Guanylyl cyclase activity during photoperiodic flower induction in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.     

Involvement of cyclic GMP in phytochrome-controlled flowering of Pharbitis nil

Light is one of the most important environmental factors influencing the induction of flowering in plants. Light is absorbed by specific photoreceptors – the phytochromes and cryptochromes system – which fulfil a sensory and a regulatory function in the process. The absorption of light by phytochromes initiates a cascade of related biochemical events in responsive cells, and subsequently changes plant growth and development.

Induction of flowering is controlled by several paths. One is triggered by the guanosine-3′:5′-cyclic monophosphate (cGMP) level. Thus, the aim of our study was to investigate the role of cGMP in phytochrome-controlled flowering.

It is best to conduct such research on short-day plants because the photoperiodic reactions of only these plants are totally unequivocal. The most commonly used plant is the model short-day plant Pharbitis nil.

The seedlings of P. nil were cultivated under special photoperiodic conditions: 72-h-long darkness, 24-h-long white light with low intensity and 24-h-long inductive night. Such light conditions cause a degradation of the light-labile phytochrome. Far red (FR) treatment before night causes inactivation of the remaining light-stable phytochrome. During the 24-h-long inductive darkness period, the total amount of cGMP in cotyledons underwent fluctuations, with maxima at the 4th, 8th and 14th hours. When plants were treated with FR before the long night, fluctuations were not observed. A red light pulse given after FR treatment could reverse the effect induced by FR, and the oscillation in the cGMP level was observed again.

Because the intracellular level of cGMP is controlled by the opposite action of guanylyl cyclases (GCs) and phosphodiesterases (PDEs), we first tested whether accumulation of the nucleotide in P. nil tissue may be changed after treatment with a GC stimulator or PDE inhibitor.

Accumulation of the nucleotide in P. nil cotyledons treated with a stimulator of cGMP synthesis (sodium nitroprusside) was markedly (approximately 80%) higher. It was highest in the presence of dipyridamole, whereas 3-isobutyl-1-methylxanthine did not significantly affect cGMP level.

These results show that the analysed compounds were able to penetrate the cotyledons’ tissue, and that they influenced enzyme activity and cGMP accumulation.

FR light applied at the end of the 24-h-long white light period inhibited flowering. Exogenous cGMP added on cotyledons could reverse the effect of FR, especially when the compound was applied in the first half of the long night. Flowering was also promoted by exogenous application of guanylyl cyclase activator and phosphodiesterase inhibitors, and in particular dipyridamole.

The results obtained suggest that an endogenous cGMP system could participate in the mechanism of a phytochrome-controlled flowering in P. nil.     

A Highlights from MBoC Selection: Forty-five years of cGMP research in Dictyostelium: understanding the regulation and function of the cGMP pathway for cell movement and chemotaxis

In Dictyostelium, chemoattractants induce a fast cGMP response that mediates myosin filament formation in the rear of the cell. The major cGMP signaling pathway consists of a soluble guanylyl cyclase sGC, a cGMP-stimulated cGMP-specific phosphodiesterase, and the cGMP-target protein GbpC. Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway. The chemoattractants stimulate heterotrimeric Gαβγ and monomeric Ras proteins. A fraction of the soluble guanylyl cyclase sGC binds with high affinity to a limited number of membrane binding sites, which is essential for sGC to become activated by Ras and Gα proteins. sGC can also bind to F-actin; binding to branched F-actin in pseudopods enhances basal sGC activity, whereas binding to parallel F-actin in the cortex reduces sGC activity. The cGMP pathway mediates cell polarity by inhibiting the rear: in unstimulated cells by sGC activity in the branched F-actin of pseudopods, in a shallow gradient by stimulated cGMP formation in pseudopods at the leading edge, and during cAMP oscillation to erase the previous polarity and establish a new polarity axis that aligns with the direction of the passing cAMP wave.     

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.     

Plant Nucleotide Cyclases: An Increasingly Complex and Growing Family

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, cGMP, has long been known to be critical to many different processes in higher plants while guanylyl cyclases (GCs), enzymes that catalyse the formation of cGMP from GTP have largely remained elusive. This is somewhat surprising considering that the unicellular green alga Chlamydomonas reinhardtii contains >90 annotated GCs. We have recently shown (PLoS ONE 2(5): e449) that a recombinant cytoplasmic domain of the Arabidopsis brassinosteroid receptor AtBRI has GC activity in vitro. This finding may suggest that other leucine-rich receptor kinases such as the phystosulfokine receptor may also confer GC activity as it has a high degree of similarity in the domain that has been delineated as essential for catalysis. In addition, the discovery of increasing complexities in the molecular architecture of higher plant nucleotide cyclases (NCs) is entirely compatible with findings in Chlamydomonas where such domains appear in >20 different combinations suggesting a role in highly diverse and complex signaling events.Key Words: nucleotide cyclase, guanylyl cyclase, cGMP, signal transduction, Arabidopsis thaliana, Chlamydomonas reinhardtii     

Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC’s (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (α/β) β1 isoform of the β subunit of NOGC (NOGCβ1) was specifically focused. NOGCβ1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCβ1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCβ1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 ± 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 ± 3.4 vs. 1.2 ± 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCβ1 abundance was reduced using specific siRNA to NOGCβ1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCβ1 (530.2 ± 141.4 vs. 26.1 ± 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCβ1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 ± 22 vs. 92 ± 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCβ1 and GC-A interact and that NOGCβ1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.     

Essential role of nitric oxide in acute ischemic preconditioning: S-Nitros(yl)ation versus sGC/cGMP/PKG signaling?

Nitric oxide (NO) plays an important role in acute ischemic preconditioning (IPC). In addition to activating soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathways, NO-mediated protein S-nitros(yl)ation (SNO) has been recently shown to play an essential role in cardioprotection against ischemia–reperfusion (I/R) injury. In our previous studies, we have shown that IPC-induced cardioprotection could be blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor) or ascorbate (a reducing agent to decompose SNO). To clarify NO-mediated sGC/cGMP/PKG-dependent or -independent (i.e., SNO) signaling involved in IPC-induced cardioprotection, mouse hearts were Langendorff-perfused in the dark to prevent SNO decomposition by light exposure. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a highly selective inhibitor of sGC) or KT5823 (a potent and selective inhibitor of PKG) did not abolish IPC-induced acute protection, suggesting that the sGC/cGMP/PKG signaling pathway does not play an important role in NO-mediated cardioprotective signaling during acute IPC. In addition, treatment with ODQ in IPC hearts provided an additional protective effect on functional recovery, in parallel with a higher SNO level in these ODQ+IPC hearts. In conclusion, these results suggest that the protective effect of NO is not related primarily to activation of the sGC/cGMP/PKG signaling pathway, but rather through SNO signaling in IPC-induced acute cardioprotection.     

Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

Differentiation of human monocytes in vitro with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression

The cytokines macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) promote differentiation of monocytes into macrophages with distinct phenotypes and unique functional abilities. In this report, we characterize how monocytes and macrophages differentiated from monocytes with M-CSF and GM-CSF regulate their cGMP levels by controlling which phosphodiesterases (PDEs) and guanylyl cyclases (GCs) are expressed. We find that PDE1B and PDE2A are expressed at low levels in monocytes, but are the major cGMP PDEs expressed in macrophages. M-CSF differentiation triggers increased expression of PDE1B and PDE2A, while GM-CSF causes a large increase only in PDE1B. Based on PDE expression, we identified THP-1 and U937 cell lines as possible models for studying the roles of PDE1B and PDE2A in macrophage function. We additionally characterized changes in expression of GCs upon differentiation. We found that GM-CSF differentiation triggers a small decrease in soluble guanylyl cyclase (sGC) and a large increase in GC-A, while M-CSF significantly decreases sGC.     

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F,Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α₁β₁ possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.     

Crystal structure of the signaling helix coiled-coil domain of the β1 subunit of the soluble guanylyl cyclase

Background  

The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner.     

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I alpha activation by NO/cGMP

cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H₂O₂ treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H₂O₂ challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H₂O₂ produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-I completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1 by NO-derived cGMP. Consequently, stimulated PKG-I may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress. nitric oxide; C-type natriuretic peptide; myosin light chain; cGMP-dependent protein kinase type I; endothelial cell barrier dysfunction     

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction

Nitric oxide (NO) diffuses as short‐lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO‐induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO‐sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO‐induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca²⁺‐free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca²⁺‐independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 337–346, 1999     

A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis inAmaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Glucagon-Like Peptide-2 (GLP-2) Modulates the cGMP Signalling Pathway by Regulating the Expression of the Soluble Guanylyl Cyclase Receptor Subunits in Cultured Rat Astrocytes

The aim of this work was to study the effect of glucagon-like peptide-2 (GLP-2) on the cyclic guanosine monophosphate (cGMP) signalling pathway and whether insulin or epidermal growth factor (EGF) might modulate the effects of GLP-2. GLP-2 produced a dose-dependent decrease in intracellular sodium nitroprusside-induced cGMP production. However, insulin induced an increase in the levels of cGMP that was dose-dependently decreased by the addition of GLP-2. By contrast, EGF induced a decrease in cGMP production, which was further reduced by the addition of GLP-2. To assess whether variations in cGMP production might be related with changes in some component of soluble guanylyl cyclase (sGC), the expression of the ??1, ??2, and ??1 subunits were determined by Western blot analysis. At 1?h, GLP-2 produced a decrease in the expression of both ??1 and ??1 in the cytosolic fraction, but at 24?h only ??1was reduced. As expected, insulin induced an increase in the expression of both subunits after 1?h of incubation; this was decreased by the addition of GLP-2. Likewise, incubation with EGF for 24?h produced a decrease in the expression of both subunits that was maximal when GLP-2 was added. In addition, incubation with insulin for 1?h produced an increase in the expression of the ??2 subunit, which was reduced by the addition of GLP-2. These results suggest that GLP-2 inhibits cGMP production by decreasing the cellular content of at least one subunit of the heterodimeric active form of the sGC, independently of the presence of insulin or EFG. This may open new insights into the actions of this neuropeptide.     

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module: MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C*

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs)⁴ have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of the extracellular domain of mammalian receptor GCs vary (less than ∼15% similarity), as would be expected given the diversity in the ligands that bind to and activate these receptors. The KHD shows ∼25–30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2, 3). The catalytic domains of mammalian receptor GCs are more conserved (∼80% sequence similarity). The gradual increase in sequence similarity across the various domains, with the extracellular domain being the most diverse and the cyclase domains sharing the maximum sequence similarity, is a reflection of the ability of these receptor GCs to converge diverse extracellular signals to a unified output of cGMP production. The guanylyl cyclase domains of receptor GCs can be classified as members of the Class III family of nucleotide cyclases (4). The recent crystal structures of a bacterial guanylyl cyclase (5) and a eukaryotic soluble guanylyl cyclase (6) show similarities in the overall three-dimensional structure of adenylyl and guanylyl cyclases and also highlight the critical residues that determine substrate utilization (either ATP or GTP) in these enzymes.Guanylyl cyclase C (GC-C) serves as the receptor for the guanylin family of endogenous peptides as well as for the exogenous heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria (7, 8). GC-C is predominantly expressed on the apical surface of epithelial cells in the intestine, although robust extra-intestinal expression is observed in the kidney and reproductive tissues of the rat (9–12). The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2, 3).A sequence of ∼70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13). This region is predicted to form an amphipathic α-helix and could also adopt a coiled coil conformation (14, 15). The linker region is also present in soluble (cytosolic) guanylyl cyclases where it connects the N-terminal heme binding regulatory domain to the C-terminal catalytic cyclase domain. The linker region is suggested to act as a dimerization module in receptor GCs (16–18) and has also been implicated in heterodimerization of the α and β subunits of soluble guanylyl cyclases (19, 20). However, there are several reports to the contrary that indicate that the linker does not affect the dimerization of receptor GCs (14, 15). Nevertheless, the critical importance of the linker in regulating the activity of receptor GCs is shown by the fact that mutations in this region of the retinal guanylyl cyclase (RetGC-1) are associated with autosomal dominant cone-rod dystrophy in humans (16, 21). We show here through extensive mutational and biochemical analysis that the linker regions in two receptor GCs, GC-C and guanylyl cyclase A (GC-A), play an important role in repressing the catalytic activity of the receptors in the absence of their ligands. In addition, our results provide for the first time a molecular explanation for detergent-enhanced guanylyl cyclase activity in this family of receptors and suggest a mechanism for this activation that could involve a hydrophobic interaction between the linker region and the guanylyl cyclase domain.