Atypical soluble guanylyl cyclases in Drosophila can function as molecular oxygen sensors

Conventional soluble guanylyl cyclases are heterodimeric enzymes that synthesize cGMP and are activated by nitric oxide. Recently, a separate class of soluble guanylyl cyclases has been identified that are only slightly activated by or are insensitive to nitric oxide. These atypical guanylyl cyclases include the vertebrate beta2 subunit and examples from the invertebrates Manduca sexta, Caenorhabditis elegans, and Drosophila melanogaster. A member of this family, GCY-35 in C. elegans, was recently shown to be required for a behavioral response to low oxygen levels and may be directly regulated by oxygen (Gray, J. M., Karow, D. S., Lu, H., Chang, A. J., Chang, J. S., Ellis, R. E., Marletta, M. A., and Bargmann, C. I. (2004) Nature 430, 317-322). Drosophila contains three genes that code for atypical soluble guanylyl cyclases: Gyc-88E, Gyc-89Da, and Gyc-89Db. COS-7 cells co-transfected with Gyc-88E and Gyc-89Da or Gyc-89Db accumulate low levels of cGMP under normal atmospheric oxygen concentrations and are potently activated under anoxic conditions. The increase in activity is graded over oxygen concentrations of 0-21%, can be detected within 1 min of exposure to anoxic conditions and is blocked by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Gyc-88E and Gyc-89Db are co-expressed in a subset of sensory neurons where they would be ideally situated to act as oxygen sensors. This is the first demonstration of a soluble guanylyl cyclase that is activated in response to changing oxygen concentrations.     

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.     

Invertebrates yield a plethora of atypical guanylyl cyclases

Invertebrate model systems have a long history of generating new insights into neuronal signaling systems. This review focuses on cyclic GMP signaling and describes recent advances in understanding the properties and functions of guanylyl cyclases in invertebrates. The sequencing of three invertebrate genomes has provided a complete catalog of the guanylyl cyclases in C. elegans, Drosophila, and the mosquito Anopheles gambiae. Using this data and that from cloned guanylyl cyclases in Manduca sexta, C. elegans, and Drosophila, plus predictions and models from vertebrate guanylyl cyclases, evidence is presented that there is a much broader array of properties for these enzymes than previously realized. In addition to the classic homodimeric receptor guanylyl cyclases, C. elegans has at least two receptor guanylyl cyclases that are predicted to require heterodimer formation for activity. Soluble guanylyl cyclases are generally recognized as being obligate heterodimers that are activated by nitric oxide (NO). Some of the soluble guanylyl cyclases in C. elegans may heterodimeric, but all appear to be insensitive to NO. The β2 soluble guanylyl cyclase subunit in mammals and similar ones in Manduca and Drosophila are active in the absence of additional subunits and there is evidence that Drosophila and Anopheles also express an additional subunit that enhances this activity.     

Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.     

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development.

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.     

Receptor guanylyl cyclases in mammalian olfactory function

The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system.     

Isolation of a Drosophila Gene Encoding a Head-Specific Guanylyl Cyclase

Abstract: We have isolated and characterized a new guanylyl cyclase gene ( dgcl) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian solubletype guanylyl cyclase α subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgcl mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya) , indicating that dgcl is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgcl may have a role in phototransduction.     

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.     

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.     

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.     

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.     

Neuron–Glia Communication via Nitric Oxide Is Essential in Establishing Antennal-Lobe Structure in Manduca sexta

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.     

Augmentation of receptor-mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a Gbetagamma subunit-independent manner.

We previously demonstrated that the mouse EP3beta receptor and its C-terminal tail-truncated receptor (abbreviated T-335) expressed in Chinese hamster ovary cells showed agonist-dependent and fully constitutive Gi activity in forskolin-stimulated cAMP accumulation, respectively. Here we examined the effect of the EP3beta receptor or T-335 receptor on adenylyl cyclase activity stimulated by the Gs-coupled EP2 subtype receptor in COS-7 cells. As a result, sulprostone, a selective EP3 agonist, dose dependently augmented butaprost-stimulated adenylyl cyclase activity in EP3beta receptor- or T-335 receptor-expressing COS-7 cells. However, such adenylyl cyclase augmentation was not attenuated by either pertussis toxin treatment or expression of the PH domain of rat betaARK1, which serves as a scavenger of Gbetagamma subunits, but was partially attenuated by treatment with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester, an intracellular Ca(2+) chelator, or W-7, a calmodulin inhibitor. These findings suggest that the C-terminal tail of the EP3beta receptor is not essentially involved in activation of EP2 receptor-stimulated adenylyl cyclase in a Ca(2+)/calmodulin-dependent but Gbetagamma subunit-independent manner.     

Age‐dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans

Age‐dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin‐based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory‐evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD‐specific guanylyl cyclase, GCY‐8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY‐8 cyclase domain reduces these age‐dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy‐8 animals. The lack of apparent correlation between age‐dependent changes in the morphology or stimuli‐evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age‐related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature‐evoked AFD responses, and experience‐based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins

The cyclic GMP-forming enzyme guanylyl cyclase exists in cytosolic and in membrane-bound forms differing in structure and regulations. Determination of the primary structures of the guanylyl cyclases revealed that the cytosolic enzyme form consists of two similar subunits and that membrane-bound guanylyl cyclases represent enzyme forms in which the catalytic part is located in an intracellular, C-terminal domain and is regulated by an extracelluar, N-terminal receptor domain. A domain of 250 amino acids conserved in all guanylyl cyclases appears to be required for the formation of cyclic nucleotide, as this homologous domain is also found in the cytosolic regions of the adenylyl cyclase. The general structures of guanylyl cyclases shows similarities with other signal transducing enzymes such as protein-tyrosine phosphatases and protein-tyrosine kinases. which also exist in cytosolic and receptor-linked forms.