Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

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.     

The adenylyl cyclase family

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase.While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.     

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.     

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.     

Use of nucleotide photoaffinity probes to study hormone action

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.     

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.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

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.     

Plant purine nucleotide cyclases

Cyclic nucleotides (cAMP and cGMP) play an essential role in many important cellular processes in prokaryotic and eukaryotic organisms. They are produced by purine nucleotide cyclases: adenylyl and guanylyl cyclases. They are classified as one of two distinct forms: soluble and bound to membranes. Beside the differences in enzyme localization, the domain structure and regulation of enzymes activity are also diverse. However, all cyclases possess three groups of important residues: substrate specifying residue, metal binding residues and transition state stabilization residues. The natural occurrence of cyclic nucleotides in plants is now established. It was shown that in higher plants cNMPs act as a second messengers in a large number of (patho)physiological responses. However, it is only recently that the first plant enzymes with AC and GC activity of the unique structure have been identified and functionally characterized. In this study a systematic analysis of all the known prokaryotic, fungal and animal cyclases was done and direct evidences for the presence AC and GC in plant cells were shown.     

Internal symmetry of the mirror type and repeats in molecules of the membrane-bound forms of adenylate cyclase]

The analysis of mirror type internal symmetry distribution in primary structures of different types of mammalian membrane-bound adenylyl cyclases was made. The transmembrane domain clusters determining enzyme topology in membrane, a highly conservative region of cytoplasmic domains forming both catalytic and regulatory centres of adenylyl cyclases, and the functionally important regions in variable parts of their molecules (in particular, calmodulin binding regions) are shown to have symmetrical structures. These data are in conformity with a hypothesis put forward by the authors: the centres of internal symmetry may commonly either coincide with sites responsible for protein biological activity, or be spaced in the immediate vicinity of these sites. In different types of adenylyl cyclases long repeating sequences were identified. The segmentary structures were established for some enzyme subdomains. The regions containing repeats usually displayed a symmetrical structure which confirms a positive correlation between internal symmetry of amino acid sequence and its repeat distribution.     

Emerging themes of cAMP regulation of the pulmonary endothelial barrier

The presence of excess fluid in the interstitium and air spaces of the lung presents severe restrictions to gas exchange. The pulmonary endothelial barrier regulates the flux of fluid and plasma proteins from the vascular space into the underlying tissue. The integrity of this endothelial barrier is dynamically regulated by transitions in cAMP (3',5'-cyclic adenosine monophosphate), which are synthesized in discrete subcellular compartments. Cyclic AMP generated in the subplasma membrane compartment acts through PKA and Epac (exchange protein directly activated by cAMP) to tighten cell adhesions, strengthen cortical actin, reduce actomyosin contraction, and decrease permeability. Confining cAMP within the subplasma membrane space is critical to its barrier-protective properties. When cAMP escapes the near membrane compartment and gains access to the cytosolic compartment, or when soluble adenylyl cyclases generate cAMP within the cytosolic compartment, this second messenger activates established cytosolic cAMP signaling cascades to perturb the endothelial barrier through PKA-mediated disruption of microtubules. Thus the concept of cAMP compartmentalization in endothelial barrier regulation is gaining momentum and new possibilities are being unveiled for cytosolic cAMP signaling with the emergence of the bicarbonate-regulated mammalian soluble adenylyl cyclase (sAC or AC10).     

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.     

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.     

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.