Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction.     

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.     

Conservation of functional domain structure in bicarbonate-regulated “soluble” adenylyl cyclases in bacteria and eukaryotes

Soluble adenylyl cyclase (sAC) is an evolutionarily conserved bicarbonate sensor. In mammals, it is responsible for bicarbonate-induced, cAMP-dependent processes in sperm required for fertilization and postulated to be involved in other bicarbonate- and carbon dioxide-dependent functions throughout the body. Among eukaryotes, sAC-like cyclases have been detected in mammals and in the fungi Dictyostelium; these enzymes display extensive similarity extending through two cyclase catalytic domains and a long carboxy terminal extension. sAC-like cyclases are also found in a number of bacterial phyla (Cyanobacteria, Actinobacteria, and Proteobacteria), but these enzymes generally possess only a single catalytic domain and little, if any, homology with the remainder of the mammalian protein. Database mining through a number of recently sequenced genomes identified sAC orthologues in additional metazoan phyla (Arthropoda and Chordata) and additional bacterial phyla (Chloroflexi). Interestingly, the Chloroflexi sAC-like cyclases, a family of three enzymes from the thermophilic eubacterium, Chloroflexus aurantiacus, are more similar to eukaryotic sAC-like cyclases (i.e., mammalian sAC and Dictyostelium SgcA) than they are to other bacterial adenylyl cyclases (ACs) (i.e., from Cyanobacteria). The Chloroflexus sAC-like cyclases each possess two cyclase catalytic domains and extensive similarity with mammalian enzymes through their carboxy termini. We cloned one of the Chloroflexus sAC-like cyclases and confirmed it to be stimulated by bicarbonate. These data extend the family of organisms possessing bicarbonate-responsive ACs to numerous phyla within the bacterial and eukaryotic kingdoms.The nucleotide sequence of rabbit sAC has been deposited (GenBank accession number AY212921)     

Plant Adenylate Cyclases

Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.     

Structure-functional organization of adenylyl cyclases of unicellular eukaryotes and molecular mechanisms of their regulation

Adenylyl cyclases, the enzymes which catalyze the formation of the second messenger cAMP, are presently known to exist in yeast and related fungi, the amoeba Dictyostelium discoideum, flagellates, plasmodium, and infusoria. However, their structure-functional organization and molecular mechanisms of regulation differ considerably. Thus, in flagellates, tens of structurally similar adenylyl cyclase one-pass transmembrane proteins performing receptor functions have been discovered. In the amoeba D. discoideum, three types of adenylyl cyclases were detected, which differ by their topology, domain organization, and sensitivity to regulatory molecules and physical factors, one of which, adenylyl cyclase-A (AC-A), is similar to mammalian membrane-bound adenylyl cyclases and regulated by extracellular cAMP. Yeasts, in turn, have been shown to possess adenylyl cyclases that do not have transmembrane domains, but are able to form intermolecular complexes stabilized by interactions between repeated regions enriched in leucine residues. The data presented in this review indicate that the main molecular mechanisms underlying the actions of vertebrate adenylyl cyclases evolved as early as in the unicellular organisms and fungi. The structures and functions of adenylyl cyclases of the lower eukaryotes are much more diverse, which might be due both to the peculiarities of their life cycles and to the development at the initial stages of evolution of different models for the functioning and regulation of cAMP-dependent signaling cascades.     

Kinetic properties of "soluble" adenylyl cyclase. Synergism between calcium and bicarbonate

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V(max) of the enzyme with little effect on the apparent K(m) for substrate, ATP-Mg(2+). Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K(m) for ATP-Mg(2+). Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels.     

Soluble adenylyl cyclase mediates nerve growth factor-induced activation of Rap1

Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC) and G protein-regulated transmembrane adenylyl cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

The class III adenylyl cyclases: multi-purpose signalling modules

cAMP serves as a second messenger in virtually all organisms. The most wide-spread class of cAMP-generating enzymes are the class III adenylyl cyclases. Most class III adenylyl cyclases are multi-domain proteins. The catalytic domains exclusively work as dimers, catalysis proceeds at the dimer interface, so that both monomers provide catalytic residues to each catalytic center. Inspection of amino acid sequence profiles suggests a division of the class III adenylyl cyclases in to four subclasses, class IIIa–IIId. Genome projects and postgenomic analysis have provided novel aspects in terms of catalysis and regulation. Alterations in the canonical catalytic residues occur in all four subclasses suggesting a plasticity of the catalytic mechanisms. The vast variety of additional, probably regulatory modules found in class III adenylyl cyclases obviously reflects a large collection of regulatory inputs the catalytic domains have adapted to. The large versatility of class III adenylyl cyclase catalytic domains remains a major scientific challenge.     

Modulation of G(ialpha(2)) signaling by the axonal guidance molecule UNC5H2

The G protein, G(ialpha(2)), regulates a number of cellular functions including cell migration, proliferation, and differentiation. The transduction of signal depends on the ability of the alpha subunit to cycle between a GDP bound and an active GTP bound state capable of interacting with intracellular enzymes. Here, we now report the novel interaction of gip2 (constitutively activated G(ialpha(2))) with the cytoplasmic domain of UNC5H2. Like G(ialpha(2)), we found that UNC5H2 is widely expressed particularly in cells which migrate. UNC5H2 binds G(ialpha(2)) when it is charged with GTP. The interaction of G(ialpha(2)) and UNC5H2 liberated adenylyl cyclase from G(ialpha(2)) inhibition. Thus, by sequestering the alpha subunit, UNC5H2 is a novel inhibitor of G(ialpha(2)) thereby increasing intracellular cAMP levels. The expression of UNC5H2 in the brain and immune system suggests that this novel inhibitor of G protein signaling may have broad significance for axonal guidance and chemotaxis.     

Dictyostelium development-socializing through cAMP

Starving Dictyostelium amoebae use cAMP as a chemoattractant to gather into aggregates, as a hormone-like signal to induce cell differentiation, and as an intracellular messenger to control stalk- and spore cell maturation and germination of spores. In this chapter we describe the respective roles of the three adenylyl cyclases ACA, ACB and ACG in controlling cAMP signaling during development and we discuss how cAMP signals are processed by the cells to trigger the large repertoire of gene regulatory events that is under control of this signal molecule.     

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.     

CO2/HCO3?- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.     

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Two domains are critical for the nuclear localization of soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC) is a newly identified source of cyclic adenosine 3',5'-monophosphate (cAMP). Unlike the well-known transmembrane adenylyl cyclases (tmACs), sAC locates to the nucleus, mitochondria and microtubules. For most cAMP-signaling microdomains, there is always an AC nearby, for example tmAC. But it was until the discovery of sAC that there was not known cAMP resource in the nucleus. sAC associates with nuclear cAMP-signaling microdomains, which were once considered to depend on the diffusion of cAMP produced by tmAC. In this report, we focus on the truncated soluble adenylyl cyclase (tsAC), the most common existence form of sAC in tissues. Two domains (145-200 aa and 257-318 aa) related with sAC nuclear localization were present here. The findings provide evidence that these two domains are critical for the nuclear localization of sAC and they collocated with the catalytic domains.     

Protective role of soluble adenylyl cyclase against reperfusion-induced injury of cardiac cells

Aims

Disturbance of mitochondrial function significantly contributes to the myocardial injury that occurs during reperfusion. Increasing evidence suggests a role of intra-mitochondrial cyclic AMP (cAMP) signaling in promoting respiration and ATP synthesis. Mitochondrial levels of cAMP are controlled by type 10 soluble adenylyl cyclase (sAC) and phosphodiesterase 2 (PDE2), however their role in the reperfusion-induced injury remains unknown. Here we aimed to examine whether sAC may support cardiomyocyte survival during reperfusion.

A structural basis for the role of nucleotide specifying residues in regulating the oligomerization of the Rv1625c adenylyl cyclase from M. tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7A. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase.     

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.