INTRODUCTION

Nature has evolved several clever mechanisms for cells to process external stimuli. Such systems incorporate a receptor at the cell surface that is activated by the stimulus and initiates signal propagation to the cell interior, recruiting several additional proteins in the process. Many of the groups of proteins involved in signal propagation exist as isoforms or closely related subtypes, as is the case for signals initiated by G-protein-coupled membrane receptors. These receptors are coupled to a variety of effectors including adenylyl cyclases, phospholipases, and protein kinases. Each of these effectors exists as isoforms with different regulatory properties allowing complex signal integration, and this capability presents opportunities for the cell to engineer highly specific responses to an external stimulus. Such appears to be the case for signals involving the enzyme adenylyl cyclase. There are nine types of adenylyl cyclase (AC)¹ that differ in their regulatory mechanisms and are expressed in a tissue-specific manner (1). All enzyme types are activated by G_s. AC I, III, and VIII are positively regulated by calcium/calmodulin, whereas types V and VI are directly inhibited by calcium. AC II and IV are stimulated by G in the presence of activated G_s. The activities of types I-III and V-VII are also influenced by phosphorylation with protein kinase C and/or A (2-9).

The ability of various AC types to respond to activated G_s and G_i, G, calcium, and/or phosphorylation places the enzyme at a central point for cross-talk between different signaling systems. The Ca²⁺/calmodulin-regulated enzymes (types I, III, and VIII) are suggested to serve as coincidence detectors for signal processing in the hippocampus (types I and VIII) and in vascular smooth muscle cells (type III) (10-13).² Similarly, the multiple regulatory inputs to the type II and IV enzymes would also allow the cell to process signals from diverse receptor systems. Many regulatory properties of the enzymes were determined following purification of the enzyme expressed in Sf9 insect cells, and it is not clear how the regulatory properties observed with the purified enzyme are integrated in the intact cell to produce a defined response to external stimuli. As an initial approach to this issue, we examined the integration of multiple signals to AC (G_s, G_i, G, and phosphorylation) following stable expression of enzyme types II, III, and IV in DDT₁-MF2 cells derived from hamster vas deferens smooth muscle.

EXPERIMENTAL PROCEDURES

[³H]cAMP (33.5 Ci/mmol) and [³²P]ATP (30 Ci/mmol) were purchased from NEN Life Science Products. Tissue culture supplies were obtained from JRH Bioscience (Lenexa, KS). Hygromycin B, isoproterenol, and forskolin were from Sigma. Phorbol 12-myristate 13-acetate (PMA) and UK14304 were provided by Research Biochemicals International (Natick, MA). GTPS was obtained from Boehringer Mannheim. Dowex 50W-X4 (100-200 mesh, hydrogen form) was from Bio-Rad, and aluminiumoxid (90 active neutral) was purchased from EM Science (Cherry Hill, NJ). The cDNAs encoding for AC types II and III were kindly provided by Dr. Randall Reed (Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD), and the AC IV cDNA was kindly provided by Dr. Alfred Gilman (Department of Pharmacology, Southwestern Medical School, Dallas, TX). Additional AC constructs were kindly provided as follows: type I, Dr. Alfred Gilman; types V and VI, Dr. Yoshihiro Ishikawa (Department of Medicine, Harvard Medical School, Boston MA); type VII, cloned from DDT₁-MF2 cells;³ type VIII, Dr. John Krupinski (Geisinger Research Clinic, Danfield PA); type IX, Dr. Richard Premont (Duke University, Durham, NC). The antiserum against cAMP and radioiodinated succinyl cyclic AMP tyrosine methyl ester were kindly provided by Dr. Jerry Webb (Department of Pharmacology, Medical University of South Carolina). Transducin  was provided by Dr. Heidi Hamm (Department of Biochemistry, University of Chicago). All other materials used were obtained as described elsewhere (15-17).

Cells were grown as monolayers on Falcon Primeria dishes at 37 °C (5% CO₂) in Dulbecco's modified Eagle's medium with high glucose (4.5 g/liter), supplemented with 10% bovine calf serum (NIH-3T3), 2.5% horse serum plus 2.5% bovine calf serum (DDT₁-MF2) or 5% horse serum plus 10% fetal bovine serum (PC-12), penicillin (100 units/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml). In some experiments, cells were pretreated with 100 ng/ml pertussis toxin for 18 h (37 °C) prior to membrane preparation or initiation of experiments in intact cells. DDT₁-MF2 cells were stably transfected with the _2A/D-AR (RG-20) as described previously (16, 17), and clones expressing receptors at a density of ~3-4 pmol/mg of membrane protein were then used for manipulation of the population of adenylyl cyclases in the cell. The coding sequences of rat AC isoforms II and IV were inserted into the pMSV expression vector (17), and the gene coding for the rat AC type III was subcloned into the pcDNA3 (Invitrogen, San Diego, CA) expression plasmid. AC II (nucleotides 1-4008, coding sequence 70-3342) in pBS.KS was digested with EcoRI and blunt-ended, and HindIII linkers were added to the 5- and 3-ends of the purified AC II insert. The modified insert was then cloned into the HindIII site of the pMSV expression vector downstream of the viral long terminal repeat promoter. AC III (nucleotides 1-4533, coding sequence 367-3801) in pBS.KS was digested with EcoRI and subcloned into the EcoRI site in pcDNA3 downstream of the cytomegalovirus promoter element. AC IV (nucleotides 1-3358, coding sequence 110-3304) in pBS.SK was digested with XhoI/BamHI and cloned into the pMSV vector at the HindIII site by blunt end ligation. DDT₁-MF2 cells expressing the _2A/D-AR were cotransfected with the AC II, AC III, or AC IV cDNA expression vector (16 µg) and the drug resistance cassette pHyg (4 µg) by calcium phosphate coprecipitation (17). Transfected cells were selected by their resistance to hygromycin B (750 µg/ml), and hygromycin B-resistant clones were screened for expression of the AC isoforms by RNA blot analysis and functional evaluation of enzyme activity. Subsequent to isolation of AC transfectants, we performed a series of preliminary experiments to characterize the enzyme activity in different clonal cell lines. These studies used three AC II, two AC III, and two AC IV clonal cell lines. The results of these experiments indicated that the cell lines for a particular AC transfectant behaved similarly relative to the interaction between the various signaling interventions, and one cell line was then used for detailed experimental analysis.

Polyadenylated RNA from control and AC transfectants was isolated using an oligo(dT) cellulose matrix (FastTrack mRNA Isolation Kit, Invitrogen). Five to ten µg of mRNA was subjected to electrophoresis on 1% agarose, 3% formaldehyde gels followed by transfer to nylon membranes. The membrane was dried in a vacuum oven at 80 °C for 2 h, processed, and hybridized with radiolabeled probes as described previously (15-18). The population of AC expressed by DDT₁-MF2, NIH-3T3, and PC12 cells was also determined by DNA amplification following reverse transcription of cellular mRNA. For amplification of AC mRNA, first strand cDNA was synthesized from 2 µg of mRNA using Superscript II RNase H reverse transcriptase (Life Technologies, Inc.) and random hexamer (50-ng) primers. The reaction mixture was incubated with RNase H and used for DNA amplification by the polymerase chain reaction. To allow detection of multiple types of AC transcripts, the amplification utilized degenerate oligonucleotide primers (5 µmol/liter) as described by Yoshimura and Cooper (19). The primers corresponded to amino acid sequences in the C2a domain of AC that are highly conserved in the different enzyme isoforms (sense primer: KIKTIGS-5-CAGAAGCTTAA(A/G)AT(T/C/A)AA(A/G)AC(T/C/ A/G)AT(T/C/A)GG(T/C/A/G)(T/A)(C/G)(T/C/A/G)AC(T/C/A/G)TA(T/C)ATGGC-3; antisense primer: YDIWGNTVNV-3-GAGGATCCAC(A/G)TT(T/C/A/G)AC(T/C/A/G)GT(A/G)TT(T/C/A/G)CCCCA(T/A/G)AT(A/G)TC(A/G)TA-5). Each primer contained a unique restriction site at its 5-end to facilitate subcloning. The reaction mixture was treated with proteinase K, phenol/chloroform-extracted, and digested with BamHI and HindIII. The restricted amplified DNA of appropriate size was purified on a 2% agarose gel, subcloned into pGEM-7Zf(+) (Promega), and characterized by DNA sequencing with dideoxy chain terminators and modified T7 DNA polymerase (Sequenase, U.S. Biochemical Corp.).

Intact cell assays utilized confluent cells in six-well Falcon Primeria plates. One hour before the assay, the medium was aspirated and replaced with 2 ml of serum-free Dulbecco's modified Eagle`s medium containing 20 mM NaHEPES-HCl, pH 7.5. Cells were preincubated with a phosphodiesterase inhibitor (350 µM) isobutyl-L-methylxanthine at 37 °C for 30 min before the addition of forskolin, isoproterenol, and/or UK14304. Reactions were terminated following a 10-min incubation by aspiration of medium and the addition of 1 ml of ice-cold 0.1 N HCl/well. In some experiments, cells were pretreated with 100 nM PMA in the presence of isobutyl-L-methylxanthine for 10 min at 37 °C before adding isoproterenol and/or UK14304. cAMP was acetylated and assayed by radioimmunoassay as described by Brooker et al. (20).

Cells at confluence (~2 × 10⁷ cells/dish) were washed twice with cell washing solution (137 mM NaCl, 2.6 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), harvested with a rubber policeman, and pelleted at 4 °C at 200 × g in a Sorvall RT6000 centrifuge. The pellet was resuspended in lysis buffer (5 ml/dish) at 4 °C (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A), and homogenized in a Dounce homogenizer. The cell homogenate was centrifuged at 17,000 × g (Sorvall RC5B, SS34 rotor) for 20 min. The pellet was resuspended in membrane buffer (50 mM NaHEPES, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µM pepstatin A), and the protein concentration was determined by the method of Lowry et al. (21). Adenylyl cyclase activity was measured as described previously (16, 17). The assay buffer contained 50 mM NaHEPES, 1 mM EDTA, 4 mM MgCl₂, 100 mM NaCl, 0.24 mM ATP, 0.1 mM GTP, 0.2 mM cAMP, 0.2% bovine serum albumin, 0.5 mM isobutyl-L-methylxanthine, [-³²P]ATP (3 × 10⁶ cpm/tube), and an ATP-regenerating system (60 mM creatinine phosphate, 10 units/ml creatinine kinase) in a final volume of 100 µl. Cyclic AMP was isolated by sequential chromatography on columns containing Dowex AG-50W-X4 resin and alumina. The 4-ml eluate from each column was equilibrated with 10 ml of Ecoscint A, and radioactivity was determined by liquid scintillation counting. In some experiments, cells were preincubated with PMA (100 nM, 10 min), and membranes were prepared as above, using lysis and assay buffer containing the phosphatase inhibitor okadaic acid (1 µM), sodium orthovanadate (100 µM), and -glycerophosphate (100 mM). In experiments with the -subunit of transducin, membranes were preincubated with transducin (200 nM) for 5 min prior to initiation of the assay.

RESULTS

We previously reported the coupling of rat ₂-AR subtypes to adenylyl cyclase following stable expression of the receptor in three cell types: DDT₁-MF2, NIH3T3, and PC-12 (16, 22). These model systems were further characterized in terms of the type of adenylyl cyclase transcripts expressed by each cell line. Poly(A)-RNA from each cell line was evaluated by RNA blot analysis using probes for enzyme types I-IX and also by RT-PCR using degenerate oligonucleotides corresponding to regions of conserved amino acid sequence in the nine enzyme types. RNA blot analysis indicated differential expression of enzyme types by the three cell lines (DDT₁-MF2, types VI, VII, VIII, and IX; NIH-3T3, type VI;⁴ and PC-12, types III, VI, VII, and IX) (Fig. 1). Brain RNA was used as a positive control for hybridization, since this tissue expressed each enzyme type. None of the cell lines expressed AC types I, II, IV, or V, whereas each cell line expressed the type VI enzyme. A similar profile of enzyme types was also observed by cDNA amplification following reverse transcriptase of mRNA (RT-PCR) from DDT₁-MF2 and NIH-3T3 fibroblasts (Table I, Fig. 1). RT-PCR using PC-12 mRNA failed to identify the AC VII and AC IX transcript visualized by RNA blot analysis (Table I, Fig. 1).⁵

Table I. Identification of adenylyl cyclases expressed in DDT1-MF2 smooth muscle cells, NIH-3T3 fibroblasts, and the pheochromocytoma cell line PC-12 by cDNA amplification Adenylyl cyclase Cell linea DDT1-MF2 NIH-3T3 PC-12 I NDb ND ND II ND ND ND III ND ND 5 IV ND ND ND V ND ND ND VI 5 11 9 VII 4 ND ND VIII 1 ND ND IX 6 ND ND a Polyadenylated RNA from each cell line was isolated and reverse transcribed using random hexamers and subsequently amplified with degenerate oligonucleotide primers as described under "Experimental Procedures." Products were isolated and subcloned, and the AC type amplified was determined by sequencing individual clones. The values in the table indicate the number of clones for each AC. The total numbers of clones sequenced were 17 (DDT1-MF2), 11 (NIH-3T3), and 14 (PC-12). b ND, not detected.

DDT₁-MF2 cells were selected to evaluate signal integration by AC before and after altering the population of enzymes expressed in the cell. AC types II, III, and IV were introduced into the cell by gene transfection. Stimulation of AC by G_s was achieved by activation of the -AR expressed in DDT₁-MF2 cells (23). To generate the inhibitory input to the enzyme, we stably expressed the AC cDNA in subclones previously transfected with the _2A/D-AR (16). The influence of G_s- and G_i-protein coupled systems on AC function for each enzyme type was determined before and after activation of protein kinase C with phorbol 12-myristate 13-acetate.

AC types II, III, and IV were stably expressed in DDT₁-MF2 cells as indicated by analysis of transfectant RNA and functional properties of the transfected cells (Fig. 2). RNA blot analysis indicated that mRNA of expected size was detected in the AC transfectants but not in control cells transfected with vector alone (Fig. 2A). Basal and maximally stimulated enzyme activity in the DDT₁-MF2 AC transfectants was compared with that of control vector-transfected cells. Relative to control cells, basal enzyme activity was not altered in AC III or AC IV transfectants, but it was increased 6-fold in AC II transfectants (Fig. 2B). Enzyme activity determined in the presence of forskolin, Mn²⁺, and GTPS was increased ~2-fold in AC III and AC IV transfectants and ~6-fold in AC II transfectants relative to the stimulation observed in control transfectants (Fig. 2B). These data indicated that each of the transfected genes was expressed in the DDT₁-MF2 cell lines.

The AC transfectants were further characterized for their responses to the diterpene forskolin and the -AR agonist isoproterenol. Basal enzyme activity in the intact cell was similar in control, AC III, and AC IV transfectants but was slightly increased in AC II transfectants (Table II). In the intact cell, the effect of forskolin on cellular cAMP levels was not altered in AC IV transfectants but was increased in AC II (1.5-fold) and AC III (1.8-fold) transfectants relative to its actions in control transfectants (Fig. 3A). The EC₅₀ of forskolin in the AC transfectants was similar to the control value. Stimulation of adenylyl cyclase via G_s in DDT₁-MF2 transfectants was achieved with the -AR agonist isoproterenol. The maximal effect of isoproterenol in the intact cell was only increased in AC II transfectants (229 ± 0.3 versus 168 ± 0.6 ng of cAMP/mg of protein) (Fig. 3B). The EC₅₀ of isoproterenol for enzyme stimulation in control, AC II, AC III, and AC IV transfectants was 99 ± 6, 146 ± 1, 24 ± 1, and 90 ± 2 nM, respectively.

Table II. Effect of protein kinase C activation on cellular cAMP in DDT1-MF2 cells stably transfected with cDNAs encoding AC II, AC III, or AC IV DDT1-MF2 transfectant Basala Isoproterenol (1 µM) Vehicle PMA (100 nM) Vehicle PMA (100 nM) Control 0.37  ± 0.01 0.39  ± 0.08 46  ± 1.4 47  ± 0.4 AC II 0.9  ± 0.09 2.45  ± 0.05 106  ± 1.6 180  ± 3.5 AC III 0.58  ± 0.01 0.72  ± 0.01 64  ± 1 75  ± 1 AC IV 0.47  ± 0.02 0.58  ± 0.01 55  ± 1 79  ± 4.2 a Data (ng of cAMP/mg of protein) are expressed as the mean ± S.E. of duplicate determinations from three experiments. The numbers under "Isoproterenol" represent the increase above basal values.

Effect of forskolin and the -AR agonist isoproterenol on cellular cAMP levels in DDT₁-MF2 cell lines stably transfected with cDNAs encoding AC II, AC III, and AC IV.

The G_s-mediated stimulation of adenylyl cyclase in the AC transfectants was also investigated after the activation of protein kinase C. Protein kinase C activation by PMA did not alter basal cellular cAMP levels in control transfectants and produced small but consistent increases in cAMP in each AC transfectant (Table II). In membrane preparations, only the AC II transfectant exhibited an elevation in basal enzyme activity (relative to control values) following PMA treatment of the cells (Table III). Protein kinase C activation also enhanced the increase in cellular cAMP elicited by the -AR agonist isoproterenol in each of the AC transfectants (Table II). Similar results were observed in enzyme activity assays using membrane preparations (Table III). These data indicate that the receptor has access to the transfected enzymes and that the sensitivity of the enzymes to G_s is increased following enzyme phosphorylation by protein kinase C. To gain insight as to how the different enzyme types process multiple stimulatory and inhibitory inputs, we determined the interaction between the G_s-coupled -AR and the G_i/G_o-coupled _2A/D-AR in the regulation of AC activity.

Table III. Effect of protein kinase C activation on adenylyl cyclase in membranes prepared from DDT1-MF2 cells stably transfected with cDNAs encoding AC II, AC III, or AC IV DDT1-MF2 transfectant Basala Isoproterenol (1 µM) Vehicle PMA (100 nM) Vehicle PMA (100 nM) Control 21.6  ± 2.5 28  ± 2.3 136  ± 2 149  ± 2 AC II 112  ± 8 145  ± 8.3 336  ± 9 421  ± 2 AC III 14.8  ± 1.3 22.3  ± 2.8 166  ± 8 193  ± 3 AC IV 14.7  ± 1.2 21.1  ± 3.4 121  ± 3 199  ± 5 a Data (pmol of cAMP/min/mg of protein) are expressed as the mean ± S.E. of duplicate determinations from three experiments. The numbers under "Isoproterenol" represent the increase above basal values.

Receptors coupled to G_i- or G_o-proteins are capable of sending both positive and negative signals to adenylyl cyclases, as is the case with the _2A/D-AR. Negative input is relayed through G_i or perhaps G_o and is commonly observed following activation of the _2A/D-AR. Positive input to adenylyl cyclases following _2A/D-AR activation is cell type-specific and may be achieved by 1) increased intracellular calcium concentration and subsequent stimulation of Ca²⁺/calmodulin-sensitive adenylyl cyclases (16), 2) receptor coupling to G_s (24), 3) altered phosphorylation status of the enzyme via activation of protein kinase C, or 4) the action of G when the enzyme is stimulated by G_s (18, 25, 26). The results of experiments in the AC transfectants indicated that the effects of _2A/D-AR receptor activation on cellular cAMP levels were dependent upon the enzyme type expressed in the cell. Basal enzyme activity in control and AC III was either not altered (intact cell) or inhibited (membranes) by the selective ₂-AR agonist UK14304 (Table IV). In AC II and AC IV transfectants, AC activity was either not altered (AC IV) or increased (AC II) by _2A/D-AR activation (Table IV). _2A/D-AR activation inhibited forskolin-stimulated enzyme activity in control and each of the AC transfectants (Fig. 4A). The effects of _2A/D-AR activation on basal and forskolin-stimulated enzyme activity in control and each AC transfectant were blocked by prior treatment of the cells with pertussis toxin⁶ and thus involved receptor coupling to G_i2 or G_i3, members of the pertussis toxin-sensitive family of G-proteins expressed in DDT₁-MF2 cells (16).

Table IV. Effect of 2-adrenergic receptor activation on adenylyl cyclase in DDT1-MF2 cells stably transfected with cDNAs encoding AC II, AC III, or AC IV DDT1-MF2 transfectant Intact cell Membranes Vehicle UK-14304 (10 µM) Vehicle UK-14304 (10 µM) ng cAMP/mg proteina pmol/cAMP/min/mg protein Control 0.64  ± 0.02 0.66  ± 0.08 18.3  ± 2.3 13  ± 1.6 AC II 0.99  ± 0.08 5.32  ± 0.3 110  ± 3.8 143  ± 2.4 AC III 0.69  ± 0.07 0.61  ± 0.1 11  ± 1.6 7.2  ± 1.2 AC IV 0.8  ± 0.03 0.9  ± 0.04 13.3  ± 1 12  ± 1.8 a Data are expressed as the mean ± S.E. of duplicate determinations from three experiments.

Effect of _2A/D-AR activation on forskolin- and isoproterenol-stimulated adenylyl cyclase in DDT₁-MF2 cell lines expressing AC II, AC III, or AC IV.

Stimulation of the three types of AC via the -AR agonist isoproterenol was differentially altered by activation of _2A/D-AR. In AC III transfectants, _2A/D-AR-mediated inhibition of isoproterenol-induced increases in cAMP was enhanced relative to control (80 versus 50% inhibition) (Fig. 4B). AC III is most closely related to AC I and AC VIII in terms of sequence homology and each of these enzymes are regulated by calcium/calmodulin (27-29). These data suggest that G_s-stimulated AC III is more sensitive to G_i-mediated inhibition compared with the enzymes coupled to _2A/D-AR in control transfectants or that perhaps there is an additional inhibitory input from G (30, 31). In AC II and AC IV transfectants, _2A/D-AR activation elicited a biphasic effect on isoproterenol-induced increases in cellular cAMP (Fig. 4B). At lower concentrations of the ₂-AR agonist, the effect of isoproterenol on enzyme activity was inhibited, whereas at higher concentrations of UK14304, the response to the -AR agonist was either unaltered or enhanced. Thus, it appears as if _2A/D-AR activation initiated both a positive and negative signal to AC II and AC IV and that the final cell response was a balance between the two types of input. Both types of input emanating from the receptor required G_i2/G_i3, since no effect of receptor activation was observed in cells pretreated with pertussis toxin.⁶ The inhibitory component represented the influence of G_i, whereas the stimulatory component probably involves G further activating the G_s-primed enzyme. Indeed, the stimulatory component was blocked when membranes were preincubated with transducin , which ties up the G released by receptor activation (Fig. 5). In control transfectants, the inhibition of AC by _2A/D-AR activation was not altered by the addition of transducin . In AC III transfectants, the inhibitory input mediated through the _2A/D-AR was actually diminished. However, in AC II and AC IV transfectants, the augmentation of isoproterenol-induced increases in AC following _2A/D-AR activation was lost in the presence of G_t, and the inhibition of the enzyme by this system was revealed (Fig. 5).

Effect of _2A/D-AR activation on isoproterenol-stimulated adenylyl cyclase activity in the presence and absence of transducin  in DDT₁-MF2 cell lines expressing AC II, AC III, or AC IV.

The preceding series of experiments indicated that the AC III and AC II/IV enzymes process information from G_i/G_o-coupled receptors in an enzyme type-specific manner. Additional differences in the integrative properties of the three enzymes were observed after activation of protein kinase C. The consequences of _2A/D-AR activation on cellular cAMP were not altered by cell treatment with PMA in either control or AC III transfectants (Fig. 6). However, although AC II and AC IV exhibit sequence homology and share several biochemical properties, the stimulatory/inhibitory input to the enzymes generated by _2A/D-AR activation were differentially modified by PMA. Treatment of DDT₁-MF2 AC II transfectants with PMA eliminated the inhibitory input and enhanced the stimulatory input to the enzyme initiated by _2A/D-AR activation (Fig. 6). PMA treatment of AC IV transfectants actually modified the effects of _2A/D-AR activation in a manner that was directly opposite to that observed in AC II transfectants. Treatment of DDT₁-MF2 AC IV transfectants with PMA eliminated the stimulatory component and enhanced the inhibitory effect of _2A/D-AR activation (Fig. 6). Similar alterations in the input to AC II and AC IV mediated by the _2A/D-AR were also observed in membranes prepared from cells pretreated with PMA (Fig. 7). Since treatment of control transfectants with PMA did not alter the effect of _2A/D-AR activation on AC activity, the effects observed in the AC II and AC IV transfectants probably reflect phosphorylation of the transfected enzyme type. These data further suggest that the AC II and AC IV enzymes received two types of input from the activated _2A/D-AR. The results observed after PMA treatment in AC II transfectants could reflect a complete elimination of the inhibitory input or a sensitization of the stimulatory component, whereas protein kinase C activation in AC IV transfectants would result in an opposite modification either enhancing the inhibitory input or preventing the stimulatory influence of G resulting from _2A/D-AR activation.

Effect of _2A/D-AR activation on isoproterenol-induced increases in cellular cAMP following cell incubation with phorbol 12-myristate 13-acetate in DDT₁-MF2 cell lines expressing AC II, AC III, or AC IV.

Effect of _2A/D-AR activation on adenylyl cyclase activity in DDT₁-MF2 cells expressing AC II, AC III, or AC IV following cell incubation with phorbol 12-myristate 13-acetate.

DISCUSSION

Most cells probably express multiple types of adenylyl cyclases. Indeed, RNA blot analysis indicated that DDT₁-MF2, NIH-3T3, and PC-12 cells express transcripts encoding distinct populations of adenylyl cyclases. None of the cell lines expressed detectable mRNA for AC types I, II, IV, or V. The absence of type I in the three cell lines is consistent with the neural specific expression of this enzyme, whereas type V is enriched in the myocardium and brain (1, 32). Types II and IV were also not detected in the three cell lines, although these enzymes are fairly widely distributed in peripheral tissues (1). AC III mRNA was identified in the PC-12 cell line, and the presence of this enzyme may explain previous observations indicating that _2A/D-AR activation in these cells augmented forskolin-stimulated enzyme activity in a calcium-dependent manner (16). Although the type III mRNA is enriched in the olfactory system, this enzyme is also expressed in several other cell types including vascular smooth muscle cells.² DDT₁-MF2 cells expressed AC VI-IX. The type VI enzyme is widely expressed, and mRNA encoding this enzyme type was also present in NIH-3T3 and PC-12 cells. The type VII enzyme exhibits highest homology to AC II/IV (9). The expression of the type VIII transcript in DDT₁-MF2 cells, which is related to AC I in terms of sequence homology and its regulation by calcium/calmodulin, was surprising, since it was thought that this enzyme was restricted to the central nervous system (33). The type IX cDNA was recently isolated and characterized by Premont et al. (34). It is not known if all of the receptors in a given cell that initiate stimulatory and/or inhibitory input to AC communicate with the same population of adenylyl cyclases nor how the different enzymes process the multiple types of input they receive. As an initial approach to these issues, we explored the function of receptors coupled to adenylyl cyclase before and after altering the population of effector enzymes in the cell. In the present study, we focused on the enzyme types II, III, and IV.

The type II and IV enzymes exhibit 76-79% amino acid homology in their conserved cytoplasmic domains, lack the C2b domain, and are both stimulated by G in the presence of activated G_s. Of the two enzymes, the type II enzyme is the best characterized, and the interaction of G with AC II involves a defined segment of the C2a domain (35). _2A/D-AR activation resulted in both stimulatory and inhibitory input to the AC II and AC IV enzymes. In AC II transfectants the stimulatory input was enhanced, and the inhibitory input was lost following treatment of the cells with PMA. Protein kinase C phosphorylation of the enzyme apparently alters its interaction with the inhibitory component, i.e. G_i. These data and the observations of Chen and Iyengar (36) are in contrast to the failure of G_i to inhibit purified AC II enzyme (37), suggesting that there are unique regulatory properties of the AC enzymes in the intact cell. In contrast to the effects of PMA on _2A/D-AR coupling to AC II, it is apparently the stimulatory input (mediated by G) to the AC IV enzyme from the _2A/D-AR that is modified by protein kinase C. This effect of protein kinase C activation is likely achieved by direct phosphorylation of the enzyme, since signaling events initiated by both ₂ and -AR were not altered by PMA in control transfectants. Although phosphorylation of AC II and AC IV by protein kinase C sensitized the enzymes to activated G_s, the input to the enzyme from G_i and from G were differentially affected by protein kinase C activation. These data support the idea that there are distinct sites on the enzyme for the action of the three types of input and suggests that these sites are independently regulated by phosphorylation.

_2A/D-AR activation initiates both positive and negative signals to AC II and AC IV, and the final cell response is apparently a balance between the two types of input. The significance of the differential regulation of these two types of inputs from the receptor to AC II and AC IV by protein kinase C would be particularly evident in cells expressing multiple types of catecholamine receptors. Thus, in cells expressing the type II enzyme, -AR, and ₂-AR, the adrenergic agonist epinephrine could exert precisely opposite effects on cellular cAMP, depending on the phosphorylation state of the enzyme. When the enzyme is not phosphorylated by protein kinase C, ₂-AR activation would inhibit enzyme activity, but following enzyme phosphorylation, receptor activation would actually increase enzyme activity. The stimulation in the latter situation reflects synergistic activation of the enzyme by G_s (via -AR stimulation) and G (via ₂-AR activation) as well as the loss of G_i inhibition subsequent to protein kinase C phosphorylation of the enzyme. A switch in the stimulatory versus inhibitory input to AC from the ₂-AR is precisely the situation that occurs in the rat myometrium over the course of pregnancy (18). Indeed, mRNA blot analysis indicates the expression of AC II in the longitudinal layer of the rat myometrium throughout the time course of pregnancy (14). At early stages of pregnancy, when it is important to maintain a relaxed state of the myometrium, ₂-AR activation augments the effects of isoproterenol on cellular cAMP, promoting smooth muscle relaxation, and this effect of ₂-AR agonists is mediated through G. At later stages of pregnancy, when contraction is important at term, ₂-AR activation inhibits stimulation of AC by the -AR agonist. Such a switch in the consequences of ₂-AR activation could be explained by a change in the phosphorylation status of the AC II enzyme. Thus, the integration of multiple inputs by AC is dynamic, and this ability of the enzyme probably plays a key role in the signaling plasticity observed in development and a wide range of physiological processes.