclase family. Synthesize cGMP, the intracelular second messenger of phototransduction, they play a crucial role in photoreceptor physiology. In recent work, it is intended to investigate the effect of adenine nucleotides and all-trans retinal on the retinal guanylate cyclase activity. Crosslinking experiments indicate, protein phosphorylation might be essential for physiological activity of cyclases. Retinal guanilat siklazlar, retGC-1 ve retGC-2, membrana bağlı guanilat siklaz ailesinin bir alt kolunu oluştururlar. Fototransdüksiyonunun intraselüler mesajcısı olan cGMP'yi sentezledikleri için fotoreseptör fizyolojisinde önemli rol oynarlar. Bu çalışmada, adenin nükleotidlerinin retinal guanilat siklazların fizyolojisine etkilerinin incelenmesi amaçlanmıştır. Çapraz bağlama çalışmaları protein fosforilasyonunun siklazın fizyolojik aktivitesinin ortaya çıkmasında önemli rol oynadığını göstermiştir.

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca2+ in the light and inhibits it in the dark when Ca2+ concentrations rise. We present the first direct evidence that Mg2+-bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP-1, three bound Ca2+ ions and could exchange Ca2+ for Mg2+. At concentrations of free Ca2+ and Mg2+ typical for the light-adapted photoreceptors, all three metal-binding EF-hands were predominantly occupied by Mg2, and the presence of bound Mg2+ in GCAP-1 was essential for its ability to stimulate RetGC-1. In the Mg2+-bound form of GCAP-1 all three Trp residues became more exposed to the polar environment compared with its apo form. The replacement of Mg2+ by Ca2+ in the EF-hands 2 and 3 further exposed Trp-21 to the solution in a non-metal-binding EF-hand domain 1 that interacts with RetGC. Contrary to that, replacement of Mg2+ by Ca2+ in the EF-hand 4 moved Trp-94 in the entering alpha-helix of the EF-hand 3 back to the non-polar environment. Our results demonstrate that Mg2+ regulates GCAP-1 not only by adjusting its Ca2+ sensitivity to the physiological conditions in photoreceptors but also by creating the conformation required for RetGC stimulation.

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and others have shown that basic fibroblast growth factor (FGF2) and angiopoietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n = 5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble beta3 (GUCY1B3), using real-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p < 0.001), FGF2 (p < 0.001), ANGPT2 (p < 0.06), and GUCY1B3 (p < 0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.

Background: The indazole derivative YC-1 has been characterized as a nitric oxide (NO)-independent and heme dependent soluble guanylate cyclase (sGC) activator, which also sensitizes sGC to NO. Objective: To examine the effects of YC-1 on vascular relaxation in newborn and 2-week-old piglet pulmonary arteries. The effect of YC-1 on the relaxation induced by exogenous NO was also analyzed. Methods: Isolated rings from third branch pulmonary arteries and fifth–seventh-generation intrapulmonary arterioles were mounted in organ chambers for isometric tension recording. Arteries were precontracted with the thromboxane A2 mimetic U46619. Results: YC-1 induced relaxation was greater in 2-week-old pulmonary arteries and was abolished by the sGC inhibitor ODQ (10 µM). YC-1 induced relaxation was similar in conduit pulmonary arteries and arterioles. In the 2-week-old conduit pulmonary arteries, the response to YC-1 was significantly reduced when the endothelium was removed or after incubation with the NO synthase inhibitor L-NAME (0.1 mM). YC-1 augmented NO-induced relaxation in 2-week-old but not in neonatal conduit pulmonary arteries. Conclusions: Our results indicate that YC-1 induced pulmonary vascular relaxation in conduit and resistance pulmonary arteries and these effects increased with postnatal age. In the 2-week-old conduit pulmonary arteries and besides being a direct activator of sGC, YC-1 produced endothelium-dependent relaxation and synergized with exogenous NO.

To describe the clinical features and genetic analysis of a family with an autosomal dominant cone dystrophy (adCD).Selected members of a family with an autosomal dominant cone dystrophy underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and genomic fragments were amplified by PCR. Linkage to locus D6S1017 was established. DHPLC mutational analysis and direct sequencing were used to identify a mutation in GUCA1A, the gene encoding the guanylate cyclase activating protein 1 (GCAP1).Of 24 individuals who are at risk of the disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinogram performed on one proband revealed profoundly subnormal and prolonged photopic and flicker responses, but preserved scotopic ERGs, consistent with a cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a novel L151F mutation in GCAP1. Like the E155G mutation, this mutation occurs in the EF4 hand domain, a region of GCAP1 critical in conferring calcium sensitivity to the protein. The leucine at this position is highly conserved among vertebrate guanylate cyclase activating proteins.A novel L151F missense mutation in the EF4 high affinity Ca2+ binding site of GCAP1 is linked to adCD in a large pedigree. The cone dystrophy in this family shares clinical and electrophysiologic characteristics with other previously described adCD caused by mutations in GUCA1A.

Purpose To characterize clinically and genetically a four-generation Italian family with autosomal dominant retinal dystrophy. Methods Thirty-seven family members underwent a detailed ophthalmologic investigation, comprising visual acuity determination, fundoscopy, electroretinogram, and electrooculogram. A genome-wide scan was performed, and three candidate genes mapping to the linked region were screened for mutations by direct sequencing. Results Nineteen individuals were affected by cone-rod dystrophy and four by cone dystrophy, whereas, in another subject, the diagnosis was compatible with central areolar choroidal dystrophy. The genome-wide search allowed mapping the disease locus to chromosome 6, region p12.2-p21.1, with a maximum lod score of 6.71. Analysis of key recombinants in affected individuals placed the locus to a 12-Mb region flanked by newly generated markers 6-41025 and 6-52969. Assuming complete penetrance, recombinations in two healthy individuals defined a smaller critical region of 3.7 Mb between markers 6-42153 and D6S459. Three genes mapping within the linked interval (RDS, GUCA1A, and GUCA1B) were considered excellent candidates because of their involvement in distinct forms of retinal dystrophies. However, mutation analyses of these genes failed to identify pathogenetic mutations. Conclusions The significant lod scores obtained and the absence of mutations in RDS, GUCA1A, and GUCA1B support the existence of a novel, yet unidentified gene responsible for retinal dystrophy within the chromosome 6 cluster.

PURPOSE: To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 4-generation family and to examine the phenotype and the degree of intrafamilial variability. DESIGN: Prospective case series. PARTICIPANTS: Six affected individuals from a nonconsanguineous British family. METHODS: Detailed ophthalmologic examination, color fundus photography, autofluorescence imaging, and electrophysiologic assessment were performed. Blood samples were taken for DNA extraction, and mutation screening of GUCA1A, the gene encoding guanylate cyclase-activating protein 1 (GCAP1), was undertaken. RESULTS: All affected subjects complained of mild photophobia and reduced central and color vision. Onset was between the third and fifth decade, with subsequent gradual deterioration of visual acuity and color vision. Visual acuity ranged between 6/9 and counting fingers. Color vision was either absent or markedly reduced along all 3 color axes. A range of macular appearances was seen, varying from mild retinal pigment epithelial disturbance to extensive atrophy. Electrophysiologic testing revealed a range of electrophysiologic abnormalities: isolated cone electroretinography abnormalities, reduced cone and rod responses (with cone loss greater than rod), and isolated macular dysfunction. The 4 coding exons of GUCA1A were screened for mutations in affected and unaffected family members. A single transition, A319G, causing a nonconservative missense substitution, Tyr99Cys, segregated uniquely in all affected subjects. CONCLUSIONS: The Tyr99Cys GUCA1A mutation has been previously shown to cause autosomal dominant progressive cone dystrophy. This is the first report of this mutation also causing both cone-rod dystrophy and isolated macular dysfunction. The phenotypic variation described here exemplifies the intrafamilial heterogeneity of retinal dysfunction that can be observed in persons harboring the same mutation and chromosomal segment.

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.

To identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation.The GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+.A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity.A novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.

Background— Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. Methods and Results— In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg · kg −1 · h −1 ) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N ω -nitro- l -arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg · kg −1 · h −1 potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619–induced pulmonary vasoconstriction before or during administration of BAY 41-2272. Conclusions— In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V.Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM-GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+-induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation.

Recessive mutations in GUCY2D, the gene encoding the retinal guanylyl cyclase protein, RetGC-1, have been shown to cause Leber Congenital Amaurosis (LCA), a severe retinal dystrophy. The purpose of this study was to determine the functional consequences of selected mutations in GUCY2Dlinked to LCA. The mutations investigated in this study map to the catalytic domain (P858S, L954P) and the extracellular domain (C105Y, L325P) of RetGC-1.All four mutations were introduced into the in vitro expression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293 cells. We assayed the abilities of the mutant cyclases to generate cGMP (basal activity), and to be activated by guanylyl cyclase activating proteins (GCAP-1 and GCAP-2). Additionally, we co-expressed the catalytic domain mutations (P858S and L954P) with a wild-type allele to test for dominant negative effects on wild-type RetGC-1.The P858S and L954P mutations, both in highly conserved residues of the catalytic domain of RetGC-1, severely impair basal, GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. In addition, when co-expressed with the wild-type allele, both catalytic domain mutations act as dominant negative proteins and reduce the activity of wild-type RetGC-1. The basal activities of the C105Y and L325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclase activities are reduced approximately 50%.GUCY2D mutations from LCA patients have distinct functional consequences on RetGC-1 catalytic activity in vitro. Our analyses showed that the catalytic domain mutations cause a marked reduction in cyclase activity, while the extracellular domain mutations moderately reduce activity. The catalytic domain mutant alleles cause dominant negative effects, indicating that the functionality of RetGC-1 is compromised even in heterozygotes. This is consistent with abnormalities in cone electroretinograms (ERGs) detected in obligate heterozygous GUCY2D parents that carry the L954P mutation.

Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca(2+)-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca(2+) and inhibit RetGC in a Ca(2+)-sensitive manner. The reported data for the RetGC inhibition by Ca(2+)/GCAPs in vitro are in disagreement with the free Ca(2+) levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854). We have found that binding of Mg(2+) dramatically affects both Ca(2+)-dependent conformational changes in GCAP-1 and Ca(2+) sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg(2+) concentrations ([Mg](f)) from 5.0 mm to 0.5 mm decreases the free Ca(2+) concentration required for half-maximal inhibition of RetGC ([Ca]((1/2))) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 microm to 0.16 and 0.03 microm, respectively. A similar effect of Mg(2+) on Ca(2+) sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]((1/2)) changes as a function of [Mg](f) in mouse retina shows that the [Ca]((1/2)) becomes consistent with the range of 23-250 nm free Ca(2+) found in mouse photoreceptors only if the [Mg](f) in the photoreceptors is near 1 mm. Our data demonstrate that GCAPs are Ca(2+)/Mg(2+) sensor proteins. While Ca(2+) binding is essential for cyclase activation and inhibition, Mg(2+) binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca(2+).

Changes in the Ca2+ concentration are thought to affect many processes, including signal transduction in a vast number of biological systems. However, only in few cases the molecular mechanisms by which Ca2+ mediates its action are as well understood as in phototransduction. In dark-adapted photoreceptor cells, the equilibrium level of cGMP is maintained by two opposing activities, such as phosphodiesterase (PDE) and guanylate cyclase (GC). Upon absorption of photons, rhodopsin-G-protein-mediated activation of PDE leads to a transient decrease in [cGMP] and subsequently to lowering of [Ca2+]. In turn, lower [Ca2+] increases net production of cGMP by stimulation of GC until dark conditions are re-established. This activation of GC is mediated by Ca2+ -free forms of Ca2+ -binding proteins termed GC-activating proteins (GCAPs). The last decade brought the molecular identification of GCs and GCAPs in the visual system. Recent efforts have been directed toward understanding the properties of GC at the physiological and structural levels. Here, we summarize the recent progress and present a list of topics of ongoing research.

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 ± 0.20 mm), GCAP2 enhanced the retGC activity 10–13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3–4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 ± 2.7 nm). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nm). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nm). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.

Mediated by guanylate cyclase-activating proteins (GCAPs), cytoplasmic Ca2+levels regulate the activity of photoreceptor guanylate cyclase (GC) and the synthesis of cGMP, the internal transmitter of phototransduction. When GCAP1 is expressed in transgenic mice on a GCAP null background, it restores the wild-type flash responses in rod photoreceptors. In this communication, we explored the role of GCAP1 in cone photoreceptors by using electroretinograms (ERGs). Under cone isolation conditions, ERGs recorded from mice lacking both GCAP1 and GCAP2 had normal amplitudes of the saturated a-wave and b-wave. However, recordings from these mice demonstrated a widened b-wave and increased sensitivity of both M- and UV-cone systems. Paired-flash ERGs revealed a delayed recovery of both the cone driven b-wave and a-wave and suggest that the delay originated from the photoreceptors. To test whether GCAP1 could restore normal cone response recovery, mice that expressed only transgenic GCAP1 in the absence of wild-type GCAP expression were tested. Immunohistochemical analysis demonstrated that cones of these mice expressed high levels of GCAP1. Paired-flash ERGs showed that the recovery of the cone-driven a-wave was restored to normal, whereas recovery of the cone-driven b-wave was slightly faster than that observed in wild-type mice. These studies reveal that, similar to rods, deletion of GCAP1 and GCAP2 delays the recovery of light responses in cones and GCAP1 restores the recovery of cone responses in the absence of GCAP2.

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.

In photoreceptor cells cGMP is the second messenger that transduces light into an electrical response. Regulation of cGMP synthesis by Ca2+ is one of the key mechanisms by which Ca2+ exerts negative feedback to the phototransduction cascade in the process of light adaptation. This Ca2+ feedback to retinal guanylyl cyclases (Ret-GCs) is conferred by the guanylate cyclase-activating proteins (GCAPs). Mutations in GCAP1 that disrupt the Ca2+ regulation of Ret-GCs in vitro have been associated with severe human vision disorders. This chapter focuses on recent data obtained from biochemical and electrophysiological studies of GCAP1/GCAP2 knockout mice and other GCAP transgenic mice, addressing: 1. the quantitative aspects of the Ca2+-feedback to Ret-GCs in regulating the light sensitivity and adaptation in intact rods; 2. functional differences between GCAP1 and GCAP2 in intact rod photoreceptors; and 3. whether GCAP mutants with impaired Ca2+ binding lead to retinal disease in vivo by constitutive activation of Ret-GCs and elevation of intracellular cGMP, as predicted from in vitro studies.

A range of cone and cone-rod dystrophies (CORD) have been observed in man, caused by mutations in retinal guanylate cyclase 1 (RetGC1) and guanylate cyclase activating protein 1 (GCAP 1). The CORD causing mutations in RetGC1 are located at a mutation "hot spot" within the dimerisation domain, where R838 is the key residue. Three disease causing mutations have been found in human GCAP1, resulting in cone or cone-rod degeneration. All three mutations are dominant in their effect although the mechanism by which the P50L mutation exerts its influence remains unclear although it might act due to a haplo-insufficiency, arising from increased susceptibility to protease activity and increased thermal instability. In contrast, loss of Ca2+ sensitivity appears to be the main cause of the diseased state for the Y99C and E155G mutations. The cone and cone-rod dystrophies that are caused by mutations in RetGC1 or GCAP1 arise from a perturbation of the delicate balance of Ca2+ and cGMP within the photoreceptor cells and it is this disruption that is believed to cause cell death. The diseases caused by mutations in RetGC1 and GCAP1 prominently affect cones, consistent with the higher concentrations of these proteins in cone cells.

Guanylyl cyclase activating proteins (GCAPs) are Ca2+-binding proteins of the EF-hand superfamily, through which the intracellular calcium regulates cGMP synthesis in vertebrate photoreceptors. GCAPs play an essential role in the calcium feedback mechanism that controls recovery and light adaptation of rods and cones. Moreover, mutations in at least one of the GCAPs have already been linked to two forms of congenital human retinal diseases. The GCAPs represent a separate small subfamily among the EF-hand proteins that are structurally similar to recoverin, but demonstrate a number of unique regulatory properties. When in the Ca2+-free conformation (as in light-adapted photoreceptors), GCAPs stimulate photoreceptor membrane guanylyl cyclase (retGC), but when the intracellular free Ca2+ concentrations ([Ca2+]free) rise (as in dark-adapted photoreceptors), GCAPs turn into retGC inhibitors. In GCAPs, site-directed mutagenesis has been successfully used to identify a number of structural elements that contribute to their specific function as guanylyl cyclase regulators. These elements include EF-hand Ca2+-binding loops and various other regions in the GCAP primary structure involved in multiple protein-protein interactions within the retGC/GCAP complex.