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.     

Non-metazoan class III nucleotidyl cyclases: novel forms and functions

Nucleotidyl cyclases synthesize cAMP or cGMP in response to various forms of sensory input. Their regulation and function in mammalian hormone action and neurotransmission has been well characterized over the past 40 years. More recently a much broader repertoire of adenylyl- and guanylyl cyclase forms is emerging from studies of more basal eukaryotes and prokaryotes. These cyclases play crucial roles in the development, physiology and pathogenicity of these organisms. This series of reviews highlights both unique and highly conserved aspects of the activation mechanisms of these enzymes, as compared to those of their mammalian descendants.     

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.     

Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.     

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.     

The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced Cgmp Increase in Caenorhabditis elegans

Adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase α1β1

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.     

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 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.     

Mycobacterial adenylyl cyclases: biochemical diversity and structural plasticity

The conversion of adenine and guanine nucleoside triphosphates to cAMP and cGMP is carried out by nucleotide cyclases, which vary in their primary sequence and are therefore grouped into six classes. The class III enzymes encompass all eukaryotic adenylyl and guanylyl cyclase, and several bacterial and archaebacterial cyclases. Mycobacterial nucleotide cyclases show distinct biochemical properties and domain fusions, and we review here biochemical and structural studies on these enzymes from Mycobacterium tuberculosis and related bacteria. We also present an in silico analysis of nucleotide cyclases found in completely sequenced mycobacterial genomes. It is clear that this group of enzymes demonstrates the tinkering in the class III cyclase domain during evolution, involving subtle structural changes that retain the overall catalytic function and fine tune their activities.     

Lineage-Specific Domain Fusion in the Evolution of Purine Nucleotide Cyclases in Cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.     

Identification of bacterial guanylate cyclases

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRP_G, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP_G can be activated by either cAMP or cGMP. Using CRP‐ and CRP_G‐dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.     

Synechocystis strain PCC 6803 cya2, a prokaryotic gene that encodes a guanylyl cyclase

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.     

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.     

Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.     

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.     

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.     

A density-sensing factor regulates signal transduction in Dictyostelium

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.     

Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications

Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and up-regulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.