Oxygen-sensitive guanylyl cyclases in insects and their potential roles in oxygen detection and in feeding behaviors

Responses to hypoxia and hyperoxia depend critically on the ability of the animal to detect changes in O2 levels. However, it has only been recently that an O2-sensing system has been identified in invertebrates. Evidence is accumulating that this molecular O2 sensor is, surprisingly, a class of soluble guanylyl cyclase (sGC) known as atypical sGCs. It has long been known that the conventional sGC alpha and beta subunits form heterodimeric enzymes that are potently activated by NO, but do not bind O2. By contrast, the Drosophila melanogaster atypical sGC subunits, Gyc-88E, Gyc-89Da and Gyc-89Db, are only slightly sensitive to NO, but are potently activated under hypoxic conditions. Here we review evidence that suggests that the atypical sGCs can function as molecular O2 sensors mediating behavioral responses to hypoxia. Sequence comparisons of other predicted O2-sensitive sGCs suggest that most, if not all, insects express two heterodimeric sGCs; an NO-sensitive isoform and a separate O2-sensitive isoform. Expression data and recent experiments that block the function of cells that express the atypical sGCs and experiments that reduce the cGMP levels in these cells also suggest a role in behavioral responses to sweet tastants.     

Ligand binding and inhibition of an oxygen-sensitive soluble guanylate cyclase, Gyc-88E, from Drosophila

Soluble guanylate cyclase (sGC) uses a ferrous heme cofactor as a receptor for NO and once bound activates the enzyme for the conversion of GTP to cGMP. The heme cofactor in sGC does not bind oxygen, thereby allowing it to selectively bind NO despite a cellular concentration of oxygen (microM) that is much higher than signaling concentrations of nitric oxide (nM). The molecular details of this ligand discrimination against oxygen have emerged and allowed for predictions regarding ligand specificity in the sGC family. The results reported here show that Gyc-88E from Drosophila is a hemoprotein that binds oxygen, as well as NO and CO. All three ligands form 6-coordinate complexes. Gyc-88E is active as a homodimer (5600 +/- 243 nmol min(-1) mg(-1)) and is inhibited by O2, CO, and NO (3.2-, 2.9-, and 2-fold, respectively). The Km for GTP was 0.66 +/- 0.15 mM in air (273 microM oxygen) and 0.82 +/- 0.15 mM under anaerobic conditions. The Ki for oxygen was calculated to be 51 +/- 28 microM. The biochemical properties of Gyc-88E are unique for guanylate cyclases and suggest a possible function as an oxygen sensor.     

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.     

A functional chimera of mammalian guanylyl and adenylyl cyclases

Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity.     

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.     

Conversion of a guanylyl cyclase to an adenylyl cyclase

Guanylyl cyclases catalyze the formation of cGMP from GTP, but display extensive identity at the catalytic domain primary amino acid level with the adenylyl cyclases. The recent solving of the crystal structures of soluble forms of adenylyl cyclase has resulted in predictions of those amino acids important for substrate specificity. Modeling of a membrane-bound homodimeric guanylyl cyclase predicted the comparable amino acids that would interact with the guanine ring. Based on these structural data, the replacement of three key residues in the heterodimeric form of soluble guanylyl cyclase has led to a complete conversion in substrate specificity. Furthermore, the mutant enzyme remained fully sensitive to sodium nitroprusside, a nitric oxide donor.     

Cyclic GMP controls Rhodospirillum centenum cyst development

Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α-proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.     

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.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.     

Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina

Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction. The decrease in the free intracellular Ca(2+) concentrations caused by illumination stimulates two isoforms of retinal membrane guanylyl cyclase (RetGC) via Ca(2+)-sensor proteins and thus contributes to photoreceptor recovery and light adaptation. Unlike other members of the membrane guanylyl cyclase family, retinal guanylyl cyclases do not have identified extracellular peptide ligands. Recoverin-like proteins, GCAP-1 and GCAP-2, interact with the intracellular portion of the cyclases and stimulate its activity through dimerization of the cyclase subunits. Several mutations that affect the function of photoreceptor guanylyl cyclase and the activator protein have been linked to various forms of congenital human retinal diseases, such as Leber congenital amaurosis, cone and cone-rod dystrophy.     

Salt and osmotic stress cause rapid increases in Arabidopsis thaliana cGMP levels

A guanylyl cyclase has been recently identified in Arabidopsis but, despite the use of pharmacological inhibitors to infer roles of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP), very few measurements of actual cGMP levels in plants are available. Here, we demonstrate that cGMP levels in Arabidopsis seedlings increase rapidly (< or =5 s) and to different degrees after salt and osmotic stress, and that the increases are prevented by treatment with LY, an inhibitor of soluble guanylyl cyclases. In addition, we provide evidence to suggest that salt stress activates two cGMP signalling pathways - an osmotic, calcium-independent pathway and an ionic, calcium-dependent pathway.     

Behavioral responses to hypoxia and hyperoxia in Drosophila larvae

The ability to detect changes in oxygen concentration in the environment is critical to the survival of all animals. This requires cells to express a molecular oxygen sensor that can detect shifts in oxygen levels and transmit a signal that leads to the appropriate cellular response. Recent biochemical, genetic and behavioral studies have shown that the atypical soluble guanylyl cyclases function as oxygen detectors in Drosophila larvae triggering a behavioral escape response when exposed to hypoxia. These studies also identified the sensory neurons that innervate the terminal sensory cones as likely chemosensors that mediate this response. Here I summarize the data that led to these conclusions and also highlight evidence that suggests additional, as yet unidentified, proteins are also required for detecting increases and decreases in oxygen concentrations.     

Behavioral responses to hypoxia and hyperoxia in Drosophila larvae: molecular and neuronal sensors

The ability to detect changes in oxygen concentration in the environment is critical to the survival of all animals. This requires cells to express a molecular oxygen sensor that can detect shifts in oxygen levels and transmit a signal that leads to the appropriate cellular response. Recent biochemical, genetic and behavioral studies have shown that the atypical soluble guanylyl cyclases function as oxygen detectors in Drosophila larvae triggering a behavioral escape response when exposed to hypoxia. These studies also identified the sensory neurons that innervate the terminal sensory cones as likely chemosensors that mediate this response. Here I summarize the data that led to these conclusions and also highlight evidence that suggests additional, as yet unidentified, proteins are also required for detecting increases and decreases in oxygen concentrations.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

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.     

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.     

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.