Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.2 (guanylate cyclase)
 8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin-like neuronal Ca2+-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca2+-binding motifs. The guanylate cyclase-activating proteins 1-3 (GCAP1-3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1-2 (GC1-2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class-specific residues for the NCBP subfamilies.
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PMID:Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man. 1186 May 7

Cyclic GMP is essential for the ability of rods and cones to respond to the light stimuli. Light triggers hydrolysis of cGMP and stops the influx of sodium and calcium through the cGMP-gated ion channels. The consequence of this event is 2-fold: first, the decrease in the inward sodium current plays the major role in an abrupt hyperpolarization of the cellular membrane; secondly, the decrease in the Ca2+ influx diminishes the free intracellular Ca2+ concentration. While the former constitutes the essence of the phototransduction pathway in rods and cones, the latter gives rise to a potent feedback mechanism that accelerates photoreceptor recovery and adaptation to background light. One of the most important events by which Ca2+ feedback controls recovery and light adaptation is synthesis of cGMP by guanylyl cyclase. Two isozymes of membrane photoreceptor guanylyl cyclase (retGC) have been identified in rods and cones that are regulated by Ca2+-binding proteins, GCAPs. At low intracellular concentrations of Ca2+ typical for light-adapted rods and cones GCAPs activate RetGC, but concentrations above 500 nM typical for dark-adapted photoreceptors turn them into inhibitors of retGC. A variety of mutations found in GCAP and retGC genes have been linked to several forms of human congenital retinal diseases, such as dominant cone degeneration, cone-rod dystrophy and Leber congenital amaurosis.
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PMID:Factors that affect regulation of cGMP synthesis in vertebrate photoreceptors and their genetic link to human retinal degeneration. 1195 89

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.
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PMID:Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes. 1215 Oct 97

Cone photoreceptors respond to light with less sensitivity, faster kinetics and adapt over a much wider range of intensities than do rods. These differences can be explained, in part, by the quantitative differences in the molecular processes that regulate the cytoplasmic free Ca2+ concentration in the outer segment of both receptor types. Ca2+ concentration is regulated through the kinetic balance between the ions' influx and efflux and the action of intracellular buffers. Influx is passive and mediated by the cyclic-GMP gated ion channels. In cones, Ca2+ ions carry about 35% of the ionic current flowing through the channels in darkness. In rods, in contrast, this fraction is about 20%. We present a kinetic rate model of the ion channels that helps explain the differences in their Ca2+ fractional flux. In cones, but not in rods, the cGMP-sensitivity of the cyclic GMP-gated ion channels changes with Ca2+ at the concentrations expected in dark-adapted photoreceptors. Ca2+ efflux is active and mediated by a Na+ and K+-dependent exchanger. The rate of Ca2+ clearance mediated by the exchanger in cones, regardless of the absolute size of their outer segment is of the order of tens of milliseconds. In rod outer segments, and again independently of their size, Ca2+ clearance rate is of the order of hundreds of milliseconds to seconds. We investigate the functional consequences of these differences in Ca2+ homeostasis using computational models of the phototransduction signal in rods and cones. Consistent with experimental observation, differences in Ca2+ homeostasis can make the cone's flash response faster and less sensitive to light than that of rods. In the simulations, however, changing Ca2+ homeostasis is not sufficient to recreate authentic cone responses. Accelerating the rate of inactivation (but NOT activation) of the enzymes of the transduction cascade, in addition, to changes in Ca2+ homeostasis are needed to explain the differences between rod and cone photosignals. The large gain and precise kinetic control of the electrical photoresponse of rod and cone retinal receptors suggested a long time back that phototransduction is mediated by cytoplasmic second messengers that, in turn, control membrane ionic conductance. (1) The unquestionable identification of cyclic GMP as the phototransduction messenger, however, did not come until the mid 1980's with the discovery that the light-regulated membrane conductance in both rods and cones is gated by this nucleotide (2-4) and is, in fact, an ion channel. (7) The cyclic nucleotide gated (CNG) channels, now we know, are not just the compliant targets of light-dependent change in cytoplasmic cGMP, but actively participate in the regulation transduction through Ca2+ feedback signals. The precise magnitude and time course of the concentration changes of cGMP and Ca2+ in either rods or cones remains controversial. It is clear, however, that whereas cGMP directly controls the opening and closing of the plasma membrane channels, Ca2+ controls the light-sensitivity and kinetics of the transduction signal. (8,9) The modulatory role of Ca2+ is particularly apparent in the process of light adaptation: in light-adapted rods or cones, the transduction signal generated by a given flash is lower in sensitivity and faster in time course than in dark-adapted cells. Light adaptation is compromised if Ca2+ concentration changes are attenuated by cytopiasmic Ca2+ buffers (8,10,11) and does not occur if Ca2+ concentration changes are prevented by manipulation of the solution bathing the cells. (2,4) Several Ca2+-dependent biochemical reactions have been identified in photoreceptors, among them: 1. ATP-dependent deactivation. (15,16) 2 Phodopsin phospshorylation, through the action of recoverin (S-modulin). (17-19) 3. Catalytic activity of guanylyl cyclase, (20-22) through the action of GCAP proteins. (23,24,25) 4. cGMP-sensitivity of the CNG channels. (26-29,30) A challenge in contemporary phototransduction research is to understand the details of these reactions and their role in the control of the phototransduction signal. Transduction signals in cone photoreceptors are faster, lower in light sensitivity, and more robust in their adaptation features than those in rods (for review see refs. 31;32). A detailed molecular explanation for these differences is not at hand. However, biochemical and electrophysiological (33) studies indicate that the elements in the light-activated pathway that hydrolyzes cGMP are quantitatively similar in their function in rods and cones and unlikely to account for the functional differences. Also, within the limited exploration completed todate, the Ca2+-dependence of guanylyl cyclase (34) and visual pigment phosphorylation (19) do not differ in rods and cones. On the other hand, data accumulated over the past few years indicate that cytoplasmic Ca2+ homeostasis, while controlled through essentially identical mechanisms it is quantitatively very different in its features in the two photoreceptor types. Both Ca2+ influx through CNG channels and the rate of Ca2+ clearance from the outer segment differ between the two receptor cells. Also, the Ca2+-dependent modulation of cGMP sensitivity is larger in extent in cones than in rods. Most significantly, the concentration range of this Ca2+ dependence overlaps the physiological range of light-dependent changes in cytoplasmic Ca2+ level in cones, but not in rods. We briefly review some of the evidence that supports these assertions and we then provide a quantitative analysis of the possible significance of these known differences. We conclude that while differences in Ca2+ homeostasis contribute importantly to explaining the differences between the two receptor types, they are alone not sufficient to explain the differences in the photoreceptor's response. It is likely that Ca2+-independent inactivation of the transduction cascade enzymes is more rapid in cones than in rods.
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PMID:Tuning outer segment Ca2+ homeostasis to phototransduction in rods and cones. 1259 22

Calcium concentration in the dark-adapted retinal rod outer segment is in the 200 to 600 nM range, and the guanylate cyclase of rod outer segments is thought to be activated in response to a fall in calcium concentration triggered by light. Calcium-binding proteins that mediate such activation, i.e., activation in the absence of or presence of low nanomolar calcium concentrations, have been identified and termed GCAPs (Guanaylate Cyclase Activating Proteins). In the course of our search for GCAP-like proteins in bovine retina, we isolated a protein fraction that stimulated rod outer segment cyclase activity at calcium concentrations higher than those in dark-adapted rod outer segments. We purified the protein responsible for this calcium-dependent stimulation of cyclase activity and found it to be of 6-7 kDa molecular weight as judged by electrophoresis under denaturing conditions and about 40 kDa by gel filtration analysis. Maximum stimulation of cyclase activity was observed at 3-4 micromolar concentration of the protein. It required about 1.5 micromolar free calcium concentration for half-maximal activation of the enzyme. Partial amino acid sequencing of peptide fragments of the activator suggested that the protein was identical with S100b, a previously described calcium-binding protein. Further characterization with antibody specific for S100b supported this possibility. However, the protein isolated in our laboratory and termed CD-GCAP (Calcium-Dependent Guanylate Cyclase Activator Protein) was found to differ significantly from commercially available S100b in the magnitude and calcium dependence of cyclase activation. It was also found to be inactivated by hydroxylamine while S100b was resistant. Investigation into these differences showed that purification methods had a significant influence on the properties of the activator, producing a less active (S100b) or more active (CD-GCAP) protein, but that it was, otherwise, one and the same protein. We conclude from this study that rod outer segment guanylate cyclase, unlike any cyclase known so far, is capable of activation by two different types of calcium-binding proteins, one that activates in response to a decrease in calcium concentration, and the other, described here, which activates in response to an increase in calcium-concentration. We hypothesize that this cyclase and others like it will be colocalized with one or the other type of activator depending upon the physiological requirement, i.e., activation in response to decreasing or increasing calcium concentration.
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PMID:Calcium-dependent activation of guanylate cyclase by S100b. 1259 34

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca(2+)-dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (kcat/Km) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on kcat/Km. The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca(2+)-sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca(2+)], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca(2+)-sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two 'GCAP modes' that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca(2+) in photoreceptor cells.
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PMID:Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity. 1295 Feb 65

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.
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PMID:Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1. 1475 May 95

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.
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PMID:Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors. 1596 91

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.
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PMID:Ca2+ and Mg2+ binding properties of GCAP-1. Evidence that Mg2+-bound form is the physiological activator of photoreceptor guanylyl cyclase. 1679 76

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.
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PMID:Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains. 1754 52


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