Functional characterization of transmembrane adenylyl cyclases from the honeybee brain

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far.Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC_50s of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC_50s of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC.In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior.     

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I_Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca²⁺ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I_Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca²⁺ influx via LTCCs on AC activity, leading to the inability to inhibit AC.     

CyaC,a redox‐regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme‐B membrane anchor domain

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane‐integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra‐histidine signature that is universally present in the membrane anchors of bacterial diheme‐B succinate‐quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme‐B protein and carried a binuclear iron‐sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme‐B in the membrane and loss of AC activity. Heme‐B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q₀ or Q₀H₂). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q₀ and O₂, or NADH and Q₀H₂ respectively. We conclude that CyaC‐like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.     

Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca²⁺-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca²⁺-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism.     

Genetic selection in<Emphasis Type="Italic"> Saccharomyces</Emphasis> of mutant mammalian adenylyl cyclases with elevated basal activities

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.     

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.     

Methionine Induces Sexual Development in the Fission Yeast Schizosaccharomyces pombe via an ste11-Dependent Signalling Pathway

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe. In heterothallic strains it induces G₁ arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.     

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells

Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.     

Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae. The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE). Insertion of two and six copies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17β-estradiol (E₂). Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde). Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner. The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2μ-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors. The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds. The estrogenic compounds, E₂, diethylstilbestrol (DES), and estrone (EST), activated expression of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells. None of the antiestrogens, at concentrations up to 1 μM, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E₂ against either form of the ER. In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentrations of 300 nM or greater to the ERcde. This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effectors of the ER.     

Transformation with Vectors Harboring the NEO Selection Marker Induces Germination-Specific Adenylylcyclase Activity in Dictyostelium Cells

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-R_M) and control R-subunits (PKA-R_C) were overexpressed in ACA null routants. Both transformations as well as transformation with an unrelated vector, carrying the same NEO^R selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEO^R selection marker induces ectopic ACG activity in Dictyostelium cells.     

The Adenylyl Cyclase Inhibitor MDL-12,330A Potentiates Insulin Secretion via Blockade of Voltage-Dependent K+ Channels in Pancreatic Beta Cells

Objective

Adenylyl cyclases (ACs) play important role in regulating pancreatic beta cell growth, survival and secretion through the synthesis of cyclic AMP (cAMP). MDL-12,330A and SQ 22536 are two AC inhibitors used widely to establish the role of ACs. The goal of this study was to examine the effects of MDL-12,330A and SQ 22536 on insulin secretion and underlying mechanisms.

Methods

Patch-clamp recording, Ca²⁺ fluorescence imaging and radioimmunoassay were used to measure outward K⁺ currents, action potentials (APs), intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion from rat pancreatic beta cells.

Results

MDL-12,330A (10 µmol/l) potentiated insulin secretion to 1.7 times of control in the presence of 8.3 mmol/l glucose, while SQ 22536 did not show significant effect on insulin secretion. MDL-12,330A prolonged AP durations (APDs) by inhibiting voltage-dependent K⁺ (K_V) channels, leading to an increase in [Ca²⁺]_i levels. It appeared that these effects induced by MDL-12,330A did not result from AC inhibition, since SQ 22536 did not show such effects. Furthermore, inhibition of the downstream effectors of AC/cAMP signaling by PKA inhibitor H89 and Epac inhibitor ESI-09, did not affect K_V channels and insulin secretion.

Conclusion

The putative AC inhibitor MDL-12,330A enhances [Ca²⁺]_i and insulin secretion via inhibition of K_V channels rather than AC antagonism in beta cells, suggesting that the non-specific effects is needed to be considered for the right interpretation of the experimental results using this agent in the analyses of the role of AC in cell function.     

Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca²⁺/calmodulin (CaM), have discrete neuronal functions. Although the Ca²⁺ sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca²⁺/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca²⁺/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca²⁺/CaM both in vivo and in vitro and explore the different role of each Ca²⁺-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca²⁺ entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca²⁺ events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca²⁺ dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.Nine membrane-bound mammalian adenylyl cyclases (ACs),² AC1–AC9, have been identified (1). They possess a common predicted structure (2)³ and are stimulated by forskolin (FSK; except AC9) and G_sα, although they are distributed and regulated differently (1, 3, 4). Four ACs are regulated by physiological concentrations of Ca²⁺ and thereby provide a critical link between the Ca²⁺- and cAMP-signaling pathways (3, 5); AC5 and AC6 are directly inhibited by Ca²⁺, whereas AC1 and AC8 are stimulated by Ca²⁺ in a calmodulin (CaM)-dependent manner (5). AC3 is also regulated by CaM in vitro, although this requires supramicromolar concentration of Ca²⁺ (6), and in vivo AC3 is inhibited by Ca²⁺ via CaM kinase II (7), unlike AC1 and AC8.AC1 is closely related in sequence to the Ca²⁺/CaM-stimulable rutabaga AC from Drosophila, which is important in Drosophila learning tasks (8–10). AC1 and the other Ca²⁺/CaM-stimulable mammalian AC, AC8, have also been implicated in learning and memory (11). As a means of establishing their proposed roles, single and/or double AC1 and AC8 knockout mice have been generated. Mouse models have demonstrated that Ca²⁺/CaM-stimulable ACs are involved in long-term potentiation and long-term memory (12). However, despite the general view that AC1 and AC8 can behave similarly, discrete physiological actions of each isoform are becoming apparent. Recent studies by Zhuo''s group demonstrated that AC1 specifically participates in N-methyl-d-aspartic acid receptor-induced neuronal excitotoxicity (13) and an increase in GluR1 synthesis induced by blocking AMPA receptors (14). Furthermore, Nicol and colleagues (15, 16) showed a contribution of AC1, but not AC8, in axon terminal refinement in the retina. On the other hand, AC8 specifically was seen to be responsible for retrieval from adaptive presynaptic silencing (17) and the acquiring of new spatial information (18). These differences in physiological roles must reflect not only differences in their distributions but also presumably in their regulatory properties. Both enzymes are expressed in brain; AC1 is neuro-specific, whereas the expression of AC8 is more widespread (1, 12). Within the central nervous system, AC1 is abundant in the hippocampus, the cerebral cortex, and the granule cells of the cerebellum, whereas AC8 has a high expression level in the thalamus and the cerebral cortex (19). Studies of mouse brain revealed that AC1 is distributed pre-synaptically and AC8 post-synaptically (18, 20).Although physiological differences in the roles of these two enzymes are suggested from the studies outlined above, the regulatory mechanisms that might underlie these differences are not. AC1 is more sensitive to Ca²⁺ than is AC8 in vitro (21, 22), yet details on how these two enzymes are regulated by Ca²⁺/CaM are sparse. In non-excitable cells, a Ca²⁺ elevation caused by capacitative Ca²⁺ entry (CCE), the mode of Ca²⁺ entry triggered by emptying Ca²⁺ from internal stores (23), preferentially stimulates AC1 and AC8 (21). Although stimulation of AC8 by CCE has been shown to be at least partially dependent on its localization at lipid rafts (24), whether AC1 is also targeted to this region of plasma membranes has never been addressed. In addition, CaM regulation of AC1 and AC8 has not been compared in detail, although CaM appears to bind to different domains of the two enzymes. AC8 utilizes two CaM binding domains: a classic amphipathic “1-5-8-14” motif at the N terminus and an IQ-like motif in the C2b domain (25). A recent study indicates that CaM pre-associates with the N terminus of AC8, where it becomes fully saturated upon a Ca²⁺ rise, and activates the enzyme via a C-terminally mediated relief of auto-inhibitory mechanisms (26). By contrast, only residues 495–522 of the C1b region of AC1 have been shown to bind CaM in a Ca²⁺-dependent manner (27, 28). With the presence of only one CaM binding domain in AC1, a simpler mechanism of CaM regulation might be expected.CaM mediates the regulation of numerous Ca²⁺-dependent processes in eukaryotic cells (29). The protein possesses N- and C-terminal lobes, both of which contain two Ca²⁺ binding EF hands (EF1 and EF2 in the N lobe, and EF3 and EF4 in the C lobe (30)). Mutations in the EF hands have been valuable for investigating the interaction of CaM with its targets. Alanine substitutions in the EF12 pair or EF34 pair have generated CaM₁₂ and CaM₃₄ to investigate the independent function of the C and N lobes of CaM, respectively (31, 32).Against the background of the distinct physiological roles carried out by AC1 and AC8, we performed a detailed comparison of the two enzymes expressed in HEK 293 cells. Their sensitivity to Ca²⁺/CaM was compared both in vitro and in vivo; the possibility that they might be expressed in different domains of the plasma membrane was addressed; and putative lobe-specific CaM regulation was assessed using Ca²⁺-binding mutants of CaM. Single cell measurements using a FRET-based cAMP sensor were performed to compare the kinetic responses of the enzymes to physiological elevations of [Ca²⁺]_i. The results demonstrate superficial similarities in the regulation of AC1 and AC8 but critical disparities in their mechanism of activation by the lobes of CaM and in the speed and pattern of their responsiveness to [Ca²⁺]_i. These discrete behaviors provide a physiological opportunity for different outcomes to elevation of [Ca²⁺]_i, depending on the AC that is expressed in particular contexts.     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.