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(The FASEB Journal. 1998;12:1019-1025.)
© 1998 FASEB

RESEARCH COMMUNICATION

Inhibition of adenylyl cyclase isoforms V and VI by various Gß{gamma} subunits

Michael L. Bayewitcha, Tomer Avidor-reissa, Rivka Levya, Thomas Pfeufferb, Igal Nevoa, William F. Simondsc, and Zvi Vogela,1

a Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel
b Department of Physiological Chemistry II, University of Düsseldorf, Düsseldorf, D-40225 Germany
c Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, 20892, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An intriguing development in the G-protein signaling field has been the finding that not only the G{alpha} subunit, but also Gß{gamma} subunits, affect a number of downstream target molecules. One of the downstream targets of Gß{gamma} is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gß{gamma} subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gß{gamma}. Here we show by transient cotransfection into COS-7 cells of either adenylyl cyclase V or VI, together with G{gamma}2 and various Gß subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gß{gamma}. In addition, we show that Gß1 and Gß5 subunits differ in their activity. Gß1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous G{gamma} subunits). On the other hand, Gß5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of G{gamma}2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gß{gamma} dimers and that Gß1 and Gß5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.—Bayewitch, M. L., Avidor-Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, W. F., Vogel, Z. Inhibition of adenylyl cyclase isoforms V and VI by various Gß{gamma} subunits. FASEB J. 12, 1019–1025 (1998)


Key Words: Gß{gamma} • signal transduction • inhibition • plasmid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADENYLYL CYCLASE (AC)2 is a major target enzyme whose activity is modulated by receptor-coupled G-proteins. AC activity can be stimulated by G{alpha}s and is inhibited by G{alpha}i/z. It has recently been shown that the wide array of AC isoforms (nine are currently known) display differential sensitivity to free Gß{gamma} subunits. Previous results have demonstrated that AC I is significantly inhibited whereas AC II, IV, and presumably VII are activated by Gß{gamma} (1–9).

Six Gß and 12 G{gamma} isoforms have been cloned to date (6). Most previous experiments investigating the role of free Gß{gamma} on AC activity were performed in cell-free systems with either baculovirus/Sf9 recombinant Gß and G{gamma} preparations (10, 11) or purified brain Gß{gamma} preparations that consist of a mixture of various Gß{gamma} heterodimers (3, 12, 13). There is little information about the possible variations between the effects of the various Gß and G{gamma} combinations in intact cells. This is important because the various Gß and G{gamma} subunits do not necessarily have the same regulatory activities. Indeed, we recently showed that activation of PLC-ß2 by Gß{gamma} is Gß isoform independent (Gß1 being equally effective as Gß5), whereas activation of MAPK/ERK and JNK/SAPK appears to be Gß isoform dependent (14).

AC V and VI represent a subfamily within the group of AC isozymes. These two isozymes share high sequence homology and are both inhibited by activated G{alpha}i, but were not found to be sensitive to free Gß{gamma} subunits when using in vitro reconstitution assays (4, 6, 7, 15, 16). In this study, we have investigated the modulation of AC V and VI by specific Gß{gamma} combinations, using cotransfection of Gß and G{gamma} together with these AC isoforms. Due to the divergence in sequence homology between Gß1 and Gß5, we have concentrated our studies on these two Gß subunits and their modulation of activity of the AC isoforms in question. We observed that the activities of AC V and VI are inhibited by Gß{gamma} and that Gß1 is more efficient than Gß5 in conferring this inhibition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures
COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Plasmids
cDNAs of AC V and VI were used in the pXMD1 vector under control of the adenovirus-2 major late promoter (17). The preparation of these plasmids as well as plasmids containing Gß1, Gß5, G{gamma}2, Gß1E10K, and G{gamma}2C68S cDNAs were described previously (9, 14, 18, 19). The cDNA for {alpha}-transducin ({alpha}T) and thyroid-stimulating hormone (TSH) receptor cDNA were described (5, 20).

Transfection of COS cells
COS-7 cells in 10 cm dishes were transfected transiently by the DEAE-dextran chloroquine method (21, 22) with the indicated cDNA. The total amount of cDNA per transfection was maintained at a constant level using vector cDNA. Twenty-four hours 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 for determination of protein expression by Western blots. Transfection efficiency was optimized and determined by staining for ß-galactosidase (23) after transfection with plasmid expressing the enzyme. Efficiency of transfections was always in the range of 60–80% as determined by microscopic visualization of stained cells.

Adenylyl cyclase assay
Cells in 24-well plates were incubated for 2 h with 0.25 ml/well of growth medium containing 5 µCi/ml of [3H]adenine. Total [3H]adenine incorporation was not affected by the transfection of various plasmid DNAs. The 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 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) and RO-20–1724 (0.5 mM). The AC stimulants forskolin (FS) or TSH were added immediately at 1 µM concentration and the cells incubated at 37°C for 10 min. Reaction was terminated with 1 ml of 2.5% perchloric acid, neutralized, and applied to a two-step column separation procedure (21, 24). The [3H]cAMP was eluted into scintillation vials and counted.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots
Transfected COS-7 cells were washed with cold phosphate-buffered saline (PBS), scraped in PBS, spun down at 5000 RPM (at 4°C for 5 min), and cell pellets (unless otherwise specified) were mixed with 100 µl of Laemmli sample buffer (25), sonicated, and frozen. Prior to application on the gel, dithiothreitol (0.1 M final) was added and the samples incubated for 5 min at 100°C (for analysis of G-protein subunits) or incubated for 2 h at 37°C (for analysis of AC isoforms). Proteins were separated on polyacrylamide gel at the concentrations indicated and blotted onto nitrocellulose. The blot was blocked in PBS containing 5% fat-free milk and 0.5% Tween-20, followed by 1.5 h incubation with either BBC-4 monoclonal antibody (against AC V) (26), RA polyclonal antibody (against Gß1), or SGS polyclonal antibody (against Gß5) (14), all diluted 1:1,000 in 5% fat-free milk and 0.5% Tween-20. Blots were washed three times with PBS containing 0.3% Tween-20 and secondary antibody (horseradish peroxidase (HRP) -coupled rat anti-mouse or HRP-coupled goat anti-rabbit; Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.) diluted 1:20,000 in 5% fat-free milk plus 0.5% Tween-20, incubated with the blot for 1 h, and the blot was washed extensively (>2 h) with PBS containing 0.3% Tween-20. Peroxidase activity was observed by the ECL chemiluminescence technique (Amersham, Arlington Heights, Ill.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AC V activity is inhibited by Gß1, Gß1/{gamma}2 and Gß5/{gamma}2
In these experiments, AC V and, where indicated, Gß and G{gamma} subunits, were transiently transfected into COS cells and the amounts of cAMP in the cells were determined after 10 min activation with FS ( Fig. 1A) or TSH ( Fig. 2). The activity of AC V was markedly inhibited by Gß1/{gamma}2 (ca. 60%) and by Gß5/{gamma}2 (ca. 40%). In addition, a significant inhibition could be observed with Gß1 but not with Gß5 transfected alone. The addition of G{gamma}2, which by itself had no activity ( Fig. 3), only slightly increased the Gß1-mediated inhibition, but significantly increased the inhibition of AC V by Gß5. Western blot analysis ( Fig. 1B) of the cotransfected cells revealed that AC V expression was not affected by the coexpression of Gß1, Gß5, and G{gamma}2. These results show that in COS cells, Gß1 is more effective in modulating AC V than Gß5 and that the addition of G{gamma}2 strengthens the efficacy of Gß5.



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  		Figure 1. AC V activity is inhibited by Gß1*sol;{gamma}2 and Gß5/{gamma}2. A) Effects of transfected Gß/{gamma} combinations on basal or FS-stimulated AC V activity. Transfections in 10 cm dishes contained 2 µg AC V, 2 µg Gß1, 2 µg Gß5, and 1 µg G{gamma}2 cDNAs. cAMP accumulation is expressed as percent of control (AC V transfected alone) and is the mean ± SE of three experiments. B) Aliquots containing 15 µg of total cellular protein from COS cells transfected as described above were analyzed by SDS-PAGE (8% acrylamide) using the anti-AC V antibody BBC-4. The two bands of 160 and 150 kDa are equivalent to the previously reported antigen recognition pattern of BBC-4 and may represent alternate glycosylated forms of AC V (41).



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  		Figure 2. Inhibition of AC V and AC VI by Gß1 and Gß5 is independent of mode of activation. Effect of Gß{gamma} combinations on the activities of A) AC V and B) AC VI under basal conditions or after TSH stimulation. COS cells were transfected with 1 µg TSH receptor cDNA and, where indicated, with 2 µg AC V or AC VI, 2 µg Gß1 or Gß5, and 1 µg G{gamma}2 cDNA. cAMP accumulation is expressed in cpm and is the average of triplicate determinations of a representative experiment repeated three times with similar results.



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  		Figure 3. Gß/{gamma} modulates AC V in a dose-dependent manner. A) Effect of increasing amounts of Gß and G{gamma} cDNA on AC V activity. COS cells were transfected with AC V cDNA (2 µg) together with the indicated amounts of Gß1, Gß5, and G{gamma}2 cDNAs. FS-stimulated cAMP accumulation is expressed as percent of control (AC V transfected alone) and is the mean ± SE of three experiments. B) Expression of Gß1 and Gß5. COS cells were transfected with 2 µg of AC V cDNA and the indicated amounts of Gß1 and Gß5 cDNAs. Aliquots containing 15 µg total cellular protein were separated by SDS-PAGE (12% acrylamide) and analyzed by Western blotting using RA polyclonal antibody (against Gß1) and SGS polyclonal antibody (against Gß5). Arrows represent positions of Gß1 and Gß5. The molecular mass of Gß5 (39 kDa) is higher than that of Gß1 (36 kDa), in agreement with the difference in amino acid number between the two proteins (340 vs. 353 for Gß1 and Gß5, respectively).

The experiment shown in Fig. 1was performed with relatively large amounts of Gß and G{gamma} cDNAs. It was therefore repeated with Gß1, Gß5, and G{gamma}2 cDNAs at various concentrations. Figure 3A demonstrates that the inhibition of AC V reached maximal values when the concentration of cDNA of Gß1 was above 1 µg (per 10 cm culture plate). A half-maximal effect was observed with ca. 250 ng of transfected Gß1 cDNA. The addition of G{gamma}2 cDNA (2 µg/plate) did not have a marked effect on the Gß1-mediated inhibition of AC V at any of the Gß1 cDNA concentrations. Expression of Gß5 at all cDNA concentrations failed to mediate any significant inhibition of AC V activity. Only when the COS cells were transfected with 2 µg of Gß5 cDNA, together with increasing levels of G{gamma}2, was inhibition of AC V observed, demonstrating the dependency of Gß5 activity on the presence of G{gamma}2. Moreover, the inhibition of AC V by Gß1 or by Gß1/{gamma}2 was much more pronounced than that observed with Gß5 together with G{gamma}2.

Western blot analysis using selective antibodies to Gß1 and Gß5 ( Fig. 3B, see also Fig. 4B) demonstrated that in agreement with our previous results (14), untransfected COS cells have endogenous Gß1 but are devoid of Gß5. Maximal expression of Gß1 was achieved at >= 500 ng of Gß1 cDNA, in agreement with the effect of Gß1 on AC activity. Western blot analysis also showed that the lack of Gß5 effect on AC V activity is not due to a low level of Gß5 protein expression and that expression of this protein was already maximal when 250 ng cDNA was used in the transfection.



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  		Figure 4. Efficacy of the Gß1E10K mutant in inhibiting AC V activity. A) Effect of cotransfection of Gß1E10K mutant on basal or 1 µM FS-stimulated AC V activity. COS cells were transfected with 2 µg AC V and either 2 µg Gß1 or 2 µg Gß1E10K cDNAs. cAMP accumulation is expressed as percent of control (AC V transfected alone) and is the mean ± SE of three experiments. B) Expression of Gß1 vs. Gß1E10K. COS cells were transfected as described above and aliquots of 5 µg of total cellular protein or 5 µg of the cholate-soluble membrane fraction prepared by solubilizing the membranes of transfected COS cells in 1% cholate (14) were separated by SDS-PAGE (12% acrylamide), and the Gß subunits detected by the RA polyclonal antibody.

Inhibition of AC V and VI by Gß1, Gß1/{gamma}2, and Gß5/{gamma}2 is independent of mode of AC stimulation
In the previous experiments, FS was used to stimulate AC V. We have found that the inhibition of AC V by Gß1, Gß1/{gamma}2, and Gß5/{gamma}2 is not dependent on the method used for AC stimulation. COS-7 cells cotransfected with the TSH receptor and stimulated with TSH to activate AC through G{alpha}s revealed ( Fig. 2) the same inhibitory pattern as established for AC V activated by FS.

AC VI is a close homologue of AC V (4, 15, 16). Indeed, as shown in Fig. 2, AC VI displayed a similar pattern of activity as AC V when cotransfected with Gß and G{gamma} subunits. AC VI activity was inhibited upon cotransfection with the Gß1 subunit alone while remaining insensitive to Gß5. The addition of G{gamma}2 in both cases contributed to further inhibition observed with Gß1 as well as with Gß5. The same pattern of inhibition by Gß1 and Gß5 (in the presence of G{gamma}2) was observed on basal activity of AC V and VI (e.g., in the absence of TSH stimulation). In addition, COS-7 cells have been shown to contain at least the AC-VII and IX isozymes (27), and the COS endogenous AC activity (stimulated by TSH or FS) was not inhibited by transfected Gß1 or Gß5 (data not shown). Thus, the inhibitions observed result from modulations in the activity of the transfected AC.

Diminished inhibition of AC V by Gß1E10K mutant
To determine whether Gß1-mediated inhibition is dependent on endogenous G{gamma} subunits, we cotransfected AC V with the previously described point mutant, Gß1E10K, shown to be defective in its capacity to associate with G{gamma}2 subunits (19). Indeed, Gß1E10K was much weaker than Gß1 in inhibiting AC V activity ( Fig. 4A). Using antibodies against Gß1 (recognizing both Gß1 and Gß1E10K), Western blot analysis of total cellular protein shows that similar expression was achieved for both Gß1 and Gß1E10K ( Fig. 4B). In contrast, immunoblot analysis of the cholate-soluble cell membrane fraction revealed lower expression of Gß1E10K compared to the wild-type Gß1, consistent with the mutant's impaired ability to interact with G{gamma}. This result indicates that proper localization of the Gß1 protein to the membrane (probably via endogenous G{gamma}) is important for the inhibitory modulation of AC V and suggests that the active inhibitory mediator is the Gß{gamma} dimer itself.

Sequestration of Gß{gamma} activates AC V
The above-described result suggests that Gß{gamma} dimers are involved in the inhibition of AC V activity. Therefore, it would follow that molecules that sequester free Gß{gamma} should relieve AC V of such tonic inhibitory activity. A number of protein molecules have been shown to interfere with Gß{gamma} activities. A mutant form of G{gamma}2 that lacks the prenylation site (G{gamma}2C68S) and therefore cannot anchor to the membrane has been shown to redirect Gß subunits into the cellular cytosol, thus reducing Gß content at the cell membrane (28, 29). In addition, G{alpha} subunits such as {alpha}T will combine with Gß{gamma} and interfere with Gß{gamma}-mediated signaling (5, 30). We have checked the effect of cotransfection of cDNAs of {alpha}T or G{gamma}2C68S on AC V activity. As shown in Fig. 5, both approaches increased the level of activation of AC V by FS. {alpha}T increased AC V activity by threefold and G{gamma}2C68S by 1.6-fold. These results suggest there is a tonic inhibition of AC V activity that is mediated by endogenous Gß{gamma}.



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  		Figure 5. Effect of Gß{gamma} sequestration on AC V activity. COS cells were transfected with 2 µg AC V and either 2 µg G{gamma}2C68S or 2 µg {alpha}T. cAMP accumulation is expressed as percent of control (AC V transfected alone) and is the mean ± SE of three experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gß{gamma} dimers are involved in the regulation of various effector systems including PLC-ß2, MAPK/ERK, JNK/SAPK, PI3-kinase, and several AC isozymes (6, 14, 30–33). AC isozymes were shown to differ in their response to Gß{gamma}. AC types II, IV, and presumably VII are stimulated by free Gß{gamma}, whereas AC type I was shown to be inhibited (1, 2, 5–7, 9, 32, 34). Here we report that Gß{gamma} inhibits the activation of AC V and AC VI.

Gß1 and Gß5 represent two distinct forms of Gß as demonstrated by their sequence, expression pattern (Gß5 being predominantly found in brain), and ability to differentially affect downstream signaling proteins such as ERK and JNK (14, 35). We have recently shown that Gß1/{gamma}2 and Gß5/{gamma}2 have distinct effects on the modulation of AC II activity (36). Our current study shows that they also differ in their potency to modulate the activity of AC V and VI in COS cells. We have observed that the Gß1 subunit efficiently inhibits AC V when it is transfected alone, whereas Gß5 was functional only upon the addition of G{gamma}2. Moreover, the Gß1E10K mutant, which was shown to be defective in its ability to couple to the G{gamma}2 subunit and possibly to other G{gamma} subunits (19, 29), was much less efficient in inhibiting AC V activity. It follows that Gß1 is more efficient than Gß5 in recruiting and coupling with endogenous G{gamma} subunits present in COS cells to form Gß{gamma} dimers. It has been shown that Gß1 can couple with a number of G{gamma} subunits equally well (37, 38), whereas Gß5 appears to have a more restrictive selectivity to G{gamma}2 (14, 35). Accordingly, we have shown that sequestration of endogenous Gß{gamma} by a Gß{gamma} scavenger molecule such as {alpha}T, or the removal of Gß{gamma} from the membrane by interfering with G{gamma} anchoring (using the prenylation-deficient mutant), enhances AC V activity. These data led us to suggest that a pool of Gß{gamma} exists in COS cells that can exert a tonic inhibition on AC V activity.

It has recently been shown that the activity of P/Q- and N-type, voltage-dependent Ca2+ channels can be modulated by Gß{gamma} subunits (39). In agreement with our results, it was shown that transfection of Glß2 alone into tsA-201 cells was almost as effective as Gß2/{gamma}3 in regulating Ca2+ channel activation in these cells. It has been speculated that the overexpression of Gß1 and Gß2 could lead to increased levels of Gß{gamma} dimers or that the Gß has an intrinsic activity on its own (39).

AC V and AC VI were reported to be insensitive to Gß{gamma} in membrane reconstitution assays (2, 16). Here we report, using COS cells transfected with AC V and AC VI, that Gß{gamma} dimers can inhibit the activity of these AC isozymes. A search of AC V amino acid sequence reveals that AC V contains the sequence QXXER in the C1 cytoplasmic loop at positions 429–433 (18). This sequence has been shown to be involved in Gß{gamma} interaction with AC II as well as with voltage-dependent N-type calcium channels (8, 40). In addition, AC VI contains a similar sequence (RXXER) at the homologous position. Additional experiments are needed to show whether the above-described sequences are involved in AC V-VI/Gß{gamma} interactions and why such interaction leads to inhibition of AC V and VI activity, whereas in AC II it allows for Gß{gamma}-mediated stimulation. We cannot rule out the possibility that the effect of Gß{gamma} on AC V and AC VI may be indirect. Such indirect effects of Gß{gamma} have been observed for the activation of MAPK/ERK, where it has been shown that free Gß{gamma} recruits PI3-kinase to the plasma membrane, which in turn leads to a cascade of events leading to increased MAPK activity (33).

In summary, we have shown that AC V and AC VI are inhibited by Gß{gamma} heterodimers, demonstrating that after agonist-mediated activation of Gi/o-coupled receptors, the activities of AC V and VI can be inhibited via activated G{alpha}i/o and Gß{gamma} dimers released from G-protein heterotrimers.


   ACKNOWLEDGMENTS
 
This work was supported by the National Institute of Drug Abuse (DA-06265), the German-Israeli Foundation for Scientific Research and Development, the Forschheimer Center for Molecular Genetics, and the Israeli Ministries of Science and Arts and of Absorption (fellowship to M.B.). Z.V. is the incumbent of the Ruth and Leonard Simon Chair for Cancer Research.


   FOOTNOTES
 
1 Correspondence: Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: bnvogel{at}weizmann.weizmann.ac.il

2 Abbreviations: AC, adenylyl cyclase; FS, forskolin; HRP, horseradish peroxidase; TSH, thyroid-stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine; {alpha}T, {alpha}-transducin; PBS, phosphate-buffered saline.

Received for publication December 5, 1997. Accepted for publication March 9, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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