Differential Modulation of Adenylyl Cyclases I and II by Various Gbeta Subunits*

Michael L. BayewitchDagger §, Tomer Avidor-ReissDagger , Rivka LevyDagger , Thomas Pfeuffer, Igal NevoDagger , William F. Simondspar , and Zvi VogelDagger **

From the Dagger  Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel, the  Department of Physiological Chemistry II, University of Düsseldorf, Düsseldorf, D-40225 Germany, and the par  Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by Gbeta gamma dimers states that the various isoforms of AC respond differently to the presence of free Gbeta gamma . It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by Gbeta gamma subunits. This result does not address the possible differences in modulation that may exist among the different Gbeta gamma heterodimers. Six isoforms of Gbeta and 12 isoforms of Ggamma have been cloned to date. We have established a cell transfection system in which Gbeta and Ggamma cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both Gbeta 1/gamma 2 and Gbeta 5/gamma 2 combinations, AC II responded differentially and was stimulated by Gbeta 1/gamma 2 and inhibited by Gbeta 5/gamma 2. This finding demonstrates differential modulatory activity by different combinations of Gbeta gamma on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The heterotrimeric G-protein has been shown to be a central molecule that connects seven-transmembrane domain receptors to the many downstream effector molecules whose activities they regulate. The G-protein superfamily has been divided into many classes, originally based on the activity that the Galpha subunit exerted on the effectors. For example, the Galpha subunit that was found to activate adenylyl cyclase (AC)1 was classified Galpha s while the Galpha subunit found to inhibit AC activity was called Galpha i (reviewed in Refs. 1-4). In the last few years, the role of free Gbeta gamma subunits (disassociated from Galpha upon receptor activation) in signal transduction has begun to be revealed (reviewed in Refs. 5-7). In vitro membrane reconstitution assays have clearly demonstrated that a number of AC isoforms are sensitive to Gbeta gamma subunits and that AC activity can either be stimulated or inhibited by Gbeta gamma subunits depending of the AC isoform in question. For example, the activity of AC type I is significantly inhibited while the activity of AC types II, IV, and presumably VII are activated by Gbeta gamma (6-13). This finding has led to the understanding of how it is possible that the classically known inhibitory receptors that are coupled to Galpha i/o can actually stimulate AC activity in situations where AC isoforms that are activated by free Gbeta gamma subunits are present (11, 12, 14).

Six Gbeta and 12 Ggamma isoforms have been cloned (reviewed in Refs. 4, 5). Most of the prior experiments investigating the role of free Gbeta gamma on AC activity were performed in cell-free systems with either baculovirus/Sf9 recombinant Gbeta and Ggamma preparations (15, 16) or with purified brain Gbeta gamma preparations that consist of a mixture of various Gbeta gamma heterodimers (8, 9). There is little information about the possible variations between the effects of various Gbeta and Ggamma subunits in the intact cell. This seems to be important since the various Gbeta and Ggamma subunits do not necessarily have the same regulatory activities. Due to the recent cloning of various Gbeta and Ggamma isoforms, it is now possible to study the activity of the various Gbeta gamma combinations. Indeed, it has recently been shown by us that activation of PLC-beta 2 by Gbeta gamma is Gbeta isoform-independent (Gbeta 1/gamma 2 being equally effective as Gbeta 5/gamma 2) while MAPK/ERK and JNK/SAPK activation appeared to be Gbeta isoform-dependent and was much more efficiently activated with Gbeta 1/gamma 2 than with Gbeta 5/gamma 2 (17).

In this study, we have characterized the modulations of two AC isoforms by specific Gbeta gamma combinations. Utilizing cotransfection of Gbeta and Ggamma together with AC isoforms I and II, we observed differential modulation of AC activities by specific combinations of Gbeta gamma . We found that AC type I was inhibited by both Gbeta 1/gamma 2 and Gbeta 5/gamma 2. On the other hand, AC II activity was stimulated by one isoform of Gbeta (beta 1) while inhibited by another (beta 5).

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

Cell Cultures-- COS-7 cells were obtained from ATCC (Bethesda, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 units/ml penicillin and 100 µg/ml streptomycin.

Plasmids-- cDNAs of AC I and II were used in the pXMD1 vector under control of the adenovirus-2 major late promoter (18). AC I cDNA was released from pSK-AC-I (19) using HindIII and XbaI and ligated after "fill-in" into the SmaI site of pXMDI. AC II cDNA was released from pSK-AC-II (20) by EcoRI and ligated to the EcoRI site of pXMDI. Gbeta 1 and Gbeta 5 cDNAs in pcDNAIII and Ggamma 2 in pcDM8 were described earlier (17, 21). Ggamma 2C68S in pcDM8 (a mutant that cannot undergo prenylation) was described earlier (21, 22). cDNA for Galpha -transducin (alpha T) was provided by Dr. J. S. Gutkind. Constitutively active Galpha s (Galpha sQ227L) was obtained from Dr. H. Bourne (23).

Transfection of COS Cells-- COS-7 cells in 10-cm dishes were transfected with the indicated cDNAs by the DEAE-dextran chloroquine method (24). Vectors were added to complement the amount of cDNA in the transfection mixture to 6-7 µg. 24 h later, the cells were trypsinized and cultured for an additional 24 h in 24-well plates for AC activity assay or in 10-cm dishes to check protein expression by Western blots. Transfection efficiency, as determined by staining for beta -galactosidase (25) activity following transfection with the plasmid expressing the enzyme, was 60-80%.

Adenylyl Cyclase Assay-- The assays were performed in triplicate as described (26). Cells cultured in 24-well plates were incubated for 3 h with 0.25 ml/well of fresh growth medium containing 5 µCi/ml [3H]adenine. This medium was replaced with Dulbecco's modified Eagle's medium containing 20 mM Hepes (pH 7.4), 1 mg/ml bovine serum albumin, and the phosphodiesterase inhibitors isobutylmethylxanthine (IBMX) (0.5 mM) and RO-20-1724 (0.5 mM). Unless otherwise indicated, the AC stimulants forskolin (FS) or ionomycin (which increases intracellular Ca2+ and thus increases the activity of AC I) were immediately added at 1 µM concentrations, and the cells were incubated at 37 °C for 10 min. Reaction was terminated with 1 ml of 2.5% perchloric acid containing 0.1 mM unlabeled cAMP. Aliquots of 0.9 ml were neutralized and applied to a two-step column separation procedure (27). The [3H]cAMP was eluted into scintillation vials and counted.

SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blots-- Cells were washed with cold phosphate-buffered saline (PBS), scraped in PBS, and spun down at 5000 rpm (4 °C for 5 min), and cell pellets were mixed with 100 µl of Laemmli sample buffer (28), sonicated, and frozen. Prior to application on the gel, dithiothreitol (0.1 M final) was added, and the samples were incubated for 2 h at 37 °C. Proteins were separated on polyacrylamide gels (8% for AC and 12% for Gbeta ) and transferred to nitrocellulose. Nitrocellulose was blocked in PBS containing 5% fat-free milk and 0.5% Tween-20 for 1 h followed by 1.5 h of incubation with either BBC-1 monoclonal antibody (against AC I), BBC-4 monoclonal antibody (against AC II) (29, 30), RA polyclonal antibody (against Gbeta 1), or SGS polyclonal antibody (against Gbeta 5) (17). Blots were washed 3 times with PBS containing 0.3% Tween-20 and secondary antibodies (horseradish peroxidase (HRP)-coupled rat anti-mouse or HRP-coupled goat anti-rabbit, Jackson Immunoresearch Laboratories, Inc.) diluted 1:20,000 in 5% fat-free milk plus 0.5% Tween-20 and incubated with the blot for 1 h, and the blot was extensively washed (>2 h) with PBS containing 0.3% Tween-20. Peroxidase activity was observed by the ECL chemiluminescence technique (Amersham Corp.).

    RESULTS
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Procedures
Results
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References

AC I Activity Is Inhibited by Gbeta 1/gamma 2 and Gbeta 5/gamma 2-- We have performed cotransfection experiments of AC I together with Gbeta 1 or Gbeta 5 and Ggamma 2. Activation of AC I is known to be dependent on the presence of Ca2+ ions (8). In the experiment shown in Fig. 1, we have assayed the activity of this isozyme in the presence of the Ca2+ ionophore ionomycin (1 µM) together with FS (1 µM). This combination was shown to synergistically stimulate AC I activity (11). The endogenous AC activity present in COS cells was not significantly affected by ionomycin and thus contains very little Ca2+-stimulated AC isozymes. As shown in Fig. 1A, AC I activity was significantly inhibited upon cotransfection with either Gbeta 1 or Gbeta 5 together with Ggamma 2. The individual Gbeta 1, Gbeta 5, and Ggamma 2 subunits had no inhibitory activity on their own. Western blots of AC I protein (see Fig. 1B) reveals that the levels of AC I were not affected by the cotransfection of the other cDNAs. Our results thus indicate that the inhibitory effect mediated by Gbeta gamma subunits, originally found in membrane assays, can also be observed in a whole cell system and that both Gbeta 1 and Gbeta 5 are effective in the inhibition of AC I, provided that Ggamma 2 is present.


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Fig. 1.   AC I activity is inhibited by Gbeta 1/gamma 2 and Gbeta 5/gamma 2. A, effects of cotransfection of Gbeta /gamma combinations on the basal or stimulated (1 µM FS + 1 µM ionomycin) AC I activity. Transfections in 10-cm dishes contained 2 µg AC I, 2 µg Gbeta 1, 2 µg Gbeta 5, and 1 µg Ggamma 2 cDNAs, as indicated. cAMP accumulation is expressed as percent of control (AC I transfected alone) and is the mean ± S.E. of three experiments performed in triplicate. 100% cAMP accumulation is equivalent to 12,000-14,000 cpm (depending on the experiment). The significance of inhibition of AC I activity was determined by Student's t test (*, p < 0.001). B, aliquots of 15 µg of protein of COS cells transfected as above were analyzed by Western blotting using the anti-AC I antibody BBC-1. Numbers show positions of molecular weight markers.

AC II Activity Is Stimulated by Gbeta 1 and Gbeta 1/gamma 2 but Is Inhibited by Gbeta 5/gamma 2-- The AC II subtype is known to be stimulated by free Gbeta gamma (8). We have performed cotransfection of AC II with constitutively active Galpha s (Galpha sQ227L) and, where indicated, with Gbeta 1 or Gbeta 5 and with Ggamma 2. The amounts of cAMP in these cells were determined 2 days after transfection. In addition, we have treated the cells for 10 min with a mixture of the phosphodiesterase inhibitors, IBMX and RO-20-1724, and assayed the amounts of cAMP following this incubation. From the results shown in Fig. 2, it is clear that transfection with AC II increased the amounts of cAMP present in the cells under both conditions. As expected, transfection with Gbeta 1 caused a further increase in cAMP accumulation. This is due to AC II activation since it was not observed when Gbeta 1 was transfected to COS cells without the addition of AC II plasmid (data not shown) or when it was transfected with AC I (Fig. 1). The increase of AC II activity mediated by Gbeta 1 was not significantly affected by the addition of Ggamma 2, which by itself had no effect. Interestingly, in contrast to Gbeta 1, Gbeta 5 caused a siginificant inihibition of AC II activity, and the addition of Ggamma 2 increased this inhibition. These results show that in contrast to AC I, AC II is differentially affected by Gbeta 1 and Gbeta 5, thus demonstrating a level of specificity within the Gbeta family members in regards to their ability to affect AC II activity. Western blot analysis of the AC II protein levels under the various transfection conditions revealed that AC II expression was not affected by the cotransfection with Gbeta and Ggamma subunits.


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Fig. 2.   AC II activity is stimulated by Gbeta 1 and Gbeta 1/gamma 2 and is inhibited by Gbeta 5 and Gbeta 5/gamma 2. A, cAMP was determined under basal conditions or after a 10-min exposure to IBMX and RO-20-1724 (0.5 mM each). Transfections contained 0.4 µg of Galpha sQ227L cDNA and, where indicated, 2 µg of AC II, 2 µg of Gbeta 1, 2 µg of Gbeta 5, and 1 µg of Ggamma 2 cDNAs. cAMP accumulation is expressed as percent of control (transfection with Galpha sQ227L and AC II) and is the mean ± S.E. of three experiments performed in triplicate. 100% cAMP accumulation is equivalent to 20,000-23,000 cpm (depending on the experiment). The significance of stimulation or inhibition of AC II activity was determined by Student's t test (*, p < 0.001). B, aliquots of 15 µg of protein of COS cells transfected as above were analyzed by Western blotting using the anti-AC II antibody BBC-4.

Gbeta Subunits Modulate AC I and AC II in a Dose-dependent Manner-- The experiments shown in Figs. 1 and 2 were performed with relatively large amounts of Gbeta and Ggamma cDNAs. They were, therefore, repeated with Gbeta 1 and Gbeta 5 cDNAs at various concentrations. Fig. 3A demonstrates that the inhibition of AC I reaches maximal values when concentrations of cDNAs of Gbeta 1 or Gbeta 5 are above 2.5 µg. The efficiencies of inhibition by transfected Gbeta 1 and Gbeta 5 (in the presence of Ggamma 2) were similar. Half-maximal effect was observed with ~1 µg of Gbeta 1 and 2 µg of Gbeta 5 cDNAs. The inhibition observed for AC I activity was dependent on the presence of Ggamma 2 (2 µg/plate) for both Gbeta 1 and Gbeta 5.


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Fig. 3.   Dose dependence of the modulation of AC I and AC II by Gbeta /gamma combinations. A, effect of increasing amounts of Gbeta cDNA on AC I activity. COS cells in 10-cm plates were transfected with 2 µg of AC I cDNA and, where indicated, with Gbeta 1, Gbeta 5, and 2 µg of Ggamma 2 cDNA. cAMP accumulation was measured 10 min after the addition of 1 µM FS and 1 µM ionomycin (see Fig. 1). B, the effect of increasing amounts of Gbeta cDNA on AC II activity. COS cells were transfected with 0.4 µg of Galpha sQ227L and 2 µg of AC II cDNA and, where indicated, with Gbeta 1, Gbeta 5, and 2 µg of Ggamma 2 cDNA. cAMP accumulation was measured 10 min after the addition of IBMX and RO-20-1724. Data are expressed as percent of controls (AC I transfected alone or AC II transfected together with Galpha sQ227L) and are the mean ± S.E. of three experiments performed in triplicate. 100% cAMP accumulation is equivalent to 10,000-12,000 cpm for AC I and 18,000-21,000 cpm for AC II (depending on the experiment).

As shown in Fig. 3B, the level of stimulation of AC II by Gbeta 1 is dependent on the amount of Gbeta 1 cDNA used, reaching a maximum above ~1 µg of transfected cDNA. The addition of 2 µg Ggamma 2 cDNA to the transfection mixture increased the effect observed with Gbeta 1 cDNA alone by only a marginal value of approximately 10%. Conversely, transfection of Gbeta 5 cDNA caused a marked inhibition of AC II activity, and this inhibition was greatly enhanced by the presence of transfected Ggamma 2. Maximal inhibition (~30%) was observed with 5 µg of Gbeta 5 cDNA/10-cm culture dish, while in the presence of Ggamma 2, maximal inhibition (of the same level of 30%) was already observed with ~1 µg of Gbeta 5 cDNA.

Endogenous Gbeta gamma in COS Has a Role in AC II Stimulation-- Since we have demonstrated that the Gbeta 5/gamma 2 combination can be inhibitory to AC II activity, it became of interest to characterize the role of endogenous Gbeta gamma in COS-7 cells with respect to AC II modulation. To investigate this question, we utilized a number of molecular tools that were shown to interfere with Gbeta gamma activity. A mutant form of Ggamma 2 that lacks the prenylation site (Ggamma 2C68S) and which therefore cannot anchor to the membrane has been shown to redirect Gbeta subunits into the cellular cytosol (21, 22). Additionally, wild-type Galpha subunits such as alpha T combines with Gbeta gamma and interferes with Gbeta gamma -mediated signaling (11, 14). Fig. 4A demonstrates the effect of cotransfection of the above mentioned proteins on AC II activity in COS-7 cells. A marked inhibition of AC II activity was observed in cells cotransfected with alpha T. Ggamma 2C68S also produced a significant inhibition of AC II activity although the efficacy of inhibition compared with alpha T was noticeably less. The cotransfection of alpha T and Ggamma 2C68S with AC II did not have any effect on the expression of AC II (data not shown). These results suggest that Gbeta gamma in COS cells has a role in the stimulation of AC II activity. Fig. 4B demonstrates that COS cells do indeed express endogenous Gbeta 1, but appear to be devoid of Gbeta 5. Transfection of COS cells with Gbeta 1 or Gbeta 5 cDNAs led to a significant increase in the levels of Gbeta 1 and Gbeta 5 protein in the cell membranes.


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Fig. 4.   Endogenous Gbeta gamma of COS 7 cells is stimulatory for AC II. A, effect of cotransfection with alpha T or Ggamma 2C68S on basal and Galpha sQ227L-stimulated AC II activity. Transfections contained 0.4 µg of Galpha sQ227L and, where indicated, 2 µg of AC II, 2 µg of Ggamma 2C68S (gamma *), or 2 µg of alpha T cDNAs. cAMP accumulation (in cpm) is from one representative experiment out of three which gave similar results. The significance of inhibition of AC II activity was determined by Student's t test (*, p < 0.001). B, expression of Gbeta 1 and Gbeta 5 in transfected COS cells. Membranes were prepared from untransfected cells as well as from cells transfected with 2 µg of Gbeta 1 or Gbeta 5 cDNAs and solubilized with 1% cholate. Aliquots of 5 µg of protein of the cholate particulate (P) and soluble (S) fractions were separated by SDS-polyacrylamide gel electrophoresis (12% polyacrylamide), and the Gbeta subunits were detected by the RA polyclonal antibody (against Gbeta 1) or SGS polyclonal antibody (against Gbeta 5) (17). The procedure for the preparation of membranes and cholate solubilization was described previously (17).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

There are many reports that show involvement of the Gbeta gamma dimer complex in the regulation of various effectors including PLC-beta 2, MAPK/ERK, JNK/SAPK, phosphatidylinositol 3-kinase, and several AC isozymes (5, 8, 14, 17, 31, 32). Most of these experiments were performed either in whole cells using Gbeta gamma scavengers (i.e. molecules that strongly interact with Gbeta gamma and block its ability to modulate effectors) or with cell membranes. In the latter case, the Gbeta gamma used for most of the experiments has been with either baculovirus/Sf9 recombinant Gbeta and Ggamma preparations or with a mixture of a large repertoire of Gbeta gamma heterodimers, such as in Gbeta gamma preparations purified from bovine brain. Therefore, there has been little information regarding the possible differences in activities between the various Gbeta and Ggamma subunits composing the Gbeta gamma dimers in intact cells. Following the cloning of specific Gbeta and Ggamma isoforms, it has become possible to dissect the individual Gbeta and Ggamma combinations that affect AC activity. In a previous study, using transfection of intact cells with Gbeta 1 or Gbeta 5 cDNAs, we showed that Gbeta 1 and Gbeta 5 differ in their ability to activate MAPK/ERK and JNK/SAPK but not in their capacity to activate PLC-beta 2 (17). Here, we have used the same approach to investigate the role of Gbeta 1 and Gbeta 5 in the modulation of two types of AC isozymes: AC I, previously shown to be inhibited by Gbeta gamma heterodimers, and AC II, previously reported to be stimulated by Gbeta gamma (8, 15, 16).

Our results demonstrate that indeed, as reported using in vitro reconstitution assays, Gbeta gamma inhibits AC I activity. We found that Gbeta 1 and Gbeta 5 do not markedly differ in their capacity to inhibit AC I and that in both cases, the inhibition was dependent on the cotransfection of a Ggamma subunit. Our observations with AC II showed that while Gbeta 1/gamma 2 yielded stimulation of this isoform, in agreement with previously reported activities of Gbeta gamma (7, 8, 11, 13, 14). Gbeta 5/gamma 2 caused a marked inhibition of AC II activity, demonstrating selective effects of Gbeta subunits on the activity of this AC isoform. Interestingly, transfected Gbeta 1 was by itself sufficient in stimulating AC II activity. This is probably due to its capacity for recruiting endogenous Ggamma present in the cell. Gbeta 5 also had some effect on its own, although its effect on the inhibition of AC II was significantly enhanced by cotransfection of Ggamma 2. As previously proposed (17, 22), these results suggest a difference in the capacity of Gbeta 1 and Gbeta 5 to interact with Ggamma subunits.

It is known that of the six cloned Gbeta isoforms, Gbeta 5 shares only a 53% homology compared with the other Gbeta isoforms (5). The unique sequence of Gbeta 5 may be the source of the different effects on AC II activity that we have observed. It should be noted that this specific activity could have been missed in prior studies in which Gbeta gamma was tested in in vitro membrane reconstitution assays due to the possibility that the concentration of Gbeta 5 in the pool of Gbeta gamma (usually purified from bovine brain) may have been too small. The activity of Gbeta 5 may have been masked by other Gbeta subunits present in the preparation, and therefore, the levels of Gbeta 5 were not high enough to observe any inhibitory effects on AC II activity.

In summary, Gbeta 1 and Gbeta 5 represent two distinct forms of the Gbeta subunit, as based on their sequence, expression pattern, and ability to affect downstream signaling proteins (17, 33). Our results show that they also differ in their capacity to affect the activity of AC type II. Future studies should allow complete characterization of the various Gbeta gamma combinations influencing the activity of each of the AC isozymes and elucidate the finer regulation of Gbeta gamma signaling within the AC system.

    FOOTNOTES

* This work was supported in part by the National Institute on Drug Abuse (DA-06265), the German-Israeli Foundation for Scientific Research and Development, and the Forschheimer Center for Molecular Genetics.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.

§ Recipient of an Israeli Ministries of Science and Arts and of Absorption Fellowship.

** Incumbent of the Ruth and Leonard Simon Chair for Cancer Research and to whom correspondence should be addressed. Tel.: 972-8-934-2402; Fax: 972-8-934-4131; E-mail: bnvogel{at}weizmann.weizmann.ac.il.

1 The abbreviations used are: AC, adenylyl cyclase; FS, forskolin; HRP, horseradish peroxidase; Galpha sQ227L, constitutively active Galpha s subunit; IBMX, isobutylmethylxanthine; PBS, phosphate-buffered saline; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase.

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