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Research ArticleCELLULAR AND MOLECULAR

Novel Regulatory Properties of Human Type 9 Adenylate Cyclase

Medhane G. Cumbay and Val J. Watts
Journal of Pharmacology and Experimental Therapeutics July 2004, 310 (1) 108-115; DOI: https://doi.org/10.1124/jpet.104.065748

Abstract

Nine membrane-bound members of the mammalian adenylate cyclase family have been identified. The least characterized and most divergent in sequence of the nine adenylate cyclase isoforms is AC9. Stimulation by Gαs and inhibition by Ca2+/calcineurin are two modes of regulation that have been reported for AC9. We explored the possibility of additional modes of regulation of human AC9. We now report that quinpirole activation of the inhibitory G protein-coupled D2L dopamine receptor inhibits Gαs stimulation of AC9 by approximately 50%. The effects of quinpirole were reversed by the D2 antagonist spiperone and by pertussis toxin pretreatment. We also report the first evidence for regulation of AC9 by protein kinase C (PKC). Specifically, phorbol ester activation of PKC significantly attenuated (∼50%) Gαs-stimulated AC9 activity. The effect of PKC activation on AC9 was reversed by the PKC inhibitor bisindolylmaleimide. Gαs-stimulated cyclic accumulation was reduced more by simultaneous addition of both quinpirole and phorbol 12-myristate 13-acetate than by either drug alone. Additional studies investigated the role of glycosylation on AC9 activity. The results show that blocking glycosylation of AC9 significantly attenuates Gαs stimulation. In contrast, the ability of PKC and Gαi/o to negatively regulate AC9 did not seem to be affected by the glycosylation state of AC9. These observations demonstrate the diverse regulatory features of AC9 and the ability of AC9 to integrate multiple signals.

Adenylate cyclases (EC 4.6.1.1) produce the second messenger cyclic AMP in response to activation of multiple signal transduction pathways. Currently, nine membrane-bound members of the mammalian adenylate cyclase family (AC1–9) have been identified. Various agents regulate the activity of adenylate cyclases, including G proteins, protein kinases, Ca2+, Mg2+, and the plant-derived diterpene forskolin (Taussig and Zimmermann, 1998; Chern, 2000; Defer et al., 2000; Sunahara and Taussig, 2002). All nine membrane-bound mammal adenylate cyclases are sensitive to stimulation by Gαs and Mg2+, and all but AC9 are potently activated by forskolin. The additional factors that regulate adenylate cyclase activity are isoform-specific and mechanistically distinct between isoforms. For example, AC1, AC3, and AC8 are stimulated by Ca2+/calmodulin, whereas AC5 and AC6 are inhibited by Ca2+ directly acting on these enzymes (Cooper et al., 1995; Guillou et al., 1999). A number of adenylate cyclase isoforms are also differentially regulated by protein kinases. For instance, phorbol 12-myristate 13-acetate (PMA) activation of protein kinase C (PKC) stimulates the activity of AC2 and AC7 but inhibits the activity of AC4 and AC6 (Yoshimura and Cooper, 1993). The diversity in regulation among the different isoforms of adenylate cyclases coupled with the ability of each isoform to integrate numerous signals can make prediction of cyclic AMP signals difficult.

Essential to interpreting and predicting the cyclic AMP response in cells that have a complex pattern of adenylate cyclase expression is identifying the regulatory characteristics of each isoform. The least characterized and most divergent in sequence of the adenylate cyclase isoforms is AC9 (Premont et al., 1996; Patel et al., 2001). Northern blot, immunocytochemistry, in situ hybridization, and RNA protection analysis indicate that AC9 is expressed widely in the central nervous system as well as in other major organs (Antoni et al., 1998; Hacker et al., 1998; Paterson et al., 2000; Sosunov et al., 2001). Of particular interest is the relatively abundant expression of AC9 message and protein in the hippocampus, a region important for learning and memory (Antoni et al., 1998). Stimulation by Gαs and inhibition by Ca2+/calcineurin are two modes of regulation that have been reported for AC9 (Paterson et al., 1995, 2000; Antoni et al., 1998; Hacker et al., 1998). Considering the complex regulation of other adenylate cyclase isoforms, we hypothesized that additional modes of regulation exist for AC9.

In the present study, we identified novel regulatory properties of human AC9. We report that Gαs stimulation of AC9 is inhibited by the activation of inhibitory G proteins that couple to dopamine D2L receptor. This inhibition is blocked by the D2 receptor-selective antagonist spiperone or by pertussis toxin pretreatment. We also report the first evidence for negative regulation of AC9 by PKC. Additional studies investigated the effect of glycosylation on AC9 activity using biochemical and genetic approaches. The results show that blocking glycosylation of AC9 significantly attenuates Gαs stimulation, whereas the inhibitory effects of PKC and Gαi/o on AC9 were not altered. These observations demonstrate that AC9 acts as a coincidence detector in a manner similar to that of other adenylate cyclase isoforms and displays unique regulatory properties.

Materials and Methods

Materials. [3H]Cyclic AMP was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Forskolin, (-)-quinpirole, and isoproterenol were purchased from Sigma/RBI (Natick, MA). PMA and calcimycin (A23187) were purchased from Calbiochem (La Jolla, CA). Fetal clone serum and bovine calf serum were purchased from Hyclone Laboratories (Logan, UT). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.

Cell Culture and Transient Transfection. HEK-D2L or HEK-D2L cells coexpressing AC1, AC2, or AC9 were derived and maintained as described previously (Cumbay and Watts, 2001). For transient transfections, cells were grown in 24-well plates until approximately 90% confluent. Cells were transfected using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. A ratio of 2 μl of LipofectAMINE 2000/1 μg of cDNA construct was used for all transfections. Cells were assayed 24 to 48 h after transfection.

Mutagenesis and Epitope Tagging of AC9. Glycosylation-deficient mutants (N955A and N964A) of AC9 were generated using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) in pcDNA3-AC9 construct. The primers 5′-CCC-CTTGACGCAGTACAGGCTTTCAGTTCCGAGAGG-3′/5′CCTCTCGGAACTGAA-AGCCTGTACTGCGTCAAGGGG-3′ (for N955A) and 5′-GAGAGGAACCCGTGCG-CTAGTTCGGTGCCGCGTGAC-3′/5′-GCACGCGGCACCGAACTAGCGCACGG-GTTCCTCTC-3′ (for N694A) were used with slight modification to the cycling protocol: 95°C for 1 min; 55°C for 0.5 min; 68°C for 20 min times 20 cycles. The mutations were confirmed by sequencing, and functional properties were determined by transient expression in HEK293 cells. Epitope tagging of AC9 was performed by polymerase chain reaction amplification of the 5′ end of AC9, from the ATG start codon to the internal MluI restriction site, using the primers 5′-GCCAGTGAATCGATGGCTTCC-CCGCCCCACCAGCTG-3′/5′-CAGGGCGAACACCAGCAGGGTGAG-3′, which introduced a 5′-ClaI restriction site integrated into the ATG codon. Similarly, the primers 5′-GTGCTGTCTAGAGAACCCAGAGATGTCTTGTCC-3′/5′-CAACATGCTGTGGTCCAACTTCAAGCTCCGCGTC-3′ were used to amplify the 3′ end, from internal NcoI site to the 3′ end of AC9 coding sequence, generating an XbaI site at the 3′ end of the coding sequence. The polymerase chain reaction products were digested to produce a 5′-ClaI/MluI fragment and a 3′-NcoI/XbaI fragment, and along with the MluI/NcoI fragment excised from pcDNA3-AC9 were subcloned into the pPICZα vector (Invitrogen) in frame with a 3′ end c-myc epitope tag (pPICZα-AC9-cmyc). The epitope-tagged AC9 construct was excised from pPICZα-AC9-cmyc by digestion with ClaI/BamHI and subcloned into pBluescript SK(-). The construct was further subcloned into pcDNA3 after excision with NotI/KpnI from pBluescript SK(-).

Western Blotting and Immunodetection. Transiently transfected cells were washed once with ice-cold phosphate-buffered saline (137 mM NaCl, 2.68 mM KCl, 10 mM Na2PO4, and 1.76 mM KH2PO4, pH 7.4) and placed on ice. Cells were washed once again in membrane isolation buffer (15 mM Na+/Hepes, pH 7.4, 250 mM sucrose, 1 mM dithiothreitol, and 0.3 mM phenylmethylsulfonyl fluoride) and resuspended in the same buffer. Glass-Teflon homogenizers were used to homogenize cells on ice (8–10 strokes). Homogenates were centrifuged at 750g for 10 min at 4°C. The supernatant was transferred to a fresh tube and centrifuged at 48,900g for 10 min at 4°C to collect the membrane fractions. Pellets were resuspended in sucrose-free membrane isolation buffer. Proteins were separated by polyacrylamide gel electrophoresis on a 7.5% precast Ready-Gels on Mini-PROTEAN 3 Cell electrophoresis systems from Bio-Rad (Hercules, CA). Approximately 50 μg of total protein was loaded per sample. The proteins were transferred onto polyvinylidene difluoride membranes for immunodetection with c-myc antibody (Cell Signaling Technology Inc., Beverly, MA).

Membrane Adenylate Cyclase Assay. HEK293 cells stably expressing AC9 were washed once with ice-cold phosphate-buffered saline (as defined above) and placed on ice. Cells were resuspended in deglycosylation buffer (15 mM Na+/Hepes, pH 7.4, 5 mM MgCl2, 1 μg/ml apoprotinin, and 2 μg/ml leupeptin) to produce an approximately 2 mg of protein/ml of cell suspension. Glass-Teflon homogenizers were used to homogenize (8–10 strokes) the suspended cells on ice. Total protein levels were determined with a BCA protein assay kit (Pierce Chemical, Rockford, IL). Approximately 1 mg of total protein was incubated with 2500 U of peptide:N-glycosidase F (PNGase F) or an equivalent volume of PNGase F storage buffer (50 mM NaCl, 20 mM Tris/HCl, pH 7.4, 5 mM Na+/EDTA, and 50% glycerol) for 45 min at 30°C. Membranes were placed on ice and allowed to cool for at least 15 min. For cyclic AMP accumulation assays, microcentrifuge tubes were placed on ice and reaction buffer was added to each tube (15 mM Na+/Hepes, pH 7.4, 5 mM MgCl2, 1 mM 3-isobutyl-1-methyl xanthine, 1 mM ATP, 20 mM phosphocreatine, 125 U of creatine phosphokinase) along with increasing concentrations of the nonhydrolyzable GTP analog 5′-guanylylimidodiphosphate (GppNHp). Approximately 30 to 40 μg of total protein from chilled membranes was added to each reaction tube on ice bringing the total volume of the reaction mixture to 100 μl. The tubes were transferred to 30°C water bath and incubated for 15 min. The reaction was terminated with 200 μl of ice-cold 3% trichloroacetic acid, and cyclic AMP levels were quantified as described below.

Cyclic AMP Accumulation Assay. Cells were seeded at densities between 100,000 and 150,000 cells/well in 24-well cluster plates and grown to confluence. The cells were preincubated for 10 min with 200 μl/well of assay buffer (Earle's balanced salt solution containing 0.02% ascorbic acid and 2% bovine calf serum). The cells were then placed on ice, and the indicated drugs were added. The cells were then incubated in a 37°C water bath for 15 min. After the incubation, the stimulation media were decanted, and the reaction was terminated with 200 μl/well of ice-cold 3% trichloroacetic acid. The 24-well cluster plates were stored at 4°C for up to 1 week before analysis.

Quantification of Cyclic AMP. Cyclic AMP accumulation was quantified using a competitive binding assay as described previously (Watts and Neve, 1996) with minor modifications. Duplicate samples of trichloroacetic acid cell extracts (15 μl) were added to reaction tubes. [3H]Cyclic AMP (∼1 nM final concentration) and cyclic AMP binding protein (∼150 mg) were diluted in cyclic AMP assay buffer (100 mM Tris/HCl, pH 7.4, 100 mM NaCl, and 5 mM EDTA) and then added to each well for a total volume of 550 ml. The tubes were incubated on ice for 2 h and harvested by filtration (Unifilter GF/C; PerkinElmer Life and Analytical Sciences) using a 96-well Filtermate cell harvester (PerkinElmer Life and Analytical Sciences). The filters were allowed to dry, and Microscint O scintillation fluid was added. Radioactivity on the filters was determined using a TopCount scintillation/luminescence detector (PerkinElmer Life and Analytical Sciences). Cyclic AMP concentrations in each sample were estimated in duplicate from a standard curve ranging from 0.1 to 300 pmol of cyclic AMP per assay.

Data Analysis. One-way ANOVA followed by Bonferroni's post hoc analysis was used for statistical comparison between multiple stimulation, transfection, and treatment conditions. Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA)

Results

D2L Dopamine Receptor Inhibition of AC9 Is Mediated by Gi/o Proteins. Dopamine D2L receptors couple to inhibitory heterotrimeric G proteins (Gi/o) and have been shown to inhibit drug-stimulated cyclic AMP accumulation in HEK293 cells (Watts and Neve, 1996). We used HEK293 cells stably expressing D2L receptors alone (HEK-D2L) or D2L in combination with AC9 (AC9/D2L) to examine the ability of inhibitory G proteins to regulate the activity of AC9. Activation of endogenous stimulatory G protein-coupled β-adrenergic receptors in HEK293 cells with isoproterenol (1 μM) produced a marked increase in cyclic AMP accumulation in AC9/D2L cells compared with HEK-D2L cells (HEK-D2L, 5.7 ± 1.1 pmol/well, n = 4; AC9/D2L, 104 ± 18 pmol/well, n = 10). The addition of the D2L agonist quinpirole significantly reduced cyclic AMP accumulation in AC9/D2L cells but did not completely block Gαs stimulation of AC9. Quinpirole activation of D2L receptors inhibited Gαs-activated cyclic AMP accumulation by approximately 50% (Fig. 1). Consistent with the proposed D2L-Gi/o-mediated inhibition of AC9, the addition of D2 receptor-selective antagonist spiperone (Fig. 1A) or pretreatment with pertussis toxin (Fig. 1B), an agent that uncouples Gi/o proteins from receptors, reversed the effects of quinpirole on Gαs stimulation of AC9. These data indicate that D2L receptor activation attenuates Gαs stimulation of AC9 in a Gi/o-dependent mechanism.

Fig. 1.
Fig. 1.

D2L dopamine receptor-mediated inhibition of cyclic AMP accumulation in HEK-AC9 cells. Cyclic AMP accumulation was measured under basal conditions, in presence of 1 μM Iso, or isoproterenol plus 10 μM quinpirole (+Quin). Where indicated, cells were stimulated in the presence of 1 μM spiperone (+ Quin + Spip) (A) or pretreated with 200 ng/ml pertussis toxin (+ Quin + PTX) (B) for 2 h before stimulation. Data shown are mean ± standard error of the mean of three to four independent experiments. *, p < 0.05 compared with isoproterenol stimulated cyclic AMP accumulation (one-way ANOVA with Bonferroni's post hoc test).

PKC Inhibits Gαs-Stimulated AC9 Activity. A number of adenylate cyclases are regulated by protein kinases. Our initial studies aimed at elucidating the regulatory properties of AC9 suggested a possible regulation by PKC. To further characterize PKC regulation of AC9, we explored the effects of the PKC activator PMA on Gαs-stimulated cyclic AMP accumulation in AC9/D2L cells. Additional studies examined PKC regulation of two other adenylate cyclase isoforms that exhibit differential regulation by PKC; AC2 (AC2/D2L), a PKC-stimulated isoform of adenylate cyclase, and AC1 (AC1/D2L), a PKC-insensitive isoform of adenylate cyclase. Using AC1 and AC2 for comparisons with AC9 provides an additional advantage because these adenylate cyclase isoforms can be selectively stimulated in HEK293 cells by the Ca2+ ionophore A23187 (AC1) and PMA (AC2) (Cumbay and Watts, 2001). We first characterized the ability of the individual agents to activate adenylate cyclases in the three cell lines. Isoproterenol treatment significantly increased cyclic AMP accumulation in AC1/D2L, AC2/D2L, and AC9/D2L cells (Fig. 2, A–C). A23187 stimulation produced significant increases in cyclic AMP accumulation in AC1/D2L cells (Fig. 2A) but did not alter cyclic AMP levels in AC2/D2L or AC9/D2L cells (Fig. 2, B and C). Similarly, PMA selectively elevated cyclic AMP levels in AC2/D2L cells (Fig. 2B) without altering cyclic AMP levels in AC1/D2L or AC9/D2L cells (Fig. 2, A and C). Having established the effects of A23187 or PMA alone on each of three cell lines, we next explored the effect of A23187 or PMA in combination with isoproterenol. Consistent with previous observations, isoproterenol in combination with A23187 or PMA synergistically enhanced cyclic AMP accumulation in AC1/D2L and AC2/D2L cells, respectively (Fig. 2, D and E) (Cumbay and Watts, 2001). To examine whether PKC could regulate Gαs-stimulated AC9 activity, we measured the effect of PMA on isoproterenol-stimulated cyclic AMP accumulation in AC9/D2L cells. The addition of PMA produced an approximately 60% reduction in the total isoproterenol-stimulated cyclic AMP accumulation (Fig. 2F). The effects of PMA on isoproterenol-stimulated cyclic AMP accumulation in AC2/D2L and AC9/D2L cells were blocked by the PKC inhibitor bisindolylmaleimide (Bis) (Fig. 2, E and F). Bisindolylmaleimide had no significant effect on isoproterenol-stimulated cyclic AMP levels in HEK cells stably expressing AC9 (Iso, 430 ± 36 pmol/well, n = 4; Iso + Bis, 487 ± 52 pmol/well, n = 3). Additional studies revealed that isoproterenol-stimulated cyclic AMP accumulation in HEK-D2L cells (Iso, 5.7 ± 1.1 pmol/well, n = 4; Iso + PMA, 7.5 ± 1.4 pmol/well, n = 4) and AC1/D2L cells was not affected by PMA (Fig. 2D). The ability of bisindolylmaleimide to attenuate the effect of PMA on AC9 is consistent with the ability of PMA to activate PKC and indicates that PKC can inhibit AC9 activity. That PMA activates both conventional and novel PKCs prompted us to examine the effects of Go6976, an inhibitor of conventional PKCs on AC9 activity. The results of these experiments revealed that concentrations up to 1 μM Go6976 failed to inhibit the effects of PMA on AC9 (Table 1). In contrast, 1 μM Go6976 prevented completely PMA-stimulated AC2 activity, suggesting different classes of PKC isoforms have differential specificity for individual adenylate cyclases (Table 1). Together, these observations suggest that AC9 is negatively regulated by a novel PKC isoform in HEK293 cells.

Fig. 2.
Fig. 2.

PKC modulation of Gαs-stimulated adenylate cyclase activity. Cyclic AMP accumulation in HEK-D2L cells expressing AC1, AC2, or AC9 was stimulated with 1 μM Iso, 3 μM A23187, 100 nM PMA, or 1 μM Bis alone or in combination. Data shown are mean ± standard error of the mean of four to nine independent experiments. *, p < 0.05 compared with vehicle (A–C) or isoproterenol-stimulated (D–F) cyclic AMP accumulation; †, p < 0.05 compared with isoproterenol plus PMA-stimulated (+PMA) cyclic AMP accumulation (one-way ANOVA with Bonferroni's post hoc test).

View this table:
TABLE 1

Effects of Go6976 on PKC inhibition of Gas-stimulated cyclic AMP accumulation in HEK-AC9 cells Cyclic AMP accumulation in HEK293 cells expressing AC2 or AC9 was stimulated with 1 μM Iso. Where indicated, isoproterenol-stimulated cyclic AMP accumulation was measured in the presence of 100 nM PMA (+PMA) or PMA combined with varying concentrations of Go6976. Data shown are mean ± standard error of the mean of four independent experiments.

Because adenylate cyclases can integrate multiple signaling pathways, we explored the ability of AC9 to integrate both Gi/o and PKC signals. Isoproterenol-stimulated cyclic AMP accumulation in AC9/D2L cells was determined in the presence of 10 μM quinpirole alone (a concentration that produces maximal inhibition), PMA alone, or both drugs combined. The simultaneous addition of both quinpirole and PMA further reduced the Gαs-stimulated cyclic accumulation compared with each drug individually (Fig. 3). The ability of PMA to enhance further maximal inhibition by quinpirole is consistent with independent mechanisms for the concurrent actions of Gi/o and PKC on AC9.

Fig. 3.
Fig. 3.

Simultaneous D2L dopamine receptor and PKC inhibition of AC9. Cyclic AMP accumulation in HEK-D2L cells expressing AC9 was measured in the presence of 1 μM Iso alone or with 10 μM quinpirole (+Quin), 100 nM PMA (+PMA), or quinpirole plus PMA (+ Quin + PMA). All data sets were normalized to isoproterenol-stimulated cyclic AMP accumulation (100%) for each independent experiment. Data shown are mean ± standard error of the mean of four independent experiments. The average value for isoproterenol-stimulated cyclic AMP accumulation was 205 ± 66 with a range of 127 to 404 pmol/well. *, p < 0.05 compared with isoproterenol alone; †, p < 0.05 compared with + Quin or + PMA condition (one-way ANOVA with Bonferroni's post hoc test).

Glycosylation State of AC9 Does Not Influence Its Sensitivity to Activation by Forskolin but Determines Its Relative Sensitivity to Activation by Gαs. Preventing the glycosylation of AC6 has recently been shown to alter its forskolin sensitivity without affecting its regulation by Gαs (Wu et al., 2001). We speculated that the relative insensitivity of AC9 to forskolin stimulation could be a consequence of its glycosylation state. To explore this possibility, we first studied the effects of tunicamycin treatment, an agent that blocks N-glycosylation of proteins, on subsequent AC9 activity. In HEK293 cells stably expressing AC9 (HEK-AC9), pretreatment with tunicamycin (10 μg/ml) for 24 h did not significantly alter forskolin-stimulated cyclic accumulation (Fig. 4A). Because tunicamycin blocks the N-glycosylation of all proteins, the nonspecific effects of tunicamycin may mask the influence of glycosylation on forskolin-stimulated AC9 activity. To directly address the role of glycosylation, we eliminated two potential N-glycosylation sites (N955 and N964) in extracellular loop 6 of AC9 by site-directed mutagenesis. The putative glycosylation sites were mutated to alanine to produce individual (N955A and N964A) and double (N955A/N964A) glycosylation-deficient mutants of AC9. Western blot analysis of transiently expressed wild-type and glycosylation mutants of AC9 in HEK293 cells revealed two immunoreactive bands (∼160 and ∼130 kDa) for AC9 wild type, N955A, and N964A corresponding to the glycosylated and nonglycosylated forms of AC9 (Fig. 5). In contrast, examination of HEK293 cells transiently transfected with N955A/N964A revealed only one immunoreactive band (∼130 kDa) equivalent to the nonglycosylated form of AC9 (Fig. 5). Furthermore, the single glycosylation mutants of AC9 (N955A and N964A) seemed to migrate slightly faster than wild-type AC9, indicating that both putative sites are glycosylated (Fig. 5). Comparison of forskolin-stimulated cyclic AMP accumulation in HEK293 cells transfected with AC9, N955A, N964A, or N955A/N964A demonstrated no significant differences in cyclic AMP levels among the different transfection conditions (Fig. 4B).

Fig. 4.
Fig. 4.

Effect of glycosylation on forskolin-stimulated AC9 activity. A, HEK293 cells stably expressing AC9 were grown to confluence and treated with vehicle or 10 μg/ml tunicamycin for 24 to 36 h in growth media. B, wild type (AC9) or a single (N955A, N964A) or double (N955A/N964A) glycosylation-deficient mutant of AC9 was transiently expressed in HEK293 cells. Cyclic AMP accumulation was measured in the presence of 10 μM forskolin (FSK). Data shown are mean ± standard error of the mean of four independent experiments.

Fig. 5.
Fig. 5.

Immunodetection of glycosylation-deficient mutants of AC9. HEK293 cells were transiently transfected with pcDNA3 vector or c-myc epitope-tagged wild type (AC9) or a single (N955A, N964A) or double (N955A/N964A) glycosylation mutants of AC9. Cells were harvested 36 h post-transfection, and membranes were isolated as described under Materials and Methods. Approximately 50 μg of protein was loaded in each well, and proteins were separated on a 7.5% polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membrane and immunoblotted with c-myc antibody. Immunoblot shown is a representative of three independent experiments. Arrows indicate the glycosylated (Gly) and nonglycosylated (non-Gly) forms of AC9.

Although the above-mentioned observations suggest that the glycosylation state of AC9 does not influence its sensitivity to forskolin activation, we sought to investigate the role of glycosylation on other functional features of AC9 (i.e., Gαs, PKC, and Gi/o). Preventing the glycosylation of AC9 by pretreating cells with tunicamycin dramatically reduced isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells (Fig. 6A). Transient transfection of wild-type and glycosylation-deficient mutants of AC9 in HEK293 cells followed by isoproterenol stimulation revealed a significantly reduced cyclic AMP accumulation in cells expressing N955A/N964A, but no significant reduction in the cyclic AMP levels were observed in N955A- or N964A-expressing cells compared with wild-type AC9-expressing cells (Fig. 6, B and C). Dose-response curves for isoproterenol stimulation of AC9 activity indicated a reduced maximal cyclic AMP accumulation in tunicamycin-treated or N955A/N964A-expressing cells compared with vehicle-treated or AC9-expressing cells, respectively (Fig. 6, A and B). However, the EC50 (wild type AC9 33 ± 5.2 nM, n = 6; N955A/N964A 38 ± 3.8 nM, n = 6) for isoproterenol-stimulated activity did not seem to be affected by the glycosylation state of AC9. We also sought to examine how the glycosylation state of AC9 could affect the two novel modes of regulation identified in the current study. Because glycosylation alters Gαs responsiveness (Fig. 6), the data examining PKC and Gi/o regulation were normalized to isoproterenol-stimulated cyclic AMP accumulation observed after transfection with AC9, N955A, N964A, or N955A/N964A as indicated. The addition of PMA to HEK293 cells transiently expressing wild-type or glycosylation-deficient AC9 mutants inhibited isoproterenol-stimulated cyclic AMP accumulation to the same extent (ca. 50%) independent of the glycosylation state (Fig. 7A). Similarly, maximal D2L receptor-mediated inhibition of Gαs-activated N955A/N964A activity was commensurate with that of wild-type AC9 (Fig. 7B).

Fig. 6.
Fig. 6.

Effect of glycosylation on Gαs-stimulated AC9 activity. A, HEK-AC9 cells were treated with vehicle or 10 μg/ml tunicamycin for 24 to 36 h in growth media. B and C, wild type (AC9) or a single (N955A, N964A) or double (N955A/N964A) glycosylation-deficient mutant of AC9 was transiently expressed in HEK293 cells. Cyclic AMP accumulation was measured in the presence of 1 μM isoproterenol. A and B, representative dose-response curves for three to four independent experiments. C, data shown are mean ± standard error of the mean of four independent experiments. *, p < 0.05 compared with isoproterenol-stimulated cyclic AMP accumulation in wild-type AC9-transfected HEK293 cells (one-way ANOVA with Bonferroni's post hoc test).

Fig. 7.
Fig. 7.

Inhibition of glycosylation-deficient mutants of AC9 by PKC and Gi/o proteins. Cyclic AMP accumulation in HEK293 cells transiently expressing wild type (AC9) or a single (N955A, N964A) or double (N955A/N964A) glycosylation-deficient mutant of AC9 was measured in the presence of 1 μM Iso alone or with 100 nM PMA (+PMA) (A) or 10 μM quinpirole (+Quin) (B). Data are normalized to isoproterenol-stimulated cyclic AMP accumulation (100%) observed after transfection with AC9, N955A, N964A, or N955A/N964A as indicated. Data are presented as the mean ± standard error of the mean of four independent experiments. *, p < 0.05 compared with isoproterenol-stimulated cyclic AMP accumulation for each transfection condition (one-way ANOVA with Bonferroni's post hoc test).

Apart from the direct effect of glycosylation, reduced trafficking to the plasma membrane may explain the reduced Gαs-stimulated cyclic AMP accumulation in N955A/N964A-expressing cells. Decreased trafficking to the plasma membrane would reduce membrane adenylate cyclase levels and consequently reduce Gαs-stimulated cyclic AMP levels. To address this possibility, we used membranes from HEK-AC9 cells, circumventing the potential for reduced trafficking of glycosylation-deficient mutants of AC9. This method also allows for the direct examination of the role of glycosylation with the use of a degylcosidase. Treating membranes with peptide:PNGase F for 45 min before stimulating cyclic AMP accumulation with 5′-GppNHp significantly attenuated Gαs-stimulated cyclic AMP accumulation compared with vehicle-treated membranes (Fig. 8). Combined, the data presented here indicate that the glycosylation state of AC9 selectively influences its activation by Gαs.

Fig. 8.
Fig. 8.

Gαs stimulation of AC9 after treatment with PNGase F. Membranes from HEK293 cells stably overexpressing AC9 were collected as described under Materials and Methods. Approximately 1 mg of total protein was incubated with 2500 U of PNGase F or an equivalent volume of PNGase F storage buffer (Vehicle) for 45 min at 30°C. Cyclic AMP accumulation was stimulated by adding increasing concentrations of the nonhydrolyzable GTP analog GppNHp and incubating membranes for 15 min in 30°C water bath. The reaction was terminated with 200 μl/well of ice-cold 3% trichloroacetic acid and cyclic AMP accumulation was determined as described under Materials and Methods. *, p < 0.05 compared with GppNHp-stimulated cyclic AMP accumulation in PNGase F-treated membranes (one-way ANOVA with Bonferroni's post hoc test).

Discussion

Adenylate cyclase type 9 is a unique member of the mammalian adenylate cyclase family. It is the most divergent in sequence and unlike the other isoforms, AC9 is relatively insensitive to activation by forskolin (Hacker et al., 1998; Yan et al., 1998). Little is known about the regulatory properties of AC9 apart from activation by Gαs and inhibition by calcineurin (Antoni et al., 1998; Hacker et al., 1998; Paterson et al., 2000). The data presented in this study demonstrate that activation of inhibitory G protein-coupled D2L dopamine receptor significantly inhibits Gαs-stimulated AC9 activity. We also provide evidence that phorbol ester activation of a novel PKC significantly attenuates Gαs-stimulated AC9 activity. Furthermore, examining the consequence of glycosylation on AC9 activity reveals that the glycosylation state of AC9 can selectively influence regulation by Gαs.

Although Gαs activation is common to all adenylate cyclase isoforms, regulation by inhibitory G proteins (Gi/o) is variable among the different isoforms. The α-subunit of Gi/o potently inhibits AC5 and AC6 but does not alter the activity of AC2 or AC8 (Chen and Iyengar, 1993; Taussig et al., 1993; Nielsen et al., 1996). Further illustrating the complication of Gi/o regulation of adenylate cyclases, AC2 can be conditionally activated by βγ-subunits released from Gi/o proteins (Tang and Gilman, 1991; Lustig et al., 1993). Initial evaluation, by Hacker et al. (1998), of the ability of Gi/o to inhibit isoproterenol-stimulated AC9 activity suggested that AC9 is insensitive to modulation by inhibitory G proteins. These initial studies of AC9 regulation by Gi/o were characterized in HEK293 cells expressing the human clone of AC9 used in the current study. They demonstrated that activation of endogenously expressed somatostatin receptors does alter Gαs-stimulated AC9 activity. Although endogenously expressed somatostatin receptors have been shown to inhibit Ca2+-stimulated AC1 activity in HEK293 cells, the extent of inhibition observed was notably lower than that produced by dopamine D2L receptors overexpressed in the same cell line (Nielsen et al., 1996). One plausible explanation for the difference in the degree of inhibition is that the receptor levels of an exogenously expressed receptor are likely to be much higher relative to the endogenous receptors. We reasoned that if AC9 is not potently inhibited by Gi/o, expression of D2L receptors would allow us to detect inhibition of AC9. In the current study, we used HEK293 cells expressing both AC9 and D2L to investigate Gi/o inhibition of AC9. We show that activation of D2L receptors significantly attenuates Gαs-stimulated AC9 activity. Inhibition by D2L receptors does not completely block stimulated AC9 activity but reduces cyclic AMP to approximately 50% of maximal levels. This is consistent with the view that AC9 is not potently inhibited by Gi/o. The ability of pertussis toxin to block inhibition of AC9 suggests that the D2L receptor acts through a Gi/o pathway to mediate its inhibition of AC9. It should be noted, however, that this set of experiments does not distinguish between the α- or βγ-subunits in mediating the inhibitory effects of D2L. Although, reconstitution studies with membranes from mouse AC9-infected Sf9 cells, purified constitutively active Gαs, and purified βγ-subunits indicate that βγ-subunits do not significantly alter Gαs-stimulated AC9 activity (Premont et al., 1996). These data imply that the inhibitory effects of D2L on AC9 activity are the consequence of the α-subunit of Gi/o acting on AC9.

Protein phosphorylation is a significant regulatory mechanism for adenylate cyclases. All adenylate cyclases, with the exception of AC9, have previously been shown to be regulated by one of three classes of protein kinases: protein kinase A, Ca2+/calmodulin-dependent protein kinases, and PKC (Sunahara and Taussig, 2002). Evidence from several reports demonstrates that protein kinase regulation of adenylate cyclase can occur through direct phosphorylation (Jacobowitz and Iyengar, 1994; Wayman et al., 1996; Wei et al., 1996; Chen et al., 1997; Lai et al., 1999; Lin et al., 2002). We have identified in this study an inhibitory mode of regulation of AC9 by PKC. To demonstrate that the PMA effects on AC9 resulted from the activation of PKC, we included in this study adenylate cyclases that exhibit differential regulation by PKC, AC1 and AC2. Consistent with the potent and relatively specific activation of PKC by PMA (100 nM), no PKC-independent effects of PMA on cyclic AMP accumulation were observed. Furthermore, the results obtained with the PKC inhibitor bisindolylmaleimide agree with these observations.

One of the well characterized regulatory features of AC9 is inhibition of basal activity by Ca2+ in calcineurin-dependent manner (Paterson et al., 1995, 2000). Calcineurin is a protein phosphatase that has been shown to be coexpressed with AC9 in postsynaptic terminals of some hippocampal and cortical neurons (Sosunov et al., 2001). Observations that a protein kinase and phosphatase can regulate AC9 activity could indicate that both calcineurin and PKC target the same residues on AC9. This seems unlikely because both activation of PKC and calcineurin results in the inhibition of AC9. It is probable that another protein kinase(s) can also act on AC9 to modulate its activity. Considering that calcineurin inhibits basal AC9 activity it is likely that AC9 is tonically phosphorylated by a yet-to-be identified protein kinase.

Coincidence detection is an important feature of adenylate cyclases and is essential for cells that receive numerous signals simultaneously (Anholt, 1994; Mons and Cooper, 1994; Xia and Storm, 1997; Mooney et al., 1998). The cyclic AMP signaling pathway is present in a wide range of cell types, and expression of multiple isoforms of adenylate cyclase within a cell can facilitate the integration of numerous signals (Chern, 2000). Having identified two novel modes of regulation for AC9, we examined the ability of this adenylate cyclase to perform as a coincidence detector. Gαs-stimulated AC9 activity in HEK-AC9 cells was less when treated with quinpirole and PMA than when treated with quinpirole or PMA alone. Analogous to the regulatory features of other adenylate cyclase isoforms, AC9 exhibits the ability to integrate multiple signals.

Glycosylation of proteins has been shown to influence stability, transport to the plasma membrane, and protein function (Boege et al., 1988; Khanna et al., 2001; Martinez-Maza et al., 2001). An alignment of the nine membrane-bound adenylate cyclase isoforms indicates that potential N-glycosylation sites, localized to the fifth and/or sixth extracellular loops, are conserved in each isoform (Wu et al., 2001). A number of adenylate cyclase isoforms (i.e., AC2, AC3, AC6, AC8, and AC9) have been shown to be glycosylated; however, the functional consequence of glycosylation seems to differ between isoforms (Cali et al., 1996; Premont et al., 1996; Wei et al., 1996; Bol et al., 1997; Wu et al., 2001). Blocking the glycosylation of AC6 reduces inhibition by PKC phosphorylation and alters the response to inhibitory G protein-coupled receptors without significantly affecting the maximal response to Gαs stimulation (Wu et al., 2001). In contrast, AC9 stimulation by Gαs is attenuated, whereas PKC and Gi/o inhibition seem to be unaffected by the glycosylation state of AC9 under the conditions used in this study. Despite the functional differences in response to deglycosylation, glycosylation likely mediates its effects by influencing the structure of adenylate cyclases, although the location of the glycosylation sites could lead to different structural consequences. The two N-glycosylation sites of AC9 are located on the sixth extracellular loop, whereas AC6 has glycosylation sites on the fifth and sixth loops. We have shown in this study that both sites on AC9 are glycosylated; however, single-site glycosylation deficient mutants of AC9 do not exhibit significant changes in response to Gαs or PKC. Unfortunately, similar studies examining the role of the individual glycosylation sites on the fifth and sixth extracellular loop of AC6 have not been reported. The current study provides additional evidence for unique functional consequences of glycosylation on adenylate cyclase function.

Cyclic AMP has been demonstrated to have an essential role in the learning and memory process. High-frequency stimulation of hippocampal neurons establishes long-term potentiation, a molecular model for learning and memory, and causes Ca2+ influx resulting in Ca2+/calmodulin-dependent increases in cyclic AMP (Mons et al., 1999; Chern, 2000). This increase in cyclic AMP has been demonstrated to be required for long-term memory. Mice lacking the two Ca2+/calmodulin-activated adenylate cyclases expressed in the hippocampus, AC1 and AC8, do not exhibit normal late-phase long-term potentiation and long-term memory (Mons et al., 1999). In addition to the two Ca2+-stimulated adenylate cyclases, two Ca2+-inhibited isoforms are also expressed in the hippocampus: AC6, which is directly inhibited by Ca2+, and AC9, which is inhibited by Ca2+ in a calcineurin-dependent manner. It has been suggested that fine-tuning the cyclic AMP pathway by Ca2+-dependent or -independent factors, possibly Gi/o or PKC, may play a role in facilitating learning and memory (Chern, 2000). That AC6 and now AC9 are known to be inhibited by PKC and Gi/o presents an opportunity for fine-tuning of cyclic AMP signaling that should be explored in vivo.

Footnotes

  • This work was supported by the National Institutes of Health Grants T32GM008737 (to M.G.C.) and MH60397 (to V.J.W.).

  • DOI: 10.1124/jpet.104.065748.

  • ABBREVIATIONS: AC, adenylate cyclase; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; HEK, human embryonic kidney; PNGase F, peptide:N-glycosidase F; GppNHp, 5′-guanylylimidodiphosphate; ANOVA, analysis of variance; Iso, isoproterenol; Bis, bisindolylmaleimide; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole.

    • Received January 19, 2004.
    • Accepted March 2, 2004.

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Research ArticleCELLULAR AND MOLECULAR

Novel Regulatory Properties of Human Type 9 Adenylate Cyclase

Medhane G. Cumbay and Val J. Watts
Journal of Pharmacology and Experimental Therapeutics July 1, 2004, 310 (1) 108-115; DOI: https://doi.org/10.1124/jpet.104.065748
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