Inhibition of adenylyl isoforms V and VI by

various Gbg subunits

MICHAEL L. BAYEWITCH, TOMER AVIDOR-REISS, RIVKA LEVY, THOMAS PFEUFFER,* IGAL NEVO, WILLIAM F. SIMONDS,† AND ZVI VOGEL1 Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel; *Department of Physiological Chemistry II, University of Du¨sseldorf, Du¨sseldorf, D-40225 Germany; and †Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, 20892, USA

ABSTRACT An intriguing development in the G- and Gg preparations (10, 11) or purified brain Gbg signaling field has been the finding that not preparations that consist of a mixture of various Gbg only the Ga subunit, but also Gbg subunits, affect a heterodimers (3, 12, 13). There is little information number of downstream target molecules. One of the about the possible variations between the effects of downstream targets of Gbg is , and it the various Gb and Gg combinations in intact cells. has been demonstrated that a number of isoforms of This is important because the various Gb and Gg sub- adenylyl cyclase can be either inhibited or stimulated units do not necessarily have the same regulatory ac- by Gbg subunits. Until now, adenylyl cyclase type I has tivities. Indeed, we recently showed that activation of been the only isoform reported to be inhibited by PLC-b2 by Gbg is Gb isoform independent (Gb1 being free Gbg. Here we show by transient cotransfection equally effective as Gb5), whereas activation of into COS-7 cells of either adenylyl cyclase V or VI, MAPK/ERK and JNK/SAPK appears to be Gb isoform together with Gg2 and various Gb subunits, that these dependent (14). two adenylyl cyclase isozymes are markedly inhibited AC V and VI represent a subfamily within the by Gbg. In addition, we show that Gb1 and Gb5 subunits group of AC isozymes. These two isozymes share high differ in their activity. Gb1 transfected alone mark- and are both inhibited by acti- edly inhibited adenylyl cylcase V and VI (probably by vated Gai, but were not found to be sensitive to free recruiting endogenous Gg subunits). On the other Gbg subunits when using in vitro reconstitution assays hand, Gb5 produced less inhibition of these isozymes, (4, 6, 7, 15, 16). In this study, we have investigated and its activity was enhanced by the addition of Gg2. the modulation of AC V and VI by specific Gbg com- These results demonstrate that adenylyl cyclase types binations, using cotransfection of Gb and Gg together V and VI are inhibited by Gbg dimers and that Gb1 and with these AC isoforms. Due to the divergence in se- Gb5 subunits differ in their capacity to regulate these quence homology between Gb1 and Gb5, we have con- adenylyl cyclase isozymes.—Bayewitch, M. L., Avidor- centrated our studies on these two Gb subunits and Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, their modulation of activity of the AC isoforms in W. F., Vogel, Z. Inhibition of adenylyl cyclase iso- question. We observed that the activities of AC V and forms V and VI by various Gbg subunits. FASEB J. 12, VI are inhibited by Gbg and that Gb1 is more efficient 1019–1025 (1998) than Gb5 in conferring this inhibition.

Key Words: Gbg · · inhibition · plasmid MATERIALS AND METHODS ADENYLYL CYCLASE (AC)2 is a major target whose activity is modulated by receptor-coupled G- cultures . AC activity can be stimulated by Gas and is inhibited by Gai/z. It has recently been shown that the COS-7 cells were cultured in Dulbecco’s modified Eagle’s me- wide array of AC isoforms (nine are currently known) dium supplemented with 5% fetal calf serum, 100 U/ml pen- icillin, and 100 mg/ml streptomycin. display differential sensitivity to free Gbg subunits. Pre- vious results have demonstrated that AC I is signifi- cantly inhibited whereas AC II, IV, and presumably 1 Correspondence: Department of Neurobiology, Weiz- VII are activated by Gbg (1–9). mann Institute of Science, Rehovot 76100, Israel. E-mail: Six G and 12 G isoforms have been cloned to date [email protected] b g 2 Abbreviations: AC, adenylyl cyclase; FS, ; HRP, (6). Most previous experiments investigating the role horseradish peroxidase; TSH, -stimulating hormone; of free Gbg on AC activity were performed in cell-free IBMX, 3-isobutyl-1-methylxanthine; aT, a-; PBS, systems with either baculovirus/Sf9 recombinant Gb phosphate-buffered saline.

0892-6638/98/0012-1019/$02.25 ᮊ FASEB 1019

/ 382d 0008 Mp 1019 Friday Jun 26 11:33 AM LP±FASEB 0008 Plasmids idase activity was observed by the ECL chemiluminescence technique (Amersham, Arlington Heights, Ill.). 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 RESULTS Gb1,Gb5,Gg2,Gb1E10K, and Gg2C68S cDNAs were described previously (9, 14, 18, 19). The cDNA for a-transducin (aT) and thyroid-stimulating hormone (TSH) receptor cDNA were AC V activity is inhibited by Gb1,Gb1/g2and Gb5/g2 described (5, 20).

Transfection of COS cells In these experiments, AC V and, where indicated, Gb and Gg subunits, were transiently transfected into COS-7 cells in 10 cm dishes were transfected transiently by the COS cells and the amounts of cAMP in the cells were DEAE-dextran chloroquine method (21, 22) with the indi- determined after 10 min activation with FS (Fig. 1A) cated cDNA. The total amount of cDNA per transfection was or TSH (Fig. 2). The activity of AC V was markedly maintained at a constant level using vector cDNA. Twenty-four inhibited by Gb1/g2 (ca. 60%) and by Gb5/g2 (ca. 40%). hours later, the cells were trypsinized and cultured for an ad- In addition, a significant inhibition could be ob- ditional 24 h in 24-well plates for AC activity assay or in 10 cm dishes for determination of protein expression by Western served with Gb1 but not with Gb5 transfected alone. blots. Transfection efficiency was optimized and determined The addition of Gg2, which by itself had no activity by staining for b-galactosidase (23) after transfection with plas- (Fig. 3), only slightly increased the Gb1-mediated in- mid expressing the enzyme. Efficiency of transfections was al- hibition, but significantly increased the inhibition of ways in the range of 60–80% as determined by microscopic ACVbyG . Western blot analysis (Fig. 1B) of the visualization of stained cells. b5 cotransfected cells revealed that AC V expression was Adenylyl cyclase assay

Cells in 24-well plates were incubated for 2 h with 0.25 ml/ well of growth medium containing 5 mCi/ml of [3H]adenine. Total [3H]adenine incorporation was not affected by the transfection of various plasmid DNAs. The medium was re- placed with Dulbecco’s modified Eagle’s medium containing 20 mM Hepes (pH 7.4), 1 mg/ml bovine serum albumin, and the inhibitors 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) and RO-20–1724 (0.5 mM). The AC stim- ulants forskolin (FS) or TSH were added immediately at 1 mM concentration and the cells incubated at 37ЊC for 10 min. Re- action was terminated with 1 ml of 2.5% perchloric acid, neu- tralized, and applied to a two-step column separation proce- dure (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-buf- fered saline (PBS), scraped in PBS, spun down at 5000 RPM (at 4ЊC for 5 min), and cell pellets (unless otherwise specified) were mixed with 100 ml of Laemmli sample buffer (25), soni- cated, and frozen. Prior to application on the gel, dithio- threitol (0.1 M final) was added and the samples incubated for 5 min at 100ЊC (for analysis of G-protein subunits) or in- cubated for 2 h at 37ЊC (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- Figure 1. AC V activity is inhibited by Gb1/g2 and Gb5/g2. A) 20, followed by 1.5 h incubation with either BBC-4 monoclo- Effects of transfected Gb/g combinations on basal or FS-stim- nal antibody (against AC V) (26), RA polyclonal antibody ulated AC V activity. Transfections in 10 cm dishes contained (against Gb1), or SGS polyclonal antibody (against Gb5) (14), 2 mgACV,2mgGb1,2mgGb5, and 1 mgGg2 cDNAs. cAMP all diluted 1:1,000 in 5% fat-free milk and 0.5% Tween-20. accumulation is expressed as percent of control (AC V trans- Blots were washed three times with PBS containing 0.3% fected alone) and is the mean { SE of three experiments. B) Tween-20 and secondary antibody (horseradish peroxidase Aliquots containing 15 mg of total cellular protein from COS (HRP) -coupled rat anti-mouse or HRP-coupled goat anti-- cells transfected as described above were analyzed by SDS- bit; Jackson Immunoresearch Laboratories, Inc., West Grove, PAGE (8% acrylamide) using the anti-AC V antibody BBC-4. Pa.) diluted 1:20,000 in 5% fat-free milk plus 0.5% Tween-20, The two bands of 160 and 150 kDa are equivalent to the pre- incubated with the blot for 1 h, and the blot was washed ex- viously reported antigen recognition pattern of BBC-4 and tensively (ú2 h) with PBS containing 0.3% Tween-20. Perox- may represent alternate glycosylated forms of AC V (41).

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/ 382d 0008 Mp 1020 Friday Jun 26 11:33 AM LP±FASEB 0008 Figure 2. Inhibition of AC V and AC VI by Gb1 and Gb5 is independent of mode of activation. Effect of Gbg 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 mg TSH receptor cDNA and, where indicated, with 2 mgACVorACVI,2mgGb1 or Gb5, and 1 mgGg2 cDNA. cAMP accumulation is expressed in cpm and is the average of triplicate determinations of a representative experiment repeated three times with similar results.

not affected by the coexpression of Gb1,Gb5, and Gg2. the effect of Gb1 on AC activity. Western blot analysis These results show that in COS cells, Gb1 is more ef- also showed that the lack of Gb5 effect on AC V activity fective in modulating AC V than Gb5 and that the is not due to a low level of Gb5 protein expression and addition of Gg2 strengthens the efficacy of Gb5. that expression of this protein was already maximal The experiment shown in Fig. 1 was performed when 250 ng cDNA was used in the transfection. with relatively large amounts of Gb and Gg cDNAs. It was therefore repeated with Gb1,Gb5, and Gg2 cDNAs Inhibition of AC V and VI by Gb1,Gb1/g2, and Gb5/g2 at various concentrations. Figure 3A demonstrates is independent of mode of AC stimulation that the inhibition of AC V reached maximal values when the concentration of cDNA of Gb1 was above In the previous experiments, FS was used to stimulate 1 mg (per 10 cm culture plate). A half-maximal effect AC V. We have found that the inhibition of AC V by was observed with ca. 250 ng of transfected Gb1 Gb1,Gb1/g2, and Gb5/g2 is not dependent on the cDNA. The addition of Gg2 cDNA (2 mg/plate) did method used for AC stimulation. COS-7 cells cotrans- not have a marked effect on the Gb1-mediated inhi- fected with the TSH receptor and stimulated with bition of AC V at any of the Gb1 cDNA concentra- TSH to activate AC through Gas revealed (Fig. 2) the tions. Expression of Gb5 at all cDNA concentrations same inhibitory pattern as established for AC V acti- failed to mediate any significant inhibition of AC V vated by FS. activity. Only when the COS cells were transfected AC VI is a close homologue of AC V (4, 15, 16). with 2 mgofGb5 cDNA, together with increasing lev- Indeed, as shown in Fig. 2, AC VI displayed a similar els of Gg2, was inhibition of AC V observed, demon- pattern of activity as AC V when cotransfected with strating the dependency of Gb5 activity on the pres- Gb and Gg subunits. AC VI activity was inhibited upon ence of Gg2. Moreover, the inhibition of AC V by Gb1 cotransfection with the Gb1 subunit alone while re- or by Gb1/g2 was much more pronounced than that maining insensitive to Gb5. The addition of Gg2 in observed with Gb5 together with Gg2. both cases contributed to further inhibition observed Western blot analysis using selective antibodies to with Gb1 as well as with Gb5. The same pattern of in- Gb1 and Gb5 (Fig. 3B, see also Fig. 4B) demonstrated hibition by Gb1 and Gb5 (in the presence of Gg2) was that in agreement with our previous results (14), un- observed on basal activity of AC V and VI (e.g., in the transfected COS cells have endogenous Gb1 but are absence of TSH stimulation). In addition, COS-7 cells devoid of Gb5. Maximal expression of Gb1 was have been shown to contain at least the AC-VII and achieved at ¢ 500 ng of Gb1 cDNA, in agreement with IX isozymes (27), and the COS endogenous AC ac-

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/ 382d 0008 Mp 1021 Friday Jun 26 11:33 AM LP±FASEB 0008 tivity (stimulated by TSH or FS) was not inhibited by transfected Gb1 or Gb5 (data not shown). Thus, the inhibitions observed result from modulations in the activity of the transfected AC.

Diminished inhibition of AC V by Gb1E10K mutant

To determine whether Gb1-mediated inhibition is de- pendent on endogenous Gg subunits, we cotrans- fected AC V with the previously described point mu- tant, Gb1E10K, shown to be defective in its capacity to associate with Gg2 subunits (19). Indeed, Gb1E10K was much weaker than Gb1 in inhibiting AC V activity (Fig. 4A). Using antibodies against Gb1 (recognizing both Gb1 and Gb1E10K), Western blot analysis of total cellular protein shows that similar expression was achieved for both Gb1 and Gb1E10K (Fig. 4B). In con- trast, immunoblot analysis of the cholate-soluble cell membrane fraction revealed lower expression of Gb1E10K compared to the wild-type Gb1, consistent with the mutant’s impaired ability to interact with Gg. This result indicates that proper localization of the Gb1 protein to the membrane (probably via endoge- nous Gg) is important for the inhibitory modulation of AC V and suggests that the active inhibitory me- diator is the Gbg dimer itself.

Sequestration of Gbg activates AC V

The above-described result suggests that Gbg dimers are involved in the inhibition of AC V activity. There- fore, it would follow that molecules that sequester free Gbg should relieve AC V of such tonic inhibitory activity. A number of protein molecules have been shown to interfere with Gbg activities. A mutant form Figure 3. Gb/g modulates AC V in a dose-dependent manner. of Gg2 that lacks the prenylation site (Gg2C68S) and A) Effect of increasing amounts of Gb and Gg cDNA on AC V therefore cannot anchor to the membrane has been activity. COS cells were transfected with AC V cDNA (2 mg) shown to redirect Gb subunits into the cellular cyto- together with the indicated amounts of Gb1,Gb5, and Gg2 c- sol, thus reducing Gb content at the cell membrane DNAs. FS-stimulated cAMP accumulation is expressed as per- { (28, 29). In addition, Ga subunits such as aT will com- cent of control (AC V transfected alone) and is the mean bine with G and interfere with G -mediated signal- SE of three experiments. B) Expression of Gb1 and Gb5. COS bg bg cells were transfected with 2 mg of AC V cDNA and the indi- ing (5, 30). We have checked the effect of cotrans- cated amounts of Gb1 and Gb5 cDNAs. Aliquots containing 15 fection of cDNAs of aTorGg2C68S on AC V activity. mg total cellular protein were separated by SDS-PAGE (12% As shown in Fig. 5, both approaches increased the acrylamide) and analyzed by Western blotting using RA poly- level of activation of AC V by FS. aT increased AC V clonal antibody (against Gb1) and SGS polyclonal antibody (against Gb5). Arrows represent positions of Gb1 and Gb5. The activity by threefold and Gg2C68S by 1.6-fold. These molecular mass of Gb5 (39 kDa) is higher than that of Gb1 (36 results suggest there is a tonic inhibition of AC V ac- kDa), in agreement with the difference in amino acid number tivity that is mediated by endogenous Gbg. between the two proteins (340 vs. 353 for Gb1 and Gb5, respec- tively).

DISCUSSION I was shown to be inhibited (1, 2, 5–7, 9, 32, 34). Gbg dimers are involved in the regulation of various Here we report that Gbg inhibits the activation of AC effector systems including PLC-b2, MAPK/ERK, V and AC VI. JNK/SAPK, PI3-, and several AC isozymes (6, Gb1 and Gb5 represent two distinct forms of Gb as 14, 30–33). AC isozymes were shown to differ in demonstrated by their sequence, expression pattern their response to Gbg. AC types II, IV, and presum- (Gb5 being predominantly found in brain), and abil- ably VII are stimulated by free Gbg, whereas AC type ity to differentially affect downstream signaling pro-

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/ 382d 0008 Mp 1022 Friday Jun 26 11:33 AM LP±FASEB 0008 defective in its ability to couple to the Gg2 subunit and possibly to other Gg subunits (19, 29), was much less efficient in inhibiting AC V activity. It follows that Gb1 is more efficient than Gb5 in recruiting and coupling with endogenous Gg subunits present in COS cells to form Gbg dimers. It has been shown that Gb1 can cou- ple with a number of Gg subunits equally well (37, 38), whereas Gb5 appears to have a more restrictive selectivity to Gg2 (14, 35). Accordingly, we have shown that sequestration of endogenous Gbg byaGbg scav- enger molecule such as aT, or the removal of Gbg from the membrane by interfering with Gg anchoring (using the prenylation-deficient mutant), enhances AC V activity. These data led us to suggest that a pool of Gbg exists in COS cells that can exert a tonic inhi- bition 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 Gbg subunits (39). In agreement with our results, it was shown that transfection of Glb2 alone into tsA-201 cells was almost as effective as 2/ Gb2/g3 in regulating Ca channel activation in these cells. It has been speculated that the overexpression of Gb1 and Gb2 could lead to increased levels of Gbg dimers or that the Gb has an intrinsic activity on its own (39). AC V and AC VI were reported to be insensitive to Gbg in membrane reconstitution assays (2, 16). Here we report, using COS cells transfected with AC V and AC VI, that Gbg 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 Gbg

Figure 4. Efficacy of the Gb1E10K mutant in inhibiting AC V activity. A) Effect of cotransfection of Gb1E10K mutant on basal or 1 mM FS-stimulated AC V activity. COS cells were transfected with 2 mg AC V and either 2 mgGb1 or 2 mgGb1E10K cDNAs. cAMP accumulation is expressed as percent of control (AC V transfected alone) and is the mean { SE of three experiments. B) Expression of Gb1 vs. Gb1E10K. COS cells were transfected as described above and aliquots of 5 mg of total cellular protein or 5 mg 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 Gb subunits detected by the RA polyclonal antibody. teins such as ERK and JNK (14, 35). We have recently shown that Gb1/g2 and Gb5/g2 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. Figure 5. Effect of Gbg sequestration on AC V activity. COS We have observed that the Gb1 subunit efficiently in- cells were transfected with 2 mg AC V and either 2 mgGg2C68S hibits AC V when it is transfected alone, whereas Gb5 or 2 mg aT. cAMP accumulation is expressed as percent of was functional only upon the addition of Gg2. More- control (AC V transfected alone) and is the mean { SE of three over, the Gb1E10K mutant, which was shown to be experiments.

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/ 382d 0008 Mp 1023 Friday Jun 26 11:33 AM LP±FASEB 0008 interaction with AC II as well as with voltage-depen- 12. Tang, W., and Gilman, A. (1991) Type-specific regulation of ade- nylyl cyclase by bg subunits. Science 254, 1500–1503 dent N-type calcium channels (8, 40). In addition, AC 13. Tang, W. J., Krupinski, J., and Gilman, A. G. (1991) Expression VI contains a similar sequence (RXXER) at the ho- and characterization of -activated (type I) adenylyl- mologous position. Additional experiments are cyclase. J. Biol. Chem. 266, 8595–8603 14. Zhang, S., Coso, O., Lee, C., Gutkind, J., and Simonds, W. (1996) needed to show whether the above-described se- Selective activation of effector pathways by brain-specific G pro- quences are involved in AC V-VI/Gbg interactions and tein b5. J. Biol. Chem. 271, 33575–33579 why such interaction leads to inhibition of AC V and 15. Katsushika, S., Chen, L., Kawabe, J.-I., Nilakantan, R., Halnon, N. J., Homcy, C. J., and Ishikawa, Y. (1992) Cloning and char- VI activity, whereas in AC II it allows for Gbg-mediated acterization of a sixth adenylyl cyclase isoform: types V and VI stimulation. We cannot rule out the possibility that constitute a subgroup within the mammalian adenylyl cyclase family. Proc. Natl. Acad. Sci. USA 89, 8774–8778 the effect of Gbg on AC V and AC VI may be indirect. 16. Premont, R. T., Chen, J., Ma, H. W., Ponnapalli, M., and Iyengar, Such indirect effects of Gbg have been observed for R. (1992) Two members of a widely expressed subfamily of hor- the activation of MAPK/ERK, where it has been mone-stimulated adenylyl cyclase. Proc. Natl. Acad. Sci. USA 89, 9809–9813 shown that free Gbg recruits PI3-kinase to the plasma 17. Kluxen, F. W., and Lu¨bbert, H. (1993) Maximal expression of membrane, which in turn leads to a cascade of events recombinant cDNAs in COS cells for use in expression cloning. leading to increased MAPK activity (33). Anal. Biochem. 208, 352–356 In summary, we have shown that AC V and AC VI 18. Wallach, J., Droste, M., Kluxen, F. W., Pfeuffer, T., and Frank, R. (1994) Molecular cloning and expression of a novel type V ade- are inhibited by Gbg heterodimers, demonstrating nylyl cyclase from rabbit myocardium. FEBS Lett. 338, 257–263 that after agonist-mediated activation of Gi/o-coupled 19. Garritsen, A., Gallan, P. J. M. V., and Simonds, W. F. (1993) The receptors, the activities of AC V and VI can be inhib- N-terminal coiled-coil domain of b is essential for g association: a model for G-protein bg subunit interaction. Proc. Natl. Acad. ited via activated Gai/o and Gbg dimers released from Sci. USA 90, 7706–7710 G-protein heterotrimers. 20. Akamizu, T., Ikuyama, S., Saji, M., Kosugi, S., Kozak, C., McBride, O. W., and Kohn, L. D. 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