The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Interaction of guanylyl cyclase C with SH3 domain of Src tyrosine kinase. Yet another mechanism for desensitization

Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.     

Primary structure and functional expression of the human receptor for Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (STa) produced by Escherichia coli induces intestinal secretion in mammals by binding to the brush border membrane of the small intestine and activating guanylyl cyclase. We report here the cloning and expression of a cDNA encoding the human receptor for STa. The receptor contains both an extracellular ligand binding site and a cytoplasmic guanylyl cyclase catalytic domain, making it a member of the same receptor family as the natriuretic peptide receptors. Stable mammalian cell lines over-expressing the STa receptor specifically bind 125I-STa (Kd approximately 1.0 nM) and respond to STa by dramatically increasing (approximately 50-fold) cellular cGMP levels. Sequence comparisons between the human and the rat STa receptors show less conservation in the extracellular domain than similar comparisons of natriuretic peptide receptors. This divergence may indicate important species differences in ligand-receptor interaction.     

Inhibition of receptor-stimulated guanylyl cyclase by intracellular calcium ions in Dictyostelium cells.

In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P3 which releases Ca2+ from internal stores. The following data indicate that intracellular Ca2+ ions inhibit guanylyl cyclase activity. 1) In vitro, Ca2+ inhibits guanylyl cyclase with IC50 = 41 nM Ca2+ and Hill-coefficient of 2.1. 2) Extracellular Ca2+ does not affect basal cGMP levels of intact cells. In electro-permeabilized cells, however, cGMP levels are reduced by 85% within 45 s after addition of 10(-6) M Ca2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca2+. 3) Receptor-stimulated activation of guanylyl cyclase in electro-permeabilized cells is also inhibited by extracellular Ca2+ with half-maximal effect at 200 nM Ca2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase.     

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium.     

High-density lipoprotein antagonizes the inhibitory effects of oxidized low-density lipoprotein and lysolecithin on soluble guanylyl cyclase.

Oxidatively modified LDL (LDLox) reduces the response of soluble guanylyl cyclase to nitrovasodilators. We now demonstrate that this desensitization can be antagonized by HDL. Similar to its protective effect against LDLox, HDL also inhibited the lysolecithin-induced desensitization of soluble guanylyl cyclase. Since the lysolecithin content of LDLox correlated with the amount of lysolecithin necessary to diminish stimulation of soluble guanylyl cyclase, our data support the hypothesis that lysolecithin may be responsible for the inhibitory effect of LDLox on smooth muscle relaxation and provide evidence that the antagonistic effect of HDL against desensitization of soluble guanylyl cyclase by atherogenic compounds could be responsible for the protective role of HDL in atherosclerosis.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.     

Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases.

The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.     

Phosphorylation of recoverin, the calcium-sensitive activator of photoreceptor guanylyl cyclase.

Recoverin, a new calcium binding protein from bovine rod photoreceptor cells, activates guanylyl cyclase below a free calcium concentration of 200 nM. We show here that recoverin is phosphorylated by an endogenous kinase and Mg-ATP at the same decreased calcium concentration. The calcium-dependent activation of guanylyl cyclase is enhanced in the presence of ATP. We suggest that phosphorylation of recoverin reinforces the stimulation of guanylyl cyclase at decreased calcium concentrations.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Inhibitory effect of Ca(2+) on ATP-mediated stimulation of NPR-A-coupled guanylyl cyclase in renal glomeruli from spontaneously hypertensive and normotensive rats.

Atrial natriuretic peptide (ANP) regulates blood pressure mainly through the occupation of the guanylyl cyclase-coupled receptor NPR-A, which requires ATP interaction for maximal activation. This study investigates the effect of extracellular Ca(2+) on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase activity in glomerular membranes from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). ATP induced a significant increase in basal and ANP(1-28)-stimulated guanylyl cyclase activity that was greater in SHR than in WKY. Extracellular Ca(2+) inhibited ATP-stimulated guanylyl cyclase activity in a concentration-dependent manner, but did not modify basal and ANP(1-28)-stimulated guanylyl cyclase activity. In the presence of ATP, NPR-A showed higher affinity for ANP(1-28) and lower Bmax. Ca(2+) did not modify NPR-A-ANP(1-28) binding properties. The different effects of extracellular Ca(2+) on ANP(1-28)- or ATP-mediated guanylyl cyclase activation suggest that these events are differentially regulated. Addition of extracellular Ca(2+) induced similar effects in hypertensive and normotensive rats, suggesting that it is not responsible for the elevated cGMP production observed in SHR.     

Urodilatin attenuates sympathetic neurotransmission in the rabbit isolated vas deferens without activating guanylyl cyclase.

Natriuretic peptides are produced in cardiovascular, renal and neural tissues and are believed to reduce arterial blood pressure by augmenting sodium and water loss in the urine. Another potential antihypertensive action of these peptides involves a suppression of adrenergic neurotransmission. Atrial, brain and C-type natriuretic peptides suppress sympathetic neurotransmission but no data are available on neuromodulatory actions of urodilatin. This study investigates the hypothesis that urodilatin and brain natriuretic peptide inhibit sympathetic neurotransmission by elevating guanylyl cyclase activity. Both brain natriuretic peptide and urodilatin suppressed force generation in response to electrical stimulation of the vas deferens. Brain natriuretic peptide accelerated the production of cyclic guanosine monophosphate equipotently with its effects on neurotransmission. However, urodilatin failed to increase guanylyl cyclase activity, thus dissociating its effects on neurotransmission from guanylyl cyclase stimulation. None of the natriuretic peptides altered contractile effects of either adenosine triphosphate or norepinephrine, the two putative neurotransmitters secreted from adrenergic nerves in the vas deferens. These data are consistent with the following conclusions: 1) all of the known endogenous natriuretic peptides suppress adrenergic neurotransmission; 2) guanylyl cyclase activation is not required for the inhibition of sympathetic neurotransmission by natriuretic peptides; and 3) inhibitory effects of the natriuretic peptides on neurotransmission result from a suppression of neurotransmitter exocytosis. The novel findings of this study include both the suppression of sympathetic neurotransmission by urodilatin and its biological activity in the absence of guanylyl cyclase activation.     

Backbone 1H, 13C, and 15N resonance assignments of guanylyl cyclase activating protein-1, GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca²⁺-dependent activation of retinal guanylyl cyclase that regulates the visual light response. GCAP1 is genetically linked to retinal degenerative diseases. We report backbone NMR chemical shift assignments of Ca²⁺-saturated GCAP1 (BMRB no. 18026).     

Guanylyl cyclases in unicellular organisms

Guanylyl cyclases in eukaryotic unicells were biochemically investigated in the ciliates Paramecium and Tetrahymena, in the malaria parasite Plasmodium and in the ameboid Dictyostelium. In ciliates guanylyl cyclase activity is calcium-regulated suggesting a structural kinship to similarly regulated membrane-bound guanylyl cyclases in vertebrates. Yet, cloning of ciliate guanylyl cyclases revealed a novel combination of known modular building blocks. Two cyclase homology domains are inversely arranged in a topology of mammalian adenylyl cyclases, containing two cassettes of six transmembrane spans. In addition the protozoan guanylyl cyclases contain an N-terminal P-type ATPase-like domain. Sequence comparisons indicate a compromised ATPase function. The adopted novel function remains enigmatic to date. The topology of the guanylyl cyclase domain in all protozoans investigated is identical. A recently identified Dictyostelium guanylyl cyclase lacks the N-terminal P-type ATPase domain. The close functional relation of Paramecium guanylyl cyclases to mammalian adenylyl cyclases has been established by heterologous expression, respective point mutations and a series of active mammalian adenylyl cyclase/Paramecium guanylyl cyclase chimeras. The unique structure of protozoan guanylyl cyclases suggests that unexpectedly they do not share a common guanylyl cyclase ancestor with their vertebrate congeners but probably originated from an ancestral mammalian-type adenylyl cyclase.