Protein Kinase C Inhibits Adenylyl Cyclase Type VI Activity during Desensitization of the A2a-Adenosine Receptor-mediated cAMP Response*

(Received for publication, July 25, 1996, and in revised form, November 26, 1996)

Hsing-Lin Lai Dagger , Te-Hsun Yang §, Robert O. Messing , Yung-Hao Ching Dagger , Shu-Chwun Lin Dagger and Yijuang Chern Dagger par

From the Dagger  Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, § Institute of Neuroscience, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China, and  Department of Neurology, E. Gallo Clinic and Research Center, University of California, San Francisco, California 94110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

We have previously reported that phosphorylation of adenylyl cyclase type VI (AC6) may result in the suppression of adenylyl cyclase activity during desensitization of the A2a-adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC12 cells. In the present study, we demonstrate that protein kinase C (PKC) is responsible for the phosphorylation and inhibition of AC6 during A2a desensitization. Inhibition of PKC by several independent methods markedly blocked the suppression of AC6 during A2a desensitization. Purified PKC from rat brain directly phosphorylated and inhibited recombinant AC6 expressed in Sf21 cells. Substantially lower AC6 activities were also observed in PC12 cells overexpressing PKCdelta or PKCepsilon . Stimulation of A2a-R in PC12 cells under the same conditions as those required for A2a desensitization resulted in an increase in Ca2+-independent PKC activity. Most importantly, exogenous PKC did not further suppress AC6 activity in A2a-desensitized membranes. In vitro PKC phosphorylation of AC6 isolated from A2a-desensitized cells was also profoundly lower than that from control cells, suggesting a specific role for PKC in regulating AC6 during A2a desensitization in PC12 cells. Taken together, our data demonstrate that a calcium-independent, novel PKC inhibits AC6 activity during A2a desensitization in PC12 cells. Independent regulation of AC6 by calcium-independent PKC and by Ca2+ provides an exquisite mechanism for integrating signaling pathways to fine-tune cAMP synthesis.


INTRODUCTION

We have recently reported that prolonged activation of A2a adenosine receptor (A2a-R)1 in PC12 cells significantly inhibits the activity of adenylyl cyclase type VI (AC6), which in turn causes a lower response to subsequent stimulation of A2a-R (A2a desensitization). In addition, there is evidence that protein phosphorylation may mediate the suppression of AC6 activity during A2a desensitization (1, 2). However, the exact molecular events underlying the phosphorylation and inhibition of AC6 during prolonged activation of A2a-R in PC12 cells remain largely uncharacterized.

Genes of at least 10 distinct mammalian adenylyl cyclases (AC), which can be further divided into five subfamilies, have been reported (3, 4). The specific tissue distribution of these different adenylyl cyclases supports biochemical evidence for distinct modes of regulation of cAMP levels (5). Although these enzymes can all be activated by the a subunit of Gs protein, each is under very distinct regulation. For example, AC6 can be inhibited by physiologically relevant concentrations of Ca2+, while other AC isozymes (adenylyl cyclase type I (AC1) and adenylyl cyclase type VIII (AC8)) are activated by Ca2+ in the presence of calmodulin (6). In addition, Ca2+- mediated inhibition has been clearly demonstrated for adenylyl cyclase type III (AC3) due to direct phosphorylation of AC3 by a calmodulin-kinase II (7, 8). The susceptibility of each individual adenylyl cyclase to inhibition by Gialpha also differs (9). Most interestingly, protein kinase C (PKC) has been implicated in stimulation of several ACs (AC1, adenylyl cyclase type II (AC2), and adenylyl cyclase type V (AC5)) (10-12), but markedly inhibits the activity of AC6 as demonstrated in the present study. Regulation of the Ca2+-inhibitable AC6 by PKC provides a very useful mechanism to detect and integrate coincidental signals mediated by Ca2+, cAMP, and PKC.

PKC is a family of serine/threonine protein kinases which is composed of at least 11 different members from 10 distinct genes. These PKC isozymes are divided into three subfamilies based on their amino acid sequences and their specific modes of activation. The "conventional PKCs" including alpha , beta , and gamma  are dependent on calcium and diacylglycerols (or phorbol esters) for activation. Members of another subfamily of PKC, novel PKCs (delta , epsilon , eta , and theta ), require diacylglycerols (or phorbol esters) but not calcium for their activation. Activation of the "atypical PKCs," including iota  and zeta , is independent of either calcium or diacylglycerols (13). Recently, two related, phospholipid-dependent kinases, PKCµ and PKD, have been identified that are phorbol ester-sensitive and calcium-independent. They are unique in having a hydrophic domain near the amino terminus, an internal pleckstrin homology domain, and a substrate specificity different from other PKCs, suggesting that they constitute a new PKC subgroup (14, 15).

In the present study, we present evidence to demonstrate that stimulation of A2a-R activates calcium-independent PKC, which phosphorylates and inhibits AC6. Suppression of AC6 by protein phosphorylation thus yields a lower response of AC6 to subsequent stimulation of A2a adenosine receptor in PC12 cells.


EXPERIMENTAL PROCEDURES

Materials

CGS21680 were obtained from Research Biochemicals, Inc. (Natick, MA). Cyclic AMP and ATP were obtained from Sigma. Okadaic acid (OKA) and protein kinase C were from Boehringer Mannheim Biochemica (Mannheim, Germany). Western blot analysis revealed that this mixture of PKCs from rat brain contains at least PKCalpha , beta I, beta II, gamma , delta , epsilon , and µ (data not shown). The monoclonal anti-FLAG antibody, M2, was obtained from Eastman Kodak Co. Purified human protein phosphatase type 2A, and the epsilon -PKC substrate were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Phorbol-12,13-didecanoate (PDD) was from Calbiochem-Novabiochem Int. (San Diego, CA).

Cell Culture

PC12 cells were originally obtained from American Type Culture Collection (Rockville, MD). The pRc/RSV, PKCdelta , and PKCepsilon -transfected clones (designated C1, delta 1, and epsilon 1, respectively) were developed and characterized as described elsewhere (16). These PC12 variants were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Life Technologies, Inc.) plus 10% horse serum (Life Technologies, Inc.) in an incubation chamber gassed with 10% CO2, 90% air at 37 °C. A123, the PKA-deficient variant of PC12 cells (17), was kindly provided by Dr. J. A. Wagner (Cornell University Medical College). A123 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus 5% horse serum in the presence of 10% CO2, 90% air at 37 °C. Sf21 cells were obtained from Pharmingen (San Diego, CA) and were grown in Grace's insect medium (Life Technologies, Inc.) supplemented with Yeastolate (0.33%), lactalbunin hydrolysate (0.33%), and 10% fetal bovine serum at 27 °C.

Adenylyl Cyclase Assay

Adenylyl cyclase activity was assayed as described previously (1). Briefly, cells were resuspended in the lysis buffer (10 mM EDTA, 20 mM Tris-HCl, 250 mM sucrose, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 40 µM leupeptin, pH 7.4), and sonicated using a W-380 sonicator (Ultrasonics Inc.) at a setting of 20% output power for a total of 45 s. The homogenate was centrifuged at 50,000 × g for 30 min to collect the P1 membrane fractions. The adenylyl cyclase activity assay was performed at 37 °C for 10 min in a 400-µl reaction mixture containing 1 mM ATP, 100 mM NaCl, 0.4 unit of adenosine deaminase, 50 mM Hepes, 0.5 mM 3-isobutyl-1-methylxanthine, 6 mM MgCl2, 1 µM GTP, and 20 µg of membrane protein. Reactions were stopped by 0.6 ml of 10% trichloroacetic acid. The cAMP formed was isolated by Dowex chromatography (Sigma) and assayed by radioimmunoassay using the 125I-labeled cAMP assay system (Amersham). No significant difference was found by adding 20 mM creatine phosphate (Sigma), or 100 units/ml creatine phosphokinase (Sigma) to the cyclase reaction. In experiments with elevated calcium concentrations, membrane preparations were washed two more times with ice-cold HDLP buffer (20 mM Hepes, 0.5 mM dithiothreitol, 0.1 mM leupeptin, 40 µM PMSF, pH 7) containing 1 mM EGTA to remove the endogenous calcium and calmodulin (10). Membrane fractions were then resuspended in a EGTA/calcium buffer to control the free calcium concentration at zero or 30 µM for adenylyl cyclase assay. Since only one calcium-inhibitable adenylyl cyclase (AC6) exists in PC12 cells (2), the AC6 activity in PC12 cells was determined as the difference between the forskolin-evoked AC activity assayed in the absence and the presence of 30 µM free calcium. Addition of calcium in the cyclase assay did not evoke any detectable phosphodiesterase activity in control or desensitized membrane. The enzyme activity was linear for up to 20 min with membrane protein up to 100 µg. All samples were assayed in triplicate.

A2a Desensitization in a Cell-free System

P1 membrane fractions were collected from PC12 cells as described above, and resuspended in DB buffer (0.4 mM EDTA, 1 mM EGTA, 0.1 mM leupeptin, 40 µM PMSF, 100 µM GTP, 1 mM ATP, 30 nM OKA, 0.2 mM sodium vanadate, 5 mM MgCl2, and 25 mM Tris, pH 7.2). A2a desensitization was triggered by incubating the P1 membrane fractions with CGS21680 (100 µM) for 30 min at 37 °C. The membranes were washed twice with ice-cold HDLP buffer to remove the excess agonist. Adenylyl cyclase activities of these treated membranes were determined as described above.

Expression of AC6 in Sf21 Cells

The rat AC6 cDNA was kindly provided by Dr. Iyengar (18). A synthetic 8-amino acid peptide, FLAG, was ligated to the N terminus of AC6 to encode a chimeric FLAG-AC6 recombinant protein (designated as F-AC6). Monoclonal antibody against FLAG was obtained from Kodak. Expression of AC6 and F-AC6 was carried out in a recombinant baculovirus-driven Sf21 cell system following the manufacturer's protocol (Pharmingen). Membrane fractions were collected as described above from Sf21 cells infected with the desired virus 68-72 h postinfection.

Preparation of Antibody

An oligopeptide corresponding to amino acids 1162 to 1181 of rat AC6 was purchased (Genosys, Woodlands, TX) and conjugated to bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester as described by Harlow and Lane (19). To produce anti-AC6 antibodies, male New Zealand White rabbits (~2.5 kg) were immunized by an intramuscular injection of the bovine serum albumin-conjugated AC6 peptide using standard procedures (19). This polyclonal AC6-specific anti-peptide antibody is designated as AC6C. A recombinant protein containing amino acid 987 ~1187 of AC6 was expressed and purified from Escherichia coli BL21-DE3, and used to raise polyclonal antibody in rabbits using standard procedures (19). The resulting anti-serum was designated as AC6D.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting

We determined protein concentration by a simple colorimetric assay based on the Bradford dye binding procedure (20) using the Bio-Rad protein assay dye reagent concentrate (Bio-Rad). For SDS-polyacrylamide gel electrophoresis, membrane fractions were combined with 2× sample buffer containing 125 mM Tris-HCl (pH 6.8), 20% glycerol, 1% SDS, 15% 2-mercaptoethanol, 200 mM dithiothreitol, and 0.01% bromphenol blue, boiled for 5 min, centrifuged to remove the insoluble material, and then separated on 8% separating gels according to the method of Laemmli (21). Following electrophoresis, the gel was transferred to a polyvinylidene difluoride membrane, blocked with 5% skim milk in PBS, then incubated with the desired antiserum at 4 °C overnight. Typically, we used a 1:1000 dilution for PKCdelta antiserum, 1:500 for PKCepsilon antiserum, 1:250 for PKCtheta antiserum, 1:1000 for AC6C, and 1:5000 for AC6D, respectively, unless stated otherwise. After three washes of 5 min in PBS, the membranes were incubated with peroxidase-conjugated donkey anti-rabbit IgG (Amersham, UK) at 1:5000 dilution for 1 h at room temperature. The membranes were washed three times with PBS, and the immunoreactive bands were stained using a light emitting nonradioactive method (ECL, Amersham, UK).

Immunoprecipitation and in Vitro Phosphorylation

To carry out the protein phosphorylation study, AC6 and F-AC6 expressed in Sf21 cells were purified by immunoprecipitation using AC6C. In brief, 1 mg of P1 membrane fractions collected from Sf21 cells infected with the indicated virus were solubilized in 0.4 ml of ice-cold radioimmune precipitation buffer (50 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8) at 4 °C for 30 min. Immunoprecipitation was initiated by addition of AC6C (1:50 dilution). The reaction mixture was incubated with gentle agitation at 4 °C overnight. Immunocomplexes were purified using Sephadex conjugated protein A (Sigma) and washed three times with ice-cold radioimmune precipitation buffer. Phosphorylation by PKC was carried out in a final volume of 0.2 ml reaction mixture containing 10 mM MgCl2, 1 mM CaCl2, 0.25% bovine serum albumin, 10 µM H89, 0.5 mM [gamma -32P]ATP (2 Ci/mole), 0.1 mM leupeptin, 40 µM PMSF, 30 nM OKA, 0.2 mM sodium vanadate, and 20 mM Tris (pH 7.5). The phosphorylation reaction was initiated by addition of protein kinase C (0.1 milliunit) purified from rat brain (Boehringer Mannheim) for 30 min at 4 °C, and terminated by the addition of 2 × SDS sample treatment buffer. The samples were then boiled for 5 min, analyzed by SDS-polyacrylamide gel electrophoresis (8%) and Western blot analysis using AC6D. To visualize the phosphorylation of AC6 by PKC, immunoblots were rinsed twice with PBS, air-dried, and autoradiographed.

To phosphorylate AC6 purified immunologically from PC12 cells using exogenous PKC, 17 mg of P1 membrane fractions collected from control or A2a desensitized cells (CGS21680, 2.5 µM, 16 h) were solubilized in 3.2 ml of ice-cold radioimmune precipitation buffer at 4 °C for 30 min. Immunological purification of AC6 using AC6C was conducted as described above. Phosphorylation by PKC was carried out in a final volume of 0.1 ml of reaction mixture containing 10 mM MgCl2, 0.25% bovine serum albumin, 11 µM [gamma -32P]ATP (35 Ci/mole), 0.1 mM leupeptin, 40 µM PMSF, 1 µM OKA, 0.2 mM sodium vanadate, 5 mM beta -glycerolphosphate, 1 µM PDD, and 25 mM Tris (pH 7.5). The phosphorylation reaction was initiated by addition of PKC (24 microunits) purified from rat brain (Boehringer Mannheim) for 5 min at 37 °C, and terminated by the addition of 2 × SDS sample treatment buffer. The samples were then boiled for 5 min and analyzed by SDS-polyacrylamide gel electrophoresis (10%) and Western blot using AC6D. To visualize the phosphorylation of AC6 by PKC, immunoblots were rinsed twice with PBS, air-dried, and autoradiographed.

Protein Kinase C Assay

PKC activities were measured under the same conditions as those for the A2a desensitization in vitro. In brief, PKC was activated by incubating the P1 membrane fractions with or without CGS21680 (100 µM) for 30 min at 37 °C in a 25-µl reaction mixture containing 0.2 µM of protein kinase A inhibitor peptide (Upstate Biotechnology Inc.), 2 µM of compound R24571 (Upstate Biotechnology Inc.), 10 µM of epsilon  peptide (Upstate Biotechnology Inc.), 0.4 mM EDTA, 1 mM EGTA, 0.1 mM leupeptin, 40 µM PMSF, 100 µM GTP, 1 mM [gamma -32P]ATP (0.2 Ci/mmol), 30 nM OKA, 0.2 mM sodium vanadate, 5 mM MgCl2, and 25 mM Tris (pH 7.2). Reactions were stopped by adding 10 µl of 30% trichloroacetic acid. A 30-µl aliquot was spotted on phosphocellulose paper (Whatman, P-81). The papers were washed five times with 0.75% phosphoric acid, once with acetone, and added to scintillation vials for counting. PKC activity was determined by subtracting the background activity measured in the absence of sample from that measured in its presence. The PKC-epsilon peptide was chosen as the substrate in our PKC assays since this 12-residue synthetic peptide (PRKRQGSVRRRV-NH2) is an effective substrate of the calcium-insensitive protein kinase C-epsilon and other isoforms (22).


RESULTS

Desensitization of A2a-R-mediated cAMP Response in a Cell-free Membrane Preparation

To trigger A2a desensitization in intact cells, PC12 cells at 70% confluency were treated with CGS21680 (CGS, 2.5 µM), an A2a-selective agonist, for 30 min. Membrane fractions were then collected from control and desensitized cells, and assayed for CGS21680- or forskolin-evoked AC activity. As we have reported earlier (1, 2), AC activities evoked by activation of A2a-R or by direct stimulation using forskolin were both significantly decreased in desensitized cells (Table I), confirming that AC is one of the major sites for the suppression of A2a-R-mediated cAMP signal during A2a desensitization. To dissect and identify the molecular events underlying the suppression of AC activity during A2a desensitization, we examined whether this phenomenon can be reproduced in a cell-free system as in intact cells. A 50,000 × g membrane fraction collected from PC12 cells was incubated with or without CGS21680 for 30 min to stimulate A2a-R and trigger A2a desensitization. As shown in Table I, significant inhibition of the A2a-R-stimulated and of the forskolin-stimulated AC activity was observed in membrane fractions pretreated with CGS21680. In addition, similar extents of AC inhibition during A2a desensitization were observed in the P1 membrane fractions and in intact cells (Table I). Therefore, molecules that are responsible for the suppression of AC activity during A2a desensitization in intact PC12 cells must exist in the P1 membrane fractions. We designated such inhibition of AC activity during prolonged exposure of PC12 membrane fractions to an A2a-selective agonist as "A2a desensitization in vitro."

Table I.

Desensitization of A2a-R-mediated cAMP response in a cell-free membrane preparation

A2a desensitization in intact PC12 cells or in P1 membrane fractions were triggered as described under "Experimental Procedures." AC activities in response to CGS21680 (CGS) (100 µM) or forskolin (FK) (5 µM) as indicated were assayed. Values represent the mean ± S.E. of nine determinations (three determinations in three independent experiments), and are expressed as percentages of the AC activity (138 ± 21 pmol/mg/min and 443 ± 71 pmol/mg/min for CGS-evoked and FK-evoked AC activity respectively) in non-desensitized cells or percentages of the AC activity (61 ± 13 and 187 ± 23 pmol/mg/min for CGS-evoked and FK-evoked AC activity, respectively) in the non-desensitized P1 membrane fractions respectively.
A2a desensitization AC activity in desensitized preparations
CGS FK

%
Intact cells 43  ± 5 60  ± 3
P1 fractions 48  ± 4 55  ± 4

We have previously reported that AC6 appears to be the major AC involved in A2a desensitization in PC12 cells (2). AC6 activity in PC12 cells was determined as the difference between the forskolin (5 µM)-evoked AC activity assayed in the absence and the presence of 30 µM free calcium (2). Similar to what we have observed in intact PC12 cells, AC6 activity was inhibited by 44 ± 4% (mean ± S.E. of 12 determinations from four independent experiments) in P1 membrane fractions pretreated with CGS21680 to trigger A2a desensitization in vitro. Therefore, AC6 is also inhibited during A2a desensitization in vitro as that observed in intact PC12 cells.

Nucleotide Dependence of A2a Desensitization in Vitro

To further characterize A2a desensitization in vitro, we examined the nucleotide dependence of this phenomenon. As shown in Fig. 1A, removal of GTP from the desensitization reaction significantly reduced inhibition of AC activity, indicating that A2a desensitization is dependent upon GTP and may be mediated through a GTP-binding protein.


Fig. 1. Nucleotide dependence of A2a desensitization in vitro. A, P1 membrane fractions were treated with or without CGS21680 (100 µM) for 30 min in the presence or absence of GTP (100 µM) as indicated to trigger desensitization. AC activity in response to forskolin (5 µM) in these treated membranes was then assayed. Values represent the mean ± S.E. of nine determinations (three determinations in three independent experiments) and are expressed as percentages of adenylyl cyclase activity in nondesensitized membranes in the presence of GTP (416 ± 51 pmol/mg/min) or absence of GTP (206 ± 21 pmol/mg/min). B, dose response curve of ATP on AC6 activity during A2a desensitization. P1 membrane fractions were treated with or without CGS21680 (100 µM) for 30 min to trigger A2a desensitization in the presence of ATP of the indicated concentration. Membrane fractions were collected and assayed for AC6 activity, which was determined as the difference between the forskolin (5 µM)-evoked AC activity assayed in the absence and the presence of 30 µM free calcium. Values represent the mean ± S.E. of nine determinations (three determinations in three independent experiments), and are expressed as percentages of the AC activity in the nondesensitized membrane at the indicated concentration of ATP (461 ± 45 pmol/mg/min; 280 ± 41 pmol/mg/min; and 95 ± 15 pmol/mg/min for 1 mM ATP, 0.1 mM ATP and 0.01 mM, respectively).
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Since our previous study (2) suggested that protein phosphorylation might mediate the suppression of AC6 activity during A2a desensitization, we examined whether A2a desensitization in vitro required ATP. As shown in Fig. 1B, lowering of ATP to 0.1 mM almost completely abolished the suppression of AC6 activity during A2a desensitization in vitro, indicating that the suppression of AC6 activity may be mediated through an ATP-dependent pathway. This observation is consistent with our previous hypothesis that AC6 is regulated through protein phosphorylation during A2a desensitization. Exclusion of GTP or ATP during A2a desensitization consistently and significantly reduced the forskolin-evoked AC activity (data not shown). The physiological significance of this observation however remains unclear.

Protein Phosphatase 2A Blocked the Inhibition of AC6 during A2a Desensitization in Vitro

To further investigate the role of protein phosphorylation in suppression of AC6 during A2a desensitization in vitro, we examined the effect of protein phosphatase 2A (PP2A) on the regulation of AC6 during A2a desensitization in vitro. As shown in Table II, incubation of the P1 membrane with PP2A (0.1 unit) completely blocks the inhibition of AC6 during A2a desensitization in vitro. This observation is consistent with our previous finding that PP2A in intact cells plays a very important role in the regulation of AC6 activity during A2a desensitization (2). Furthermore, inhibition of AC6 activity may be mediated through a protein phosphorylation mechanism during A2a desensitization.

Table II.

Protein kinase C, but not protein kinase A, might mediate the inhibition of AC activity during A2a desensitization

Except for PDD treatment, P1 membranes collected from PC12 cells were treated with CGS21680 (100 µM) for 30 min to trigger A2a desensitization in the presence of the indicated reagent. For PDD treatment, PKCs in PC12 cells were down-regulated by 17-h incubation of PC12 cells with PDD (100 nM). P1 membranes were then collected from these PDD-treated cells and exposed to CGS21680 (100 µM) for 30 min to trigger desensitization. Values represent the mean ± S.E. of 9 to 33 determinations (three determinations in 3 to 11 independent experiments), and are expressed as percentages of the AC6 activity in the control (non-desensitized) membranes in the presence of the indicated reagent (395 ± 40, 460 ± 64, 220 ± 21, 390 ± 68, and 477 ± 39 pmol/mg/min for none, H89 (10 µM)-, H8 (6 µM)-, PKCalpha 19-36 (1.5 µM)-, PDD*-, and PP2A (0.1 unit)-treated membranes, respectively). The AC6 activity in PC12 cells was determined as the difference between the forskolin (5 µM)-evoked AC activity assayed in the absence and the presence of 30 µM free calcium.
Treatment AC6 activity during A2a desensitization

%
None 54  ± 2
H89 55  ± 7
H8 118  ± 6
PKCalpha 19-36 92  ± 4
PDD 100  ± 3
PP2A 103  ± 5

Protein Kinase C, but Not Protein Kinase A, May Mediate the Inhibition of AC6 Activity during A2a Desensitization

To investigate the potential role of protein kinases, inhibitors of PKC or PKA were included during A2a desensitization in vitro. As shown in Table II, an H-series compound, H8, which inhibits both PKA and PKC at micromolar concentrations (23) blocked the inhibition of AC6 activity during A2a desensitization. By contrast, a PKA-selective inhibitor (H89) which is 30 times more potent than H8 in inhibiting PKA (23), exerted no significant effect on the inhibition of AC6 during A2a desensitization. Therefore, PKA is unlikely to inhibit AC6 activity during A2a desensitization. Alternatively, PKC might mediate the reduction of AC6 activity during prolonged activation of A2a-R. To test the above hypotheses, the effect of a pseudosubstrate of PKC (PKCalpha 19-36) was examined. At 1.5 µM, PKCalpha 19-36 almost completely blocked the suppression of AC6 activity, suggesting that PKC might play a cardinal role in A2a desensitization (Table II). This peptide inhibitor of PKC has no significant effect on smooth muscle myosin light chain kinase, calcium/calmodulin-dependent protein kinase II and PKA (24) at the concentration tested. To further verify the role of PKC, PC12 cells were treated with a PKC-stimulating phorbol ester (PDD, 100 nM), for 17 h to induce proteolysis and "down-regulation" of PKC. In these PKC down-regulated cells, no inhibition of AC6 activity during A2a desensitization in vitro was observed. These results strongly suggest that PKC, but not PKA, mediates the suppression of AC6 during A2a desensitization resulting from prolonged activation of A2a-R.

PKC Suppressed AC6 Activity in PC12 and Sf21 Cells

To further confirm the role of PKC in suppression of AC6 during A2a desensitization, we examined whether addition of purified PKC which contains a mixture of PKC isozymes (alpha , beta I, beta II, gamma , delta , epsilon  and µ; data not shown) from rat brain suppressed AC6 activity. As shown in Table III, PKC caused a 54 ± 3% inhibition of AC6 activity in the P1 membranes of PC12 cells. To facilitate the study of biochemical properties of AC6, we overexpressed AC6 and F-AC6 in insect cells (Sf21) using the baculovirus expression system. F-AC6 is a chimeric AC6 with an N-terminal peptide containing an 8-amino acid epitope (DYKDDDDK, designated as FLAG). This epitope can be recognized by a commercially available antibody (M2) and has been fused to various proteins, including AC2 (11), with no conspicuous effect on their activity (25). Likewise, no difference in the enzymatic properties of AC6 and F-AC6 was detectable (data not shown). Exogenous PKC dramatically inhibited AC6 activity in membrane fractions collected from Sf21 cells overexpressing AC6 or F-AC6 as indicated (Table III).

Table III.

PKC-suppressed AC6 activity expressed in PC12 cells and in Sf21 cells

P1 membrane fractions (1 mg) collected from PC12 cells or Sf21 cells overexpressing AC6 or F-AC6 as indicated were incubated with purified PKC (0.1 milliunit) for 30 min at 4 °C. Membrane fractions were then washed once by centrifugation to remove PKC and assayed for AC6 activity. The AC6 activity in PC12 membranes was determined as the difference between the forskolin (5 µM)-evoked AC activity assayed in the absence and the presence of 30 µM free calcium. Recombinant AC6 activities in Sf21 cells were determined as the difference between the forskolin (5 µM)-evoked AC activity in the AC6-overexpressing membrane fractions and the AcNPV (the wild type virus) expressing membrane fractions. Values represent the mean ± S.E. of six to nine determinations (three determinations in two to three independent experiments), and are expressed as percentages of the AC6 activity in the control (nontreated) membrane (364 ± 48, 171 ± 39, and 140 ± 1 pmol/mg/min for PC12 cells, AC6, and F-AC6, respectively).
Membrane PKC-evoked inhibition of AC6 activity

%
PC12 54  ± 3
AC6/Sf21 74  ± 11
F-AC6/Sf21 60  ± 15

PKC Directly Phosphorylated AC6

In order to determine whether PKC phosphorylates AC6, we prepared two polyclonal antibodies (designated AC6C and AC6D) which recognize amino acid 1162 ~ 1181 of AC6 and amino acid 987 ~ 1187 of rat AC6, respectively. Both antibodies are capable of immunoprecipitating F-AC6 expressed in Sf21 cells as shown in Fig. 2. We then purified AC6 and F-AC6 from Sf21 cells expressing the indicated cDNA using AC6C by immunoprecipitation. Phosphorylation of AC6 or F-AC6 by PKC (0.1 milliunit in a 200-µl reaction) was then carried out at 4 °C for 30 min in the presence of [gamma -32P]ATP. As shown in Fig. 3, PKC phosphorylated both AC6 and F-AC6. These data illustrate that PKC not only inhibits AC6 activity, also directly phosphorylates this calcium-inhibitable AC.


Fig. 2. Immunoprecipitation of recombinant F-AC6 protein by AC6C and AC6D. Membrane fractions (100 µg) collected from Sf21 cells overexpressing F-AC6 were solubilized and immunoprecipitated with the indicated antiserum (1:50 dilution for both AC6C and AC6D). Immunocomplexes were then purified using Sephadex-conjugated protein A and examined by immunoblot analysis using M2 antibody (1:500 dilution). The arrow marks the AC6-immunoreactive bands.
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Fig. 3. PKC phosphorylated AC6. Recombinant AC6 and F-AC6 were prepared in Sf21 cells and purified using AC6C as described in the text. Purified AC6 or F-AC6 was then incubated with or without PKC (0.1 milliunit) as indicated for 30 min at 4 °C in the presence of [gamma -32P]ATP. At the end of the incubation, the samples were boiled for 5 min and analyzed by Western blot analysis (A) using AC6D (1:5000 dilution) and by autoradiography (B). The arrows mark the AC6-immunoreactive bands.
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CGS21680 Pretreatment in Intact PC12 Cells Prevents Further Inhibition of AC6 Activity and Leads to Less Phosphorylation of AC6 by Exogenous PKC

To determine that the regulation of AC6 during A2a desensitization in PC12 cells was indeed due to a PKC, PC12 cells were pretreated with CGS21680 (2.5 µM) for 16 h to obtain maximal inhibition of AC6 (1). P1 membrane fractions of control (cells pretreated with no agonists) or CGS21680-pretreated cells were then incubated with exogenous PKC for 30 min at 4 °C. Consistent with our previous observation, exogenous PKC inhibited AC6 activity in control cells by 65 ± 8%. As predicted, pretreatment of PC12 cells with CGS21680 for 16 h caused a typical 68 ± 5% reduction in AC6 activity compared to that in control cells. Most importantly, incubation of CGS21680-pretreated membrane with PKC did not further suppress the AC6 activity (Fig. 4). To demonstrate that in vitro phosphorylation by PKC is truly related to the desensitization of AC6, exogenous PKC phosphorylation of AC6 purified from control or CGS21680-pretreated cells were examined. As shown in Fig. 5, A2a desensitization profoundly reduced in vitro phosphorylation of AC6 by PKC. Taken together, pretreatment of PC12 cells with CGS21680 which leads to A2a desensitization might induce phosphorylation of AC6 at the same residues which are phosphorylated by exogenous PKC in vitro.


Fig. 4. PKC did not further inhibit AC6 activity in desensitized membranes. PC12 cells were treated with or without CGS21680 (2.5 µM) for 16 h to trigger A2a desensitization. P1 membrane fractions collected from the control cells (CON) and desensitized cells (DES) were incubated with or without PKC (0.1 milliunit) as indicated for 30 min at 4 °C. The calcium-inhibitable AC (AC6) activities evoked by CGS21680 (100 µM) or by forskolin (5 µM) were determined as the difference between AC activity assayed in the absence and the presence of 30 µM free calcium. Values represent the mean ± S.E. of three determinations. The results are from one representative experiment out of three independent experiments performed.
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Fig. 5. Exogenous PKC effectively phosphorylated AC6 isolated from control, but not A2a desensitized PC12 cells. PC12 cells were treated with or without CGS21680 (2.5 µM) for 16 h to trigger A2a desensitization. AC6 from control (CON) or desensitized (DES) cells were purified immunologically using AC6C as described in the text. Purified AC6 was then incubated with or without PKC (24 µM) as indicated for 5 min at 37 °C in the presence of [gamma -32P]ATP. At the end of the incubation, the samples were boiled for 5 min and analyzed by Western blot analysis (A) using AC6D (1:5000 dilution) and by autoradiography (B). The arrows mark the AC6-immunoreactive bands.
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Novel PKCs Might Play a Very Critical Role in the Reduction of AC6 Activity during A2a Desensitization

Since inhibition of AC6 during A2a desensitization was well demonstrated in the "cell-free" P1 membrane preparation of PC12 cells with all of the characteristics tested identical to those observed in intact cells, enzymes necessary to suppress AC6 activity during prolonged activation of A2a-R must exist in the P1 membrane fractions. Western blot analysis demonstrated the existence of at least seven different PKC isozymes (alpha , gamma , delta , epsilon , iota , µ, and zeta ) in the P1 membrane fractions of PC12 cells (data not shown). Among these seven PKC isozymes, we are most interested in the novel PKCs which are insensitive to calcium, but require diacylglycerols (or phorbol esters) for their activation based on the following two observations. First, A2a desensitization in vitro occurs in the absence of calcium, suggesting that the PKC responsible for the suppression of AC6 during A2a desensitization is likely to be calcium-independent. Second, down-regulation of PKC by pretreating PC12 cells with a PKC-stimulating phorbol ester (PDD) prevented the reduction of AC6 during A2a desensitization (Table II). Thus, the PKC involved in the regulation of AC6 must be sensitive to phorbol esters. We therefore examined the expression of novel PKCs in P1 membrane fractions after 17-h exposure of PC12 cells to PDD. As shown in Fig. 6, P1 membrane fractions contained significant amounts of PKCdelta and PKCepsilon , but not PKCtheta . As predicted, treating PC12 cells with PDD (100 nM) for 17 h almost completely eliminated the immunoreactive bands of PKCdelta and PKCepsilon in both the P1 membrane and the supernatant fractions. Under the same conditions, no suppression of AC6 activity was observed during A2a desensitization in vitro. These data imply that PKCdelta and PKCepsilon are the two most likely candidates for the reduction of AC6 activity during A2a desensitization.


Fig. 6. Down-regulation of PKCdelta and PKCepsilon in P1 membrane fractions of PC12 by PDD. Membrane fractions were collected from PC12 cells pretreated with or without PDD (100 nM) for 17 h. Cells were then resuspended in lysis buffer and sonicated as described in the text. The homogenates were centrifuged at 50,000 × g for 30 min to separate the P1 membrane fractions (P) and the supernatant fractions (S). An equal amount (40 µg) of protein from the indicated treatment was loaded to each lane for Western blot analysis using the desired PKC antiserum. Arrows mark the indicated PKC immunoreactive band(s).
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To further strengthen the above hypothesis, regulation of AC6 activity during A2a desensitization was examined in stably transfected PC12 cell lines overexpressing PKCdelta or PKCepsilon characterized elsewhere (16). A significantly lower AC6 activity was observed in both cell lines transfected with PKCdelta (delta 1) or PKCepsilon (epsilon 1) compared to that observed in control cells (cells transfected with the pRc/RSV empty vector, designated C1). Given that these PKC-overexpressing cells exhibit significantly higher PKC activities and in turn might lead to higher PKC-mediated phosphorylation, it is reasonable to hypothesize that the lower AC6 activity in these cells might be due to a higher level of AC6 phosphorylation. Intriguingly, prolonged activation of A2a-R in these PKC-overexpressing cells did not further suppress AC6 activity whereas the same treatment resulted in a 67 ± 3% inhibition in the control C1 cells (Fig. 7A), a 50 ± 7% inhibition in the parental PC12 cells and a 45 ± 11% inhibition in a PKA-deficient PC12 variant (17), A123 cells (Fig. 7B). Since higher PKC activity in novel PKC-overexpressing cells (delta 1 and epsilon 1) and prolonged activation of A2a-R both inhibited AC6 activity in a nonadditive fashion, AC6 is very likely to be regulated through a PKC-dependent pathway. These observations strongly suggest that an increase in novel PKC-mediated phosphorylation during prolonged activation of A2a-R inhibits AC6 activity in PC12 cells. In addition, PKA is not required for the regulation of AC6.


Fig. 7. Regulation of AC6 activity in PC12 cells overexpressing novel PKC (A) or deficient in PKA (B). P1 membrane fractions collected from the indicated cells were treated with or without CGS21680 (100 µM) for 30 min at 37 °C to trigger A2a desensitization. Membrane fractions were centrifuged to remove excess CGS21680 and assayed for the calcium-inhibitable AC activity (AC6 activity), which was determined as the difference between the forskolin (5 µM)-evoked AC activity assayed in the absence and the presence of 30 µM free calcium. Values represent the mean ± S.E. of three determinations. The results are from one representative experiment out of three independent experiments performed.
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Activation of A2a-R Leads to an Increase in the Calcium-insensitive PKC Activity

The question arises as to whether PKC is activated during A2a desensitization. Since inhibition of AC6 during A2a desensitization in vitro occurs in the absence of calcium, the PKC responsible for the regulation of AC6 is likely to be calcium-independent. Using a PKC-epsilon substrate peptide, we found that the calcium-independent PKC activity was considerably enhanced by treating the P1 membrane fractions with CGS21680 under the identical conditions as those for A2a desensitization in vitro. The PKC inhibitor, PKCalpha 19-36, almost completely abolished the enhanced calcium-independent PKC activity by CGS21680 (Table IV). Activation of A2a-R therefore indeed activated a calcium-insensitive PKC which phosphorylated AC6 and caused a subsequent inhibition of AC6 activity.

Table IV.

Stimulation of A2a-R results in activation of calcium-independent PKC activity in PC12 cells

PKC activities of the P1 membrane fractions were measured by incubating the P1 membrane fractions with or without CGS21680 (100 µM) for 30 min at 37 °C in the presence or absence of a PKC peptide inhibitor (PKCalpha 19-36, 1.5 µM) as indicated. Values represent the mean ± S.E. of four determinations. The results are from one representative experiment out of three independent experiments performed.
Treatment PKC inhibitor PKC activity

cpm × 10-3
CONT  - 1.72  ± 0.16
CGS  - 2.86*  ± 0.26
CONT + 1.66  ± 0.3
CGS + 1.89  ± 0.15

Statistical significance: * indicate differences between control and CGS21680-treated membranes (p < 0.001; paired Student's t test).

Effect of Pertussis Toxin (PTX) on A2a Desensitization

To elucidate the signaling pathway underlying the suppression of AC6 by PKC, we examined the effect of PTX on A2a desensitization. As shown in Fig. 8, PTX treatment almost entirely blocked the suppression of AC activity resulting from prolonged activation of A2a-R. These data indicate that a PTX-sensitive Galpha protein is involved in the inhibition of AC activity during A2a desensitization.


Fig. 8. Effect of PTX on the inhibition of adenylyl cyclase activity during A2a desensitization. PC12 cells were treated with or without PTX (200 ng/ml) for 3 h as indicated. P1 membrane were then collected, treated with CGS21680 (100 µM) for 30 min at 37 °C to trigger A2a desensitization. Values represent the mean ± S.E. of at least nine determinations (three determinations in three independent experiments), and are expressed as percentages of the adenylyl cyclase activity in the non-desensitized membrane (371 ± 19 and 116 ± 3 pmol/mg/min for control and PTX-treated cells, respectively).
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DISCUSSION

Our previous studies indicated that activity of a Ca2+-inhibitable AC (AC6) during A2a desensitization may be regulated through a phosphorylation-dependent pathway (2). In the present study, we demonstrate that suppression of AC6 activity can be reproduced in 50,000 × g membrane fractions (P1) following prolonged stimulation of A2a-R. Thus, enzymes that regulate AC6 activity during A2a desensitization must exist in the P1 membrane fractions. Various lines of evidence strongly suggest that novel PKC isozymes in the P1 membrane fractions phosphorylate and inhibit AC6 activity after prolonged stimulation of A2a-R.

The cell-free membrane system provides a very useful and convenient model to elucidate the molecular event(s) underlying the regulation of AC6 during A2a desensitization. We have previously shown that A2a desensitization in PC12 cells is heterologous (1). AC activities stimulated by adenosine agonists, GTPgamma S, and forskolin were equally impaired, suggesting that the regulation occurred at the effector enzyme itself, but not at the A2a-R nor at the Gsalpha protein. The suppression of AC6 during A2a desensitization remained largely unchanged when GDPbeta S and MnCl2 were used to replace GTP and MgCl2 in some cyclase assays. Thus, the potential influence of Gsalpha protein in the inhibition of AC6 was further ruled out (2). Although two adenosine receptors (A2a and A2b) exist in PC12 cells, we have previously demonstrated that desensitization of the A2a response in PC12 cells is due to prolonged activation of A2a-R based on the following evidence (1). First, the relative potency of three adenosine agonists to desensitize the A2a response in PC12 cells is in the same order as their potencies for activating A2a-R. Second, an adenosine antagonist (8-cyclopentyl-1,3-dipropylxanthine) is able to block the CGS21680-evoked inhibition of AC activity (1). CGS21680 is a very selective A2a adenosine agonist (26), which does not activate A2b-R in PC12 cells up to 300 µM (1). In the present study, we have shown that the degree of AC inhibition during A2a desensitization in intact cells and in the P1 membrane fractions is very similar (Table I). Marked suppression of both CGS21680- and forskolin-evoked AC activities were observed, suggesting that the reduction of A2a signaling in P1 membrane fractions by prolonged activation of A2a-R is caused largely by inhibition of AC itself as in intact cells. In addition, inhibition of AC activity during prolonged activation of A2a-R is GTP-dependent (Fig. 1A), further confirming that this phenomenon is mediated through a GTP-binding protein coupled pathway.

Stimulation of A2a-R has long been established to cause activation of AC activity via Gsalpha protein (27). However, the fact that A2a desensitization requires GTP (Fig. 1A) and was not observed in the PTX-treated PC12 cells (Fig. 8), strongly suggests that this phenomenon is mediated via a PTX-sensitive Galpha protein. Koizumi et al. (28) have recently reported that an A2-selective agonist (CGS22492), but not an A1-selective agonist (CCHA), potentiates ATP-evoked dopamine release via a pertussis toxin-sensitive mechanism in PC12 cells in a cAMP-independent manner (28). Their finding is consistent with our hypothesis that A2a-R may transduce the cellular signal not only via Gsalpha , but also through a PTX-sensitive G protein in PC12 cells.

We have previously hypothesized that protein phosphorylation of AC6 may account for the inhibition of its activity during prolonged exposure of PC12 cells to adenosine agonists. However, it was unclear whether regulation of the phosphorylation state of AC6 resulted from activation of an unknown kinase or inactivation of PP2A during A2a desensitization (2). Using the cell-free system, we now show that the inhibition of AC6 during A2a desensitization is ATP-dependent (Fig. 2B), suggesting that activation of a kinase is required. Since stimulation of A2a-R leads to an increase in intracellular cAMP in PC12 cells, PKA is an attractive candidate for the regulation of AC6 activity during A2a desensitization. However, treatment with dibutyryl-cAMP or forskolin did not reproduce the inhibition of AC activity during A2a desensitization in PC12 cells (1). Hence, PKA by itself is not sufficient to trigger inhibition of AC6. Furthermore, a very selective and potent PKA inhibitor (H89) at 10 µM did not exert any detectable effect on the inhibition of AC6 during A2a desensitization. This PKA-selective inhibitor is an H-series kinase inhibitor which blocks PKA by competing with ATP (23). At the same concentration (10 µM) as that tested in A2a desensitization (Table II), H89 almost entirely abolished the activation of phosphodiesterase activity during prolonged activation of A2a-R in PC12 cells.2 In addition, the extent of AC6 suppression during A2a desensitization in a PKA-deficient PC12 variant (A123) is comparable to that observed in the parental PC12 cells (Fig. 7B). Taken together, elevation of cellular cAMP upon stimulation of A2a-R, that subsequently leads to activation of PKA, is not responsible for the inhibition of AC6.

In contrast to PKA, PKC appears to play a very critical role in the regulation of AC6. Inhibiting PKC through two independent means (down-regulation of PKC with PDD and use of a PKC-specific inhibitor) effectively blocked the suppression of AC6 during A2a desensitization in the cell-free membrane fractions (Table II). In addition, incubation of membrane fractions expressing AC6 with purified PKC caused a marked reduction of AC6 activity (Table III). PKC also directly phosphorylated purified AC6 (Fig. 3). A specific role for PKC in regulating AC6 during A2a desensitization in PC12 cells is further suggested by the lack of effect of exogenous PKC on the activity of AC6 isolated from desensitized cells (Fig. 4). Furthermore, in vitro PKC phosphorylation of AC6 obtained from desensitized cells was markedly reduced (Fig. 5), indicating that AC6 is indeed a substrate for PKC during A2a desensitization. Most importantly, activation of A2a-R resulted in a significant increase in the calcium-independent PKC activity (Table IV). These data strongly support PKC as the kinase responsible for the suppression of AC6 during A2a desensitization in PC12 cells. This hypothesis is further strengthened by examining the regulation of AC6 activity in PC12 clones overexpressing PKCdelta or PKCepsilon . When compared to the control clone, the Ca2+-independent PKC activities in clones overexpressing PKCdelta (delta 1) and PKCepsilon (epsilon 1) were increased by 2.2- and 1.6-fold, respectively (14). Since the levels of the phosphorylation state of AC6 were expected to be higher in these PKC-overexpressing cells, it was not surprising to find that the AC6 activity in these cells was significantly lower than in the control cells (Fig. 7A). Most strikingly, no further inhibition of AC6 activity was observed in the PKC overexpressing cells after prolonged activation of A2a-R by CGS21680, whereas the same treatment resulted in a 67 ± 3% inhibition in control cells (Fig. 7A). Prolonged activation of A2a-R may therefore regulate AC6 activity through the very same mechanism used in the PKC-overexpressing cells. In addition, AC6 appears to be very sensitive to small changes in PKC activity (~2-fold). All of the strategies utilized above provide convincing evidence to support our hypothesis that PKC inhibits the activity of AC6 in PC12 cells during prolonged activation of A2a-R.

Modulation of AC isozymes by PKC has been actively examined in the past few years. In brief, PKC has been shown to stimulate AC1, AC2, and AC5 (10-12). Phosphorylation of these AC isozymes, including AC2 and AC5, affects mainly catalytic activities (11, 12). Most intriguingly, PKC-evoked phosphorylation allows AC2 to integrate multiple signals produced by Gqalpha - and Gialpha -coupled receptors in a Gsalpha -independent manner (29). An inhibitory effect of PKC on a specific, unidentified AC subtype which is expressed mainly in hepatocytes has also been reported (30). We have shown in the present study that PKC specifically inhibits AC6 activity through protein phosphorylation. Such negative influences of PKC on a Ca2+-inhibitable AC (AC6) provides an exquisite mechanism for the fine-tuning of cAMP synthesis by Ca2+ and PKC. Activation of PKC upon stimulation by extracellular signals may therefore lead to negative regulation of AC6 and a suppression of signals transduced by receptors coupled to AC6, such as A2a-R. The effect of the PKC-induced phosphorylation on the biochemical properties of AC6 is currently under investigation.

As early as 1987, Yoshimasa et al. (31) reported that the effect of a PKC-stimulating phorbol ester (12-O-tetradecanoyl phorbol-13-acetate) on AC activity was inhibitory in some cells, but stimulatory in others. Overexpressing various PKC isozymes did not convey the desired inhibitory or stimulatory effect of phorbol esters on AC activity, suggesting that the cellular selectivity of PKC on AC results from the existence of specific AC subtypes in different cells (31). In addition, results obtained from cells transiently expressing the desired AC subtype were not always consistent with those acquired from purified AC and PKC isozymes. For example, phorbol esters were found to exert "little, if any, change" in AC activity of cells transfected with AC4, AC5 and AC6 (32, 33). However, Kawabe et al. (12) have reported that PKCalpha and PKCzeta both directly phosphorylate AC5 and markedly stimulate AC5 activity using purified PKCs and AC5. In the present study, we demonstrate that purified PKC directly phosphorylated and inhibited AC6 (Table III and Fig. 3). The discrepancy between results obtained from the two systems (in vivo versus in vitro) may reflect, at least in part, the existence of distinct ACs and PKCs in different cells.

In summary, our data suggest that PKC directly phosphorylates and suppresses AC6 activity in PC12 cells during prolonged activation of A2a-R. In addition, stimulation of A2a-R results in activation of both adenylyl cyclase and calcium-insensitive, novel PKC in PC12 cells.


FOOTNOTES

*   This work was supported by grants from the National Science Council (NSC84-2331-B001-005-M10) and from Academia Sinica, Taipei, Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence and reprint requests should be addressed. Tel.: 886-2-7899028; Fax: 886-2-7853569; E-mail: BMYCHERN{at}ccvax.sinica.edu.tw.
1    The abbreviations used are: A2a-R, A2a adenosine receptor; AC, adenylyl cyclase; AC1-6, AC types I-VI; PK, protein kinase; OKA, okadaic acid; PDD, phorbol-12,13-didecanoate; F-AC6, FLAG-AC6 recombinant protein; PP2A, protein phosphatase 2A; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; PTX, pertussis toxin; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; GDPbeta S, guanosine 5'-O-2-(thio)diphosphate.
2    Y.-H. Chang, M. Conti, Y.-C. Lee, H.-L. Lai, Y.-H. Ching, and Y. Chern, manuscript in preparation.

Acknowledgments

We thank Dr. J. A. Wagner for kindly providing the PKA-deficient variant of PC12 cells. We are also grateful to Drs. L. Y. Chau, C.-M. Chiang, L.-S. Kao, and C. V. Weaver for their helpful suggestions and comments.


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