Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.     

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.     

Gbetagamma activation site in adenylyl cyclase type II. Adenylyl cyclase type III is inhibited by Gbetagamma

The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.     

Ultrastructural localization of olfactory transduction components: the G protein subunit Golf alpha and type III adenylyl cyclase.

Electron microscopy and postembedding immunocytochemistry on rapidly frozen, freeze-substituted specimens of rat olfactory epithelia were used to study the subcellular localization of the transduction proteins Golf alpha and type III adenylyl cyclase. Antibody binding sites for both of these proteins occur in the same receptor cell compartments, the distal segments of the olfactory cilia. These segments line the boundary between organism and external environment inside the olfactory part of the nasal cavity. Therefore, they are the receptor cell regions that most likely first encounter odorous compounds. The results presented here provide direct evidence to support the conclusion that the distal segments of the cilia contain the sites of the early events of olfactory transduction.     

Effects of Ca2+ and calmodulin on adenylyl cyclase activity in sheep olfactory epithelium

Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca²⁺/calmodulin, as suggested by the results obtained both in Ca²⁺/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca²⁺ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca²⁺/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca²⁺/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed.     

Amitriptyline reduces olfactory neuron adenylyl cyclase activity

Adenylyl cyclase plays an important role in olfactory signaltransduction. Recently, a novel type III adenylyl cyclase hasbeen localized in olfactory neurons (Pfeuffer et al., 1989;Bakalyar and Reed, 1990). Because amitriptyline (AMI), a tricyclicantidepressant, appears to have an inhibitory effect on adenylylcyclase activity in other in other neuronal tissue (Yamaokaet al., 1988; Wong et al., 1991), we measured the effect ofAMI on forskolin-stimulated adenylyl cyclase activity in membranepreparations of olfactory mucosa from adult rats. In the presenceof 5'-guanylyl-imidodiphosphate, AMI (0.5–8.0 µM)inhibited forskolin-stimulated adenylyl cyclase activity ina dose-dependent manner. To determine whether this effect wasspecific for olfactory neurons, as opposed to other cells inthe olfactory epithelium, rats were unilaterally bulbectomizedin order to reduce selectively the number of olfactory neuronson the side ipsilateral to the bulbectomy. In membrane preparationsfrom unilaterally bulbectomized animals we saw significantlylower adenylyl cyclase activity in ipsilateral olfactory mucosa,compared with adenylyl cyclase activity from non-bulbectomizedmucosa. These results indicate that AMI inhibition of adenylylcyclase activity is primariy localized in olfactory neurons.     

Chromosomal mapping of human adenylyl cyclase genes type III,type V and type VI

Adenylyl cyclase activity plays a central role in the regulation of most cellular processes. At least eight different adenylyl cyclases have been identified, which are endowed with various and sometimes opposing regulatory properties. Recently we have localized the human genes encoding two of these adenylyl cyclases: the gene for type 11 adenylyl cyclase is located on chromosome 2 (sub-band 2p15.3), the gene for type VIII is located on chromosome 8 (sub-band 8824.2). More recently the type I gene has been located on chromosome 7 (sub-band 7pl2–7p13). Using in situ hybridization, we have now localized the genes for three other adenylyl cyclases: the type III gene has been localized on chromosome 2 in the sub-band 2p22–2p24, the type V gene on chromosome 3 at position 3q13.2–3q21, and the type VI gene on chromosome 12 at position 12q12–12q13. It therefore appears that all adenylyl cyclase genes, known at present are located on different chromosomes and thus are likely to be independently regulated.     

The role of calmodulin recruitment in Ca2+ stimulation of adenylyl cyclase type 8

Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.     

Stimulation of heart sarcolemmal calcium pump by calmodulin

The sarcolemmal membranes obtained from rat heart by sucrose-density gradient method were found to exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase and ATP-dependent Ca²⁺ binding activities. The Ca²⁺ stimulated ATPase activity was increased by calmodulin; maximal effect was seen at 1 to 5 μg/ml concentrations of calmodulin. The observed activation of the enzyme was associated with an increase in Vmax value from 3.45 to 5.26 μmol Pi/mg protein/hr and a decrease in Ka value from 2.78 to 0.84 μM Ca²⁺. Calmodulin was also found to increase ATP-dependent Ca²⁺ binding by 1.6 to 2.2 fold. These results suggest that the activity of Ca²⁺ pump mechanism in heart sarcolemma is regulated by calmodulin.     

Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.     

Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and G(betagamma)

Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules G(betagamma) and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, G(betagamma) and calmodulin share a two-site-interaction mechanism with G(alphas) and forskolin to modulate AC activity. Forskolin and G(alphas) are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C(1) of the specifically regulated AC subtype had been ascribed for both G(betagamma) and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C(2) is necessary to render enzyme activity susceptible to G(betagamma) or calmodulin modulation. In addition to the PFAHL motif in C(1b) of ACII, G(betagamma) contacts the KF loop in C(2), whereas calmodulin requires not only the Ca2+-independent AC28 region in C(1b) but also a Ca2+-dependent domain in C(2a) of ACI containing the VLG loop to stimulate this AC isoform.     

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED₅₀=1.8±1.4 nM)>VIP(ED₅₀=25.0±7.0 nM)>PHI (ED₅₀=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of _s and _i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of¹²⁵I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.     

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.     

Calmodulin-binding sites on adenylyl cyclase type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.     

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc.     

Quantitative reduction of type I adenylyl cyclase in human alcoholics.

The amounts of adenylyl cyclase type I (AC I) were examined in various parts of the postmortem brains from alcoholics who prior to death had been abstinent from alcohol for at least 6 months and compared with controls using immunoblot analysis with anti-AC I specific antibody. It was revealed that a significant reduction of AC I was observed in both frontal and temporal cortices. On the other hand, in other areas (occipital cortex, caudate nucleus, putamen, and hippocampus) the amounts were comparable between alcoholics and controls. In the next step, we examined two subtypes of human AC mRNA levels (AC I and AC VIII) in blood cells by quantitative RT-PCR using [alpha-32P]dCTP with two sets of the synthetic oligonucleotide primers based on the DNA sequences reported elsewhere (Villacres, E.C. et al., Genomics 16 (1993) 473-478; J. Parma et al., Biochem. Biophys. Res. Commun. 179 (1991) 455-462). The amounts of amplified DNAs of both AC I and AC VIII were significantly smaller in alcoholics than in controls. On the other hand, the amounts of amplified DNA of beta-actin DNA were almost equal between alcoholics and controls. It appears from these results that a reduction in the amount of AC subtypes may be a biological marker for alcoholics.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Regulation of adenylate cyclase from Leucophaea maderae by calcium,calmodulin and forskolin

1. Adenylate cyclase was assayed in homogenates ofhindgut tissue from Leucophaea maderae (L.). The 10,000 g supernatant enzyme was stimulated by calmodulin.2. Trifluoperazine inhibited calmodulin-stimulated adenylate cyclase activity.3. Elevated calcium levels (> 100 μM) inhibited natural and calmodulin-stimulated enzymic activity.4. Forskolin (1 mM) stimulated adenylate cyclase activity by approximately 10-fold.