Substrate selection by class III adenylyl cyclases and guanylyl cyclases

The second messengers cAMP and cGMP are of central importance in signal transduction pathways. To assure pathway specificity adenylyl and guanylyl cyclases are highly selective for their substrates, ATP and GTP, respectively. The universal class III cyclases are equipped with a variety of purine-binding modes, which have been identified by structure determination and mutagenesis. Most selection mechanisms rely on a pair of residues which form hydrogen bonds to N1 and the N(6)-amino or O(6)-keto group of adenine and guanine, respectively. Furthermore, selection is supported by hydrogen bonds involving the peptide backbone and by constraints imposed by hydrophobic side-chains.     

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.     

Structure, function and evolution of microbial adenylyl and guanylyl cyclases

Cells respond to signals of both environmental and biological origin. Responses are often receptor mediated and result in the synthesis of so-called second messengers that then provide a link between extracellular signals and downstream events, including changes in gene expression. Cyclic nucleotides (cAMP and cGMP) are among the most widely studied of this class of molecule. Research on their function and mode of action has been a paradigm for signal transduction systems and has shaped our understanding of this important area of biology. Cyclic nucleotides have diverse regulatory roles in both unicellular and multicellular organisms, highlighting the utility and success of this system of molecular communication. This review will examine the structural diversity of microbial adenylyl and guanylyl cyclases, the enzymes that synthesize cAMP and cGMP respectively. We will address the relationship of structure to biological function and speculate on the complex origin of these crucial regulatory molecules. A review is timely because the explosion of data from the various genome projects is providing new and exciting insights into protein function and evolution.     

Class III nucleotide cyclases in bacteria and archaebacteria: lineage-specific expansion of adenylyl cyclases and a dearth of guanylyl cyclases

The Class III nucleotide cyclases are found in bacteria, eukaryotes and archaebacteria. Our survey of the bacterial and archaebacterial genome and plasmid sequences identified 193 Class III cyclase genes in only 29 species, of which we predict the majority to be adenylyl cyclases. Interestingly, several putative cyclase genes were found to have non-conserved substrate specifying residues. Ancestors of the eukaryotic C1-C2 domain containing soluble adenylyl cyclases as well as the protist guanylyl cyclases were found in bacteria. Diverse domains were fused to the cyclase domain and phylogenetic analysis indicated that most proteins within a single cluster have similar domain compositions, emphasising the ancient evolutionary origin and versatility of the cyclase domain.     

Pro-nucleotide inhibitors of adenylyl cyclases in intact cells

9-substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3'-phosphoryl derivatives of 2',5'-dideoxyadenosine (2',5'-dd-Ado) and beta-l-2',5'-dd-Ado, protected 5'-phosphoryl derivatives of beta-l-2',3'-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [(3)H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [(3)H]adenine. A striking selectivity for 2',5'-dd-Ado-3'-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC(50) approximately 2 microm) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2',5'-dd-Ado-3'-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [(3)H]cAMP formation in preadipocytes (IC(50) approximately 30 nm) and suppressed opening of cAMP-dependent Cl(-) channels in cardiac myocytes (IC(50) approximately 800 nm). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.     

Functional activity of adenylyl and guanylyl cyclases in human spermatozoa with different motility

The most important functional characteristic of ejaculated spermatozoa is their ability to engage in directed sustained movement, which to a large extent determines their fertility. It is assumed that enzymes with cyclase activity—adenylyl cyclase (AC) and guanylyl cyclase (GC)—soluble and membrane-bound forms of which are found in human and mammalian sperm, play the key role in regulation of motility. However, the functional activity of the cyclases in ejaculated spermatozoa with different motilities and their contribution to the regulation of this process are virtually unexplored. The goal of this work was to determine the functional characteristics of AC and GC in ejaculates of human spermatozoa with different contents of motile forms and the study of regulation of these enzymes by hormones and nonhormonal agents. We found differences in the activity and regulatory properties of AC and GC in ejaculates differing in motile forms of spermatozoa. The basal AC activity and its sensitivity to bicarbonate anions and manganese cations, activators of cytosolic AC (cAC), were increased in ejaculates with a high proportion of motile spermatozoa. At the same time, the AC effects of forskolin, GppNHp, and adrenergic receptor agonists acting via membrane-bound AC (mAC) in this case were significantly reduced. Cytosolic GC in the ejaculates with a high proportion of motile spermatozoa was more sensitive to manganese cations, but the basal activity of GC was altered slightly. An increase in the content of motile spermatozoa in ejaculate led to a decrease in the sensitivity of CNP to receptor GC, while the sensitivity to ANP was maintained, which indicates a change in the pattern of enzyme regulation with natriuretic peptides in favor of ANP, an important regulator of sperm chemotaxis. Thus, we have concluded that the change in proportion of motile spermatozoa in ejaculate induces changes of functional activity and regulatory properties of soluble and membrane-bound forms of AC and GC, which can be used to control the motility, chemotaxis, acrosomal reaction, and other processes determining fertility of male germ cells.     

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.     

Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases

Background

The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two—the channelrhodopsins—were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.

Results

Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called “two-component cyclase opsins” (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.

Conclusions

Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

     

Bicarbonate stimulated adenylyl cyclases

Bicarbonate ion is fundamental to the biology of all living organisms. HCO(3)(-) is vital to such diverse physiological processes as carbon fixation, cellular homeostasis, sperm maturation, and nucleotide synthesis. A defined subset of adenylyl cyclases identified in eukaryotes and prokaryotes are directly activated by HCO(3)(-). As such, cAMP represents the first identified biological effector for fluctuations in intracellular inorganic carbon levels. The identification of a signal transduction pathway activated by HCO(3)(-) has far reaching implications for understanding how the cell responds to fluctuations in this essential anion.     

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 adenylyl and guanylyl cyclase superfamily

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine—arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein α subunit, promote the catalytically optimal juxtaposition of the two domains.     

The study of molecular mechanisms of action of natural amino acids and serotonin on adenylyl and guanylyl cyclases of the ciliates

It has been previously shown that some amino acids and their derivatives are capable of regulating the activity of adenylyl cyclase (AC) and guanylate cyclase (GC) in free-living ciliates Dileptus anser and Tetrahymena. The aim of this work was to study the molecular mechanisms of action of methionine, tyrosine, alanine and neurohormone serotonin on the activity of enzymes-cyclases and the identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both ciliates, and the AC effect of serotonin in T. pyriformis was carried out with the participation of Ca2+-dependent form of AC and heterotrimetic G proteins. AC stimulating effect of tyrosine and alanine was expressed weakly and only detected in D. anser. Serotonin is both ciliates and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine had no effect on GC. Methionine and serotonin bind to surface receptors of the ciliates with high affinity. K(D) for [methyl-3H] methionine binding to D. anser and T. pyriformis were 7.5 and 35.6 nM, and for [3H] serotonin binding were 2.7 and 4.7 nM, respectively. Alanine and tyrosine bind to the ciliates with low affinity. Thus, ciliates D. anser and T. pyriformis have chemosignaling systems regulated by amino acids and their derivatives and including the enzymes with cyclase activity. There is an assumption that these systems are similar to hormonal signaling systems of higher eukaryotes and are their predecessors.     

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.     

Inhibition by calcium of mammalian adenylyl cyclases

Ca(2+) regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca(2+) inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca(2+)-stimulable and Ca(2+)-inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca(2+) responses, suggesting the presence of two Ca(2+) sites of high ( approximately 0.2 microM) and low affinity ( approximately 0.1 mM). With a high affinity, Ca(2+) (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca(2+) inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg(2+) site(s). A remarkable selectivity for Ca(2+) was displayed by the high affinity site, with a K(i) value of approximately 0.2 microM, in the face of a 5000-fold excess of Mg(2+). The present results show that high and low affinity inhibition by Ca(2+) can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca(2+)-inhibitable adenylyl cyclases.     

Particulate guanylyl cyclases: multiple mechanisms of activation

Cyclic GMP (cGMP), a key messenger in several signal transduction pathways, is synthesized from GTP by a family of enzymes termed guanylyl cyclases, which are found in two forms: cytosolic (soluble) and membrane-bound (particulate). The past decade has brought significant progress in understanding the molecular mechanisms that underlie the regulation of particulate guanylyl cyclases and new members of their family have been identified. It has become more evident that the basic mechanism of catalysis of guanylyl cyclases is analogous to that recognized in adenylyl cyclases. Here we review the known basic mechanisms that contribute to the regulation of particulate guanylyl cyclases.     

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.     

Regulation of prokaryotic adenylyl cyclases by CO2

The Slr1991 adenylyl cyclase of the model prokaroyte Synechocystis PCC 6803 was stimulated 2-fold at 20 mM total C(i) (inorganic carbon) at pH 7.5 through an increase in k(cat). A dose response demonstrated an EC50 of 52.7 mM total C(i) at pH 6.5. Slr1991 adenylyl cyclase was activated by CO2, but not by HCO3-. CO2 regulation of adenylyl cyclase was conserved in the CyaB1 adenylyl cyclase of Anabaena PCC 7120. These adenylyl cyclases represent the only identified signalling enzymes directly activated by CO2. The findings prompt an urgent reassessment of the activating carbon species for proposed HCO3--activated adenylyl cyclases.     

Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases

Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR-1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology.