Phosphorylation of the G Protein gamma 12 Subunit Regulates Effector Specificity*

Hiroshi YasudaDagger , Margaret A. Lindorfer, Chang-Seon Myung, and James C. Garrison§

From the Department of Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Although the G protein beta gamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma 12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta 1gamma 12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha . Their ability to support coupling of the Gi1 alpha  subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta 1gamma 12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta . The native beta 1gamma 10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha . Creation of a phosphorylation site in the N terminus of the protein (Gly4 right-arrow Lys) resulted in a beta 1gamma 10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta 1gamma 12 dimer. Thus, phosphorylation of the beta 1gamma 12 dimer on the gamma subunit with protein kinase C alpha  regulates its activity in an effector-specific fashion. Because the gamma 12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Most cells possess multiple signaling pathways to receive signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. One of the best characterized signal transduction systems is used by receptors coupled to the heterotrimeric G proteins1 (1-5). Receptors activate this system by stimulating the release of bound GDP from the G protein alpha  subunit leading to exchange of GDP for GTP in the protein's nucleotide binding site. Binding of GTP induces a conformational change in the alpha  subunit, simultaneously activating the protein and markedly decreasing its affinity for the beta gamma dimer (1). Both the GTP-bound form of the alpha  subunit and the released beta gamma subunit are capable of activating multiple effectors to generate intracellular messages (3, 4, 6, 7). The mechanisms that regulate the lifetime of the active, GTP-bound form of the alpha  subunit have been studied extensively. All alpha  subunits have an intrinsic GTPase activity, which hydrolyzes bound GTP to GDP (1, 3, 4), returning the molecule to its basal state and increasing its affinity for GDP and the beta gamma subunit (1, 3, 4, 8). Both changes induce formation of the stable, heterotrimeric form of the G protein. Interestingly, the GTPase activity of many alpha  subunits can be increased by a class of proteins termed RGS molecules (9, 10) and by certain effectors such as PLC-beta (11).

Although the activity of the alpha  subunit is regulated by multiple mechanisms, regulation of the activity of the beta gamma dimer is not well characterized. Recently, the gamma 12 subunit has been shown to be a substrate for protein kinase C (12, 13), suggesting that dimers containing this gamma  subunit may be subject to regulation by phosphorylation. The gamma 12 subunit is widely expressed (12-15) and, given the extensive role of the beta gamma subunit in cell signaling (6, 16), its phosphorylation may have important consequences. To examine the effects of gamma 12 subunit phosphorylation on its activity, we purified recombinant beta 1gamma 12 dimers from baculovirus-infected Sf9 insect cells, phosphorylated them with PKC alpha  and beta 1, and tested their activity in three assays of beta gamma function. We examined the ability of phosphorylated dimers to support coupling of the Gi1 alpha  subunit to the A1 adenosine receptor and to activate two effectors, type II adenylyl cyclase or phospholipase C-beta . Phosphorylation of the beta 1gamma 12 subunit had no effect on its ability to activate PLC-beta , but increased its potency in the receptor coupling assay and markedly inhibited its ability to activate adenylyl cyclase. These results suggest that phosphorylation reduces the ability of the beta gamma signal to increase cyclic AMP levels and favors activation of other effectors.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Recombinant Baculoviruses for the beta  and gamma  Subunits-- Full-length clones encoding the human gamma 10 and gamma 12 proteins were identified in the EST data base and obtained from Research Genetics, Inc (gamma 12, GenBankTM N42722; gamma 10, GenBankTM U31383). To minimize the length of the construct 5' from the ATG start codon, the end of the gamma 12 cDNA was modified using the polymerase chain reaction (PCR). For the gamma 12 cDNA, the primers used were: (sense primer: 5'-CCCGGGATGTCCAGCAAAACAGCA-3'; antisense primer: 5'-ATAGAGACTGCAGAGTCCAT-3'). The PCR products were subcloned into the pCNTR shuttle vector, the gamma 12 coding sequence excised from pCNTR with SmaI and XbaI, and ligated into these sites in the baculovirus transfer vector, pVL1393. The native gamma 10 cDNA was excised from the pT7T3D plasmid with EcoRI, further digested with BanII and Asp700 and subcloned into the pCNTR shuttle vector. The gamma 10 coding sequence was excised from pCNTR with BamHI and XbaI and ligated into these sites in the pVL1393 transfer vector. The N terminus of the gamma 10 protein was modified to have a protein kinase C phosphorylation site by mutagenesis of the gamma 10 cDNA using PCR. The primers used were: (sense primer: 5'-GGATCCATGTCCTCCAAGGCTAGC-3'; antisense primer: 5'-CACTTTGTGCTTGAAGGAATTCC-3'). This modification introduced a Gly4 right-arrow Lys mutation (G4K) into the protein. The products of the PCR reaction were subcloned into pCNTR, digested with BamHI and EcoRI, and ligated into these sites in the pVL1393 transfer vector. To add the hexahistidine-FLAG affinity tags to the 5' end of the beta 1 subunit, the polymerase chain reaction was used to add XbaI and BamHI restriction sites to the 5' and 3' ends of the beta 1 coding region, respectively. The primers used were: (sense primer: 5'-TCTAGAATGAGTGAGCTTGACCAGTT-3'; antisense primer: 5'-GGATCCTTAGTTCCAGATCTTGAGGA-3'). The products of the reaction were digested with XbaI and BamHI and ligated into the pDouble Trouble (pDT) vector, which adds the nucleotide sequences for the hexahistidine and FLAG affinity tags to the 5' end of the beta 1 coding region (17). The beta 1HF coding region was excised from pDT with HindIII and BamHI and subcloned into the pCNTR shuttle vector. The beta 1HF coding sequence was excised from pCNTR with BamHI and ligated into the BamHI site of pVL1393. The pVL1393 transfer vectors containing these four constructs were sequenced to verify the fidelity of the beta  and gamma  sequences. Recombinant baculoviruses were constructed by co-transfecting each transfer vector with linearized BaculoGold® viral DNA into Sf9 cells using the PharMingen BaculoGold® kit as described (18). The recombinant baculoviruses were purified by one round of plaque purification using standard techniques (19). The construction of the recombinant baculoviruses coding for the Gi1 and Gs alpha  subunits and the A1 adenosine receptor have been described (20, 21).

Expression and Purification of Recombinant G Protein alpha  and beta gamma Subunits-- G protein alpha  and beta gamma subunits were overexpressed by infecting suspension cultures of Sf9 insect cells with recombinant baculoviruses (22, 23). The Gi1 alpha  subunit was purified to homogeneity as described (22). The recombinant beta gamma dimers were extracted from Sf9 cells and purified using DEAE chromatography and an alpha  subunit affinity column (23).

Phosphorylation of Purified beta gamma Subunits by PKC-- The purified beta 1HFgamma 12 subunit was incubated for 30 min at 30 °C in 50 mM Tris, pH 7.5, 1 mM beta -mercaptoethanol, 10 mM MgCl2, 0.4 mM CaCl2, 40-100 µM ATP, recombinant PKC alpha  or beta , 40 µg/ml phosphatidylserine, and 0.8 µg/ml diolein. Usually, 25 µg of beta gamma dimer was incubated with 0.74 unit of PKC alpha  to achieve stoichiometric phosphorylation of the gamma  subunit. Control reactions contained deionized water in the place of PKC. The stoichiometry of the phosphorylation reaction was measured by including 40 µM [32P]ATP (500-2000 cpm/pmol) in the reaction mix and subjecting the phosphorylated beta gamma subunits to Tricine/SDS-PAGE (24). The resolved gamma  subunit was cut from the dried gel and the amount of radioactive phosphate incorporated estimated by scintillation counting. After the phosphorylation reaction and before use in the assays, the beta gamma subunit was repurified from the reaction mixture by loading it onto a 0.25 ml Ni2+-NTA-agarose column (Qiagen) and washing with 15 ml of 20 mM Hepes, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM beta -mercaptoethanol, 0.6% (w/v) CHAPS, and 5 mM imidazole to remove PKC. The beta gamma dimer was eluted with 1 ml of the wash buffer containing 200 mM imidazole. To ensure that no PKC activity was carried into the assays with the beta gamma dimer, its activity was monitored in the elution fractions using the kinase reaction buffer described above with 25 µg/ml histone 3S as substrate (25). As expected, the kinase activity eluted in the void volume of the column and not with the beta gamma dimer. Controls were also performed to determine whether the 30 °C incubation with PKC and subsequent re-purification reduced the activity of the beta gamma dimer in the functional assays. In these experiments, a mock-phosphorylation reaction was performed in the absence of PKC, the beta gamma dimer re-purified and its activity compared with that of dimers subjected only to the alpha -agarose column (see "Results").

Measurement of the High Affinity Ligand Binding Conformation of the A1 Adenosine Receptor-- Sf9 insect cell membranes overexpressing recombinant A1 adenosine receptors were prepared as described (21) and reconstituted with G protein alpha  and beta gamma subunits on ice for 30 min (26). The high affinity, agonist binding conformation of the receptor was measured using the agonist ligand 125I-N6-(aminobenzyl)adenosine as described (26). Each reaction tube contained 20 fmol of receptor, 6 nM Gi1 alpha  subunit, 0-10 nM beta gamma dimer, 50 nM GDP, and 0.3 nM 125I-N6-(aminobenzyl)adenosine. Because this assay was incubated for 3 h before filtration, 100 nM microcystin was included to inhibit protein phosphatases in the Sf9 cell membrane preparation (27).

Measurement of Phospholipase C-beta Activity-- Large unilamellar phospholipid vesicles were prepared by extrusion into a buffer containing 50 mM Hepes, pH 8.0, 3 mM EGTA, 80 mM KCl, and 1 mM dithiothreitol with a Avanti Polar Lipids mini-extruder (28). The phospholipid vesicles contained a 4:1 molar ratio of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate at final concentrations of 100 and 25 µM, respectively, and about 7000 cpm/assay of [inositol-2-3H]phosphatidylinositol 4,5-bisphosphate. Phospholipid vesicles and beta gamma subunits were mixed on ice in an assay buffer containing 50 mM Hepes, pH 8.0, 0.17 mM EDTA, 3 mM EGTA, 17 mM NaCl, 67 mM KCl, 0.83 mM MgCl2, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin. The final concentration of CHAPS contributed by the beta gamma preparations in each assay tube was kept below 0.01% (w/v) to eliminate effects of detergent on PLC-beta activity (29). The reaction was begun by addition of 10 ng of recombinant, turkey erythrocyte PLC-beta and 3 µM free Ca2+ to each assay tube. The mixture was incubated for 15 min at 30 °C and stopped by the addition of ice-cold 10% trichloroacetic acid followed by the addition of 10 mg/ml bovine serum albumin. Assay tubes were centrifuged at 4,000 × g and the [3H]inositol 1,4,5-trisphosphate released measured by liquid scintillation counting (30).

Measurement of Adenylyl Cyclase Activity-- Sf9 insect cell membranes overexpressing recombinant, rat type II adenylyl cyclase (31) were prepared as described (18). The Gs alpha  subunit was extracted from an Sf9 cell preparation with 0.1% (w/v) CHAPS as described (18). Cyclase containing membranes (5 µg of protein/assay tube) were reconstituted with GTPgamma S-activated Gs alpha  subunit (32) and varying concentrations of beta gamma dimer on ice for 30 min. The reaction buffer (25 mM Hepes, pH 8.0, 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, 0.4 mM 3-isobutyl-1-methylxanthine, 10 mM MgSO4, 0.5 mM ATP, and 0.1 mg/ml bovine serum albumin) was preincubated at 30 °C for 20 min. Production of cyclic AMP was initiated by addition of the reconstituted membranes to the reaction buffer and the incubation continued for 10 min at 30 °C. Reactions were stopped by the addition of 0.1 N HCl and cyclic AMP measured using an automated radioimmunoassay (33).

Electrophoresis-- Tricine/SDS-polyacrylamide gels were run according to the procedure of Schagger and von Jagow (24). The separating gel contained 16.5% total acrylamide, 0.4% bisacrylamide, and 10% (v/v) glycerol. The stacking gel contained 4% total acrylamide and 0.1% bisacrylamide. Gels were run at constant voltage (~100 volts) at 10 °C for 4-5 h. Resolved proteins were stained with silver by the method of Morrissey (34), with the modification that the dithiothreitol incubation was reduced to 15 min.

Calculations and Expression of Results-- Experiments presented under "Results" are representative of three or more similar experiments. Data expressed as dose-response curves were fit to rectangular hyperbolas using the fitting routines in the GraphPad Prizm® software. Statistical differences between the curves were determined using all the individual data points from multiple experiments to calculate the F statistic as described (35).

Materials-- All reagents used in the culture of Sf9 cells and for the expression and purification of G protein alpha  and beta gamma subunits have been described in detail (20, 23). The baculovirus transfer vector, pVL1393, was purchased from Invitrogen; the BaculoGold® kit from PharMingen; 10% GENAPOL® C-100, CHAPS, microcystin, and the alpha  and beta 1 isoforms of PKC from Calbiochem; Ni2+-NTA-agarose from Qiagen; [3H]phosphatidylinositol bisphosphate from NEN Life Science Products; PMA from Sigma; the pCNTR shuttle vector from 5 Prime right-arrow 3 Prime, Inc. (Boulder, CO). All other reagents were of the highest purity available.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Stoichiometry of Phosphorylation of the gamma 12 and the gamma 10G4K Subunits-- Recent experiments have demonstrated that the bovine gamma 12 subunit is a substrate for protein kinase C (12), but the functional significance of this phosphorylation event has not been extensively studied. As this newly discovered gamma 12 subunit is widely expressed (12-15), its phosphorylation may have important consequences. To examine the effects of gamma 12 subunit phosphorylation on its activity, we purified recombinant beta 1gamma 12 dimers from Sf9 insect cells, phosphorylated them with PKC alpha  and beta , and tested their activity in assays of beta gamma function. The human gamma 12 subunit was rapidly phosphorylated by PKC alpha  to a stoichiometry of about 1 mol/mol as shown in Fig. 1, A and B. Protein kinase C beta 1 was less effective than PKC alpha ; the stoichiometry only reached 0.5 mol/mol after 60 min of incubation. Addition of a hexahistidine-FLAG affinity tag to the beta 1 subunit (beta 1HF) facilitated removal of PKC from the reaction mixture prior to assessing beta gamma function. Thus, the phosphorylated beta 1HFgamma 12 dimer in the reaction mix can be applied to a Ni2+-NTA-agarose column and pure beta gamma dimer eluted with imidazole. The left side of Fig. 1C shows that stained PKC protein was removed by this procedure and assay of kinase activity in the column elution fractions determined that the beta gamma dimer was free of residual kinase activity (see "Experimental Procedures"). The right side of the figure shows that only the gamma 12 subunit in the dimer is phosphorylated. Addition of the hexahistidine-FLAG affinity tag to the N terminus of the beta 1 subunit did not affect the association of the beta  and gamma  subunits or purification of the dimer on an alpha -subunit affinity column (data not shown).


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Fig. 1.   Phosphorylation of beta 1HFgamma 12 by protein kinase C. A, purified beta 1HFgamma 12 dimer was incubated with protein kinase C alpha  for the indicated times. The autoradiograph shows the time course of the phosphorylation of the gamma 12 subunit by PKC alpha  (upper panel) and PKC beta 1 (lower panel). Aliquots were withdrawn from the kinase reactions at the indicated times for analysis by tricine/SDS-PAGE as described under "Experimental Procedures." B, stoichiometry of the phosphate incorporated into the gamma 12 subunit measured as described under "Experimental Procedures." C, reaction mixtures were subjected to gel electrophoresis to visualize the purity of proteins (silver stain) and the subunits phosphorylated (autoradiograph). The left panel shows the reaction mixture before and after purification of the phosphorylated beta 1HFgamma 12 on the Ni2+-NTA-agarose column. The right panel shows that only the gamma 12 subunit is phosphorylated. Experiments are representative of 10 similar experiments.

Ser1 in the N terminus of the bovine gamma 12 subunit is the site phosphorylated by protein kinase C (12, 13). Thus, the consensus phosphorylation sequence S*XK is the motif in the gamma 12 subunit recognized by the kinase (36). None of the other known gamma  subunits have this motif, but the gamma 10 subunit has a similar pair of serine residues at its N terminus (37). To further evaluate the effect of phosphorylation on the activity of gamma  subunits, we mutated residue 4 in the gamma 10 subunit from glycine to lysine (Gly4 right-arrow Lys) to introduce the SSK motif (Fig. 2A). The ability of the wild type and the mutated gamma 10 subunit to be phosphorylated by protein kinase C alpha  was examined. As expected, the wild type beta 1HFgamma 10 dimer was not phosphorylated, but the dimer containing the gamma 10G4K subunit was rapidly phosphorylated by PKC beta  (autoradiograph in Fig. 2B) with a time course similar to that seen with gamma 12 (data not shown). The silver-stained gel in Fig. 2C compares the mobilities of the wild type and mutated gamma 10G4K subunits with those of the gamma 1 and gamma 12 subunits. Clearly, the SSK motif created in the gamma 10G4K subunit can be phosphorylated effectively. The stoichiometry of phosphorylation for the gamma 10G4K protein was about 0.5 mol/mol using PKC alpha  and about 0.25 mol/mol using PKC beta 1 (data not shown). Thus, the differences in phosphorylation rates observed using gamma 12 as a substrate were also seen using the gamma 10G4K subunit.


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Fig. 2.   Phosphorylation of the mutated beta 1HFgamma 10 dimer by protein kinase C. A, comparison of the N-terminal amino acid sequences of the bovine and human gamma 12 subunits, the human gamma 10 subunit, and the Gly4 right-arrow Lys mutant gamma 10 subunit (gamma 10G4K). Alignments of the entire protein sequences were performed with the GCG programs. The N-terminal 21-25 amino acids including the first helical region are shown. The G4K mutation in the gamma 10 subunit is underlined. B, autoradiograph showing the phosphorylation of the gamma 10G4K and gamma 12 subunits by PKC alpha . The native gamma 1 and gamma 10 proteins were not phosphorylated. C, section from a silver-stained Tricine/SDS-PAGE gel showing the mobilities of the four gamma  subunits. Experiments are representative of five similar experiments.

Effect of Phosphorylation of the gamma  Subunit on Receptor Coupling-- Having established the ability of protein kinase C to phosphorylate the two recombinant gamma  subunits, we examined the effects of phosphorylation on the function of the dimer. The beta gamma subunits play several important roles in the signaling mechanisms used by receptors to activate effectors. In combination with the alpha subunit, they participate in forming the high affinity agonist binding conformation of the receptor (21, 38, 39); they stabilize the basal state of the system by increasing the affinity of the alpha  subunit for GDP (7, 8); and when released from the alpha  subunit, they activate effectors such as type II adenylyl cyclase and phospholipase C-beta (6, 7). We first examined the effect of gamma  subunit phosphorylation on the ability of the beta gamma dimer to support establishment of the high affinity, agonist binding conformation of a G protein-coupled receptor. In membranes from Sf9 cells overexpressing recombinant A1 adenosine receptors, about 90% of the receptors are in a low affinity conformation. Reconstitution of pure Gi alpha  and beta gamma subunits into these membranes establishes high affinity agonist binding and provides a sensitive assay for receptor-alpha beta gamma interactions (21). Fig. 3 shows that the dimers used in this study, beta 1gamma 12, beta 1gamma 10, beta 1gamma 10G4K, and beta 1HFgamma 12, were able to re-establish the high affinity, agonist binding conformation of the receptor with a potency and efficacy equal to the well studied and highly effective beta 1gamma 2 dimer (26). All dimers tested support coupling with a Kact of 0.5-1.0 nM (see Fig. 3 legend). Thus, the newly discovered gamma 10 and gamma 12 subunits are able to couple very effectively to the Gi1 alpha  subunit and the A1 adenosine receptor when dimerized with the beta 1 subunit. Importantly, neither the hexahistidine-FLAG tag added to the N terminus of the beta 1 subunit nor the G4K mutation made in the N terminus of the gamma 10 subunit affect the ability of these recombinant beta gamma dimers to induce the high affinity conformation of the A1 adenosine receptor.


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Fig. 3.   Ability of native and modified beta 1gamma 12 and beta 1gamma 10 dimers to support the high affinity, agonist binding state of the adenosine A1 receptor. Sf9 cell membranes expressing recombinant bovine A1 adenosine receptors were reconstituted with 10 nM Gi1 alpha  subunit, the indicated concentrations of the defined beta gamma dimers, and high affinity 125I-aminobenzyladenosine binding measured as described under "Experimental Procedures." The ratio of receptor:alpha :beta gamma was approximately 1:20:0-33. The Kact as determined by fitting each data set to a rectangular hyperbola ranged from 0.5 to 1.0 nM. The Kact values are not significantly different. The results are representative of three similar experiments performed in triplicate.

The data in Fig. 4 illustrate the ability of phosphorylated and unphosphorylated forms of the beta 1HFgamma 12 dimer to support reconstitution of the high affinity binding state of the A1 adenosine receptor. Phosphorylation of the beta 1HFgamma 12 subunit (open circles) increased the potency of the dimer in this assay from about 6.4 to 1 nM (Fig. 4A). This difference was significant (p < 0.001). Interestingly, phosphorylation of the gamma 12 subunit has been reported to increase the affinity of the beta gamma dimer for the alpha  subunit (12), a result consistent with the increased potency seen in Fig. 4A. A similar result was obtained when the phosphorylated and unphosphorylated forms of the beta 1HFgamma 10G4K dimer were tested (Fig. 4B). This small difference in potency was also significant (p < 0.05). The observation that the phosphorylated beta 1HFgamma 10G4K dimer does not shift the curve as greatly as the phosphorylated beta 1HFgamma 12 dimer may be due to the fact that the stoichiometry of phosphorylation is only about 0.5 mol/mol (see Fig. 2 and text). It is important to note that these differences were observed only when microcystin was included in the binding assay buffer, suggesting that the Sf9 cell membranes contain a protein phosphatase able to dephosphorylate gamma 12. To examine this possibility, we incubated 32PO4-labeled beta 1HFgamma 12 with the Sf9 cell membranes at 30 °C in the presence or absence of 100 nM microcystin, removed aliquots from the incubation medium over a 60-min time period, and resolved the proteins on a Tricine/SDS gel. In the absence of microcystin, the amount of radioactivity in the gamma 12 subunit decreased more than 80% over the 60-min incubation. Little dephosphorylation occurred if microcystin was included in the 30 °C incubation or if the mixture was held at 0 °C without microcystin (data not shown). This result confirms the existence of an effective phosphatase for the gamma 12 subunit. The insect cell phosphatase must be analogous to mammalian protein phosphatases 1 and 2, which are very sensitive to microcystin (27). Finally, controls were performed to determine whether the steps used to phosphorylate and re-purify the beta gamma dimers decreased their ability to couple to the adenosine receptor (see "Experimental Procedures"). The activity of a pure beta 1HFgamma 12 dimer used directly in the binding assay was about 10% higher than dimers subjected to a mock-phosphorylation incubation and re-purified on the Ni2+-NTA column prior to assay (compare maximal binding in Figs. 3 and 4).


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Fig. 4.   Comparison of the ability of phosphorylated and unphosphorylated beta 1HFgamma 12 and beta 1HFgamma 10 dimers to support high affinity agonist binding to the adenosine A1 receptor. A, Sf9 cell membranes expressing recombinant bovine adenosine A1 receptors were reconstituted with 6 nM Gi1 alpha  subunit and the indicated concentrations of unphosphorylated (closed circles) or phosphorylated (open circles) beta 1HFgamma 12 dimers. Formation of the high affinity, agonist binding state of the receptor was measured as described under "Experimental Procedures." Phosphorylation of the beta 1HFgamma 12 dimer caused a significant increase in potency from 6.4 to 1 nM (p < 0.001). B, an analogous experiment performed with unphosphorylated (closed circles) or phosphorylated (open circles) beta 1HFgamma 10G4K dimers. Phosphorylation of the beta 1HFgamma 10G4K dimer caused a significant increase in potency from 3.4 to 1.8 nM (p < 0.05). The binding reactions were performed in the presence of 100 nM microcystin. Data points are the mean ± S.D. of three independent experiments, each performed in triplicate. Rectangular hyperbolas were fit to the data and statistical differences between the curves determined as described under "Experimental Procedures."

Effect of gamma  Subunit Phosphorylation on Effector Activity-- The data in Fig. 5A show the ability of the native and modified beta gamma dimers used in this study to activate PLC-beta in an in vitro assay using [3H]PIP2 incorporated into phospholipid vesicles as substrate. Note that beta 1gamma 12, beta 1gamma 10, beta 1HFgamma 10G4K, and beta 1HFgamma 12 are equally as effective as the beta 1gamma 2 dimer. All four forms of the protein stimulated the release of [3H]inositol 1,4,5-trisphosphate with a Kact of 6-8 nM and were equally effective (~8-fold increase in activity). The data in Fig. 5B demonstrate that there is no difference in the ability of either the phosphorylated or unphosphorylated beta 1HFgamma 12 dimers to activate PLC-beta . The phosphorylated or unphosphorylated forms of the beta 1HFgamma 10G4K dimers were also tested and no differences were observed (data not shown). As can be seen by comparison of the maximal activities shown in Fig. 5, A and B, the phosphorylation protocol caused about a 40% decrement in beta gamma activity in the PLC assay.


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Fig. 5.   Comparison of the ability of phosphorylated and unphosphorylated beta gamma dimers to activate phospholipase C-beta . A, the ability of native and modified beta 1gamma 12 and beta 1gamma 10 dimers to activate phospholipase C-beta as compared with the effect of beta 1gamma 2. The indicated concentrations of beta gamma dimers were reconstituted with recombinant, turkey phospholipase C-beta in phospholipid vesicles containing [3H]phosphatidylinositol bisphosphate and phospholipase activity measured as described under "Experimental Procedures." B, comparison of the ability of unphosphorylated (closed circles) and phosphorylated (open triangles) beta 1HFgamma 12 dimers to activate phospholipase C-beta . Data are representative of six independent experiments, each performed in duplicate. Rectangular hyperbolas were fit to the data and statistical differences between the curves determined as described under "Experimental Procedures." There was no significant difference in the curves.

The beta gamma dimer causes a synergistic activation of type II adenylyl cyclase in the presence of GTPgamma S-activated Gs alpha  subunit (40). Therefore, we examined the effect of phosphorylation on the ability of the beta 1HFgamma 12 and beta 1HFgamma 10G4K dimers to stimulate recombinant, type II adenylyl cyclase in Sf9 cell membranes. Fig. 6A shows that the unphosphorylated forms of all the beta gamma dimers used in this study effectively stimulate type II adenylyl cyclase. As before, the beta 1gamma 12, beta 1HFgamma 12, and the beta 1HFgamma 10G4K dimers are all as effective as the beta 1gamma 2 dimer. The beta 1gamma 10 dimer had activity equal to the beta 1HFgamma 10G4K dimer in this assay (data not shown). The Kact for all the dimers ranges from 3 to 14 nM. Each of these dimers was purified with the alpha  subunit affinity column and assayed directly. Thus, the newly discovered gamma 12 and gamma 10 subunits are able to effectively stimulate type II adenylyl cyclase when combined with the beta 1 subunit. Moreover, neither the hexahistidine-FLAG tag on the beta 1 subunit nor the G4K mutation in the gamma 10 subunit greatly affected the ability of these subunits to stimulate adenylyl cyclase.


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Fig. 6.   Comparison of the ability of phosphorylated and unphosphorylated beta gamma dimers to stimulate type II adenylyl cyclase. A, Sf9 cells were infected with a recombinant baculovirus encoding the type II adenylyl cyclase, membranes prepared, and the cyclase reaction performed with the indicated concentrations of four recombinant beta gamma dimers as described under "Experimental Procedures." B, the effect of phosphorylation on the ability of beta 1HFgamma 12 and beta 1HFgamma 10G4K to stimulate type II adenylyl cyclase. The beta gamma dimers were phosphorylated by protein kinase C alpha  and purified as described under "Experimental Procedures" and the legend to Fig. 1. The cyclase assay was performed with unphosphorylated (closed circles) and phosphorylated (open circles) beta gamma dimers. Whereas the data does not define a complete curve, fitting the data to rectangular hyperbolas estimates phosphorylation to decrease the Vmax from 15 nmol/min/mg of protein to about 10 nmol/min/mg of protein and the Kact from 14 to 37 nM. The differences were significant (p < 0.001). The results are representative of 10 similar experiments, each performed in duplicate. Rectangular hyperbolas were fit to the data and statistical differences between the curves determined as described under "Experimental Procedures."

Interestingly, the phosphorylated forms of the beta 1HFgamma 12 and beta 1HFgamma 10 dimers were significantly less potent and effective in their ability to activate adenylyl cyclase (Fig. 6B). Note that the phosphorylated forms of the beta gamma dimers are less active than the unphosphorylated forms over the concentration range of 1-40 nM. Fitting the data to rectangular hyperbolas estimates that phosphorylation of the dimers decreases their activity about 50% and shifts their Kact from 14 to 37 nM (see legend). As before, controls were performed to determine whether the steps needed to phosphorylate and re-purify the beta gamma dimers decreased their activity. The protocol causes about a 15% decrement in activity (compare the maximal activities in Fig. 6, A and B). To verify the effect of gamma 12 subunit phosphorylation on cyclase activity, we prepared dimers with different stoichiometries of phosphorylation and assayed their activity. As shown in Fig. 7, varying the stoichiometry of gamma 12 subunit phosphorylation between 0-1 mol/mol resulted in a gradual reduction in the dimer's ability to stimulate type II adenylyl cyclase from about 17 nmol/min/mg of protein to 10 nmol/min/mg of protein. Each of the four curves in Fig. 7 is significantly different from the other (see legend). This result clearly demonstrates that phosphorylation of the serine residue at the N terminus of the gamma  subunit can reduce its ability to stimulate type II adenylyl cyclase.


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Fig. 7.   Stimulation of type II adenylyl cyclase by beta 1HFgamma 12 dimers with different stoichiometries of phosphorylation. Sf9 cell membranes infected with the recombinant baculovirus encoding type II adenylyl cyclase were used to monitor the effect of the beta gamma dimers on cyclic AMP production as described under "Experimental Procedures." The beta 1HFgamma 12 dimers phosphorylated to different stoichiometries were prepared as follows: 0 mol of PO4/mol (closed circles), the dimer was incubated without PKC at 30 °C for 30 min; 0.25 mol of PO4/mol (open diamonds), the dimer was incubated with PKC beta 1 for 15 min at 30 °C; 0.5 mol of PO4/mol (open circles), the dimer was incubated with PKC alpha  for 2 min at 30 °C; 1.0 mol of PO4/mol (open triangles), the dimer was incubated with PKC alpha  for 30 min at 30 °C. The beta gamma dimers were purified from the PKC reaction mixture and their ability to stimulate recombinant type II adenylyl cyclase measured as described under "Experimental Procedures." Rectangular hyperbolas were fit to the data and statistical differences between the curves determined as described under "Experimental Procedures." Each curve was significantly different from the others (p < 0.001). The results are representative of four similar experiments performed in duplicate.

Previous work has demonstrated that activation of PKC with phorbol 12-myristate 13-acetate (PMA) in Sf9 cells expressing type II adenylyl cyclase results in phosphorylation of the enzyme and an increase in both basal and forskolin-stimulated cyclase activity (31). Moreover, direct phosphorylation of the type II adenylyl cyclase expressed in Sf9 cell membranes with PKC alters its responsiveness to both alpha  and beta gamma subunits (41). Thus, our finding that phosphorylation of gamma 12 by the same kinase reduces the ability of the beta gamma dimer to stimulate type II adenylyl cyclase frames an interesting problem. To determine the effect of a phosphorylated beta 1gamma 12 dimer on the activity of type II adenylyl cyclase in PMA-treated cells, we infected Sf9 cells with a recombinant baculovirus encoding type II adenylyl cyclase for 48 h, added 1 µM PMA to the medium for 30 min before harvest, and prepared membranes as described under "Experimental Procedures."

The type II adenylyl cyclase activity in the control and PMA-treated membranes was compared following treatment with vehicle, 100 nM forskolin, activated Gs alpha , and Gs plus 20 nM beta gamma . The activities of the control membranes treated with these agents were 0.2, 1.0, 1.2, and 6.25 nmol of cAMP/min/mg of protein, respectively. In keeping with previous results (31), pretreatment of cyclase infected Sf9 cells with PMA did result in a 10-20% increase in the rates of basal, 100 nM forskolin, Gs alpha , and Gs plus 20 nM beta gamma -stimulated cyclic AMP synthesis relative to control membranes. Next, dose-response curves were performed with both membrane preparations using phosphorylated and unphosphorylated beta 1HFgamma 12 dimers in the presence of activated Gs alpha . Interestingly, in the membranes from PMA-treated cells, the phosphorylated dimers were still markedly less effective than unphosphorylated dimers at stimulating type II adenylyl cyclase. In membranes from control cells, phosphorylation of the dimer reduced the stimulation of cyclase by the following percentages: at 10 nM beta gamma , by 31%; at 20 nM beta gamma , by 40%; and at 40 nM beta gamma , by 45% (n = 5). In membranes from PMA treated Sf9 cells, the reductions were very similar: at 10 nM beta gamma , by 33%; at 20 nM beta gamma , by 40%; and at 40 nM by beta gamma , 55% (n = 5). Thus, in a cell expressing the gamma 12 subunit, its phosphorylation by PKC would still inhibit the ability of a beta gamma dimer to stimulate type II adenylyl cyclase activity, even though basal or Gs-stimulated cyclase activity itself might be slightly elevated by kinase activation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the diversity in the family of G protein gamma  subunits has prompted studies to determine whether the differences in these proteins translate to specificity in transmembrane signaling (42). In this regard, one major finding of this study is that, when combined with the beta 1 subunit, the newly discovered gamma 12 and gamma 10 subunits are equal in potency and efficacy to the well studied beta 1gamma 2 dimer. Both the beta 1gamma 12 and beta 1gamma 10 dimers were fully effective in the receptor coupling assay using the Gi1 alpha  subunit and the A1 adenosine receptor and able to maximally activate type II adenylyl cyclase and PLC-beta . As these gamma  subunits are widely expressed in brain and peripheral tissues (12, 14, 15, 37), they are likely to play important roles in signaling by a large number of G protein coupled receptors. A second important finding is that phosphorylation of the beta 1gamma 12 dimer with protein kinase C has distinct effects on the activity of the molecule, increasing its potency in the receptor coupling assay and inhibiting its ability to stimulate type II adenylyl cyclase. Previous studies have determined that the phosphorylation site in gamma 12 is Ser1 at the N terminus of the molecule (12, 13). This finding is consistent with our observations that the phosphorylation site created in the gamma 10G4K subunit makes it a substrate for protein kinase C and that phosphorylation regulates its activity. Although not fully explored, the finding that the dephosphorylation of gamma 12 is blocked by microcystin suggests that the protein phosphatases that dephosphorylate the protein in the intact cell are most likely protein phosphatase 1 and/or 2A. Overall, the protein kinases and phosphatases regulating the phosphorylation state of the gamma 12 subunit are those known to participate in responses to receptors generating diacylglycerol and Ca2+ (27, 43-45), suggesting an important role for this event in cell signaling.

The finding that phosphorylation of the gamma  subunit can reduce the ability of the beta gamma dimer to stimulate one effector without changing its activity on other effectors adds complexity to the regulation of this signal. Previously, the known mechanisms for regulating beta gamma activity only involved sequestration by either G protein alpha  subunits or phosducin (3, 6, 7). Because the GDP bound form of the alpha  subunit has a higher affinity for the beta gamma subunit, an important mechanism for regulating the activity of the beta gamma subunit is the return of the active, GTP-bound alpha  subunit to its basal state (1, 3, 4, 7). Indeed, overexpression of alpha  subunits is an effective means of decreasing the activity of beta gamma subunits in cultured cells (46, 47). Phosducin also has a nanomolar affinity for the beta gamma subunit (48) and, whereas its role in cell function is still emerging, it appears to inhibit the beta gamma dimer by sequestration of the protein following dissociation of the alpha :beta gamma heterotrimer (49). Because the beta gamma dimer is needed for coupling the alpha  subunit to receptors (21, 38, 39), the continued activation of alpha  subunits is decreased. Accordingly, overexpression of phosducin in cultured cells can inhibit the ability of released beta gamma to activate PLC-beta or type II adenylyl cyclase (50). Interestingly, phosphorylation of Ser73 in phosducin via the cyclic AMP-dependent protein kinase decreases its affinity for the beta gamma dimer (49, 51), and treatment of cells overexpressing phosducin with dibutyryl cyclic AMP can relieve its inhibitory effects (50). In this context, the finding that phosphorylation of the gamma 12 subunit with protein kinase C can inhibit the activity of the beta gamma dimer toward certain effectors offers new paradigms for understanding the regulation of this important signal.

The observation that phosphorylation of the gamma 12 subunit on Ser1 inhibits the ability of the dimer to stimulate type II adenylyl cyclase suggests that the N-terminal region of the gamma  subunit is important for interaction with this effector. In support of this concept, pilot experiments demonstrated that a peptide mimicking the N-terminal 21 amino acids of the gamma 2 protein can inhibit the ability of beta 1gamma 2 to stimulate cyclase.2 Thus, the negatively charged phosphate group at the N terminus of the protein may inhibit the interaction of the dimer with the cyclase molecule. Two other functional domains of the gamma  subunit have been intensively studied using both biochemical assays and site directed mutagenesis. The C-terminal 15 amino acids and the prenyl group are important for interaction with the plasma membrane, the alpha  subunit, and the receptor (4, 5, 26, 52, 53), and the central region of the molecule is important for specific interaction with the beta  subunits (54, 55). The importance of these interaction sites is clearly supported by the x-ray structures of the alpha beta gamma heterotrimer, which indicate a stretch of 15 amino acids beginning at Arg30 of gamma 1 that interact with the beta  subunit and likely contacts between the C-terminal domain of the gamma  subunit and the membrane-alpha subunit interface (56, 57). Overall, these findings are interesting because they indicate that three different domains of this small protein are used for interactions with other proteins. Comparison of the N-terminal sequences of the 11 gamma  subunits through the first helical region (Val26 in gamma 1 (57)) shows that amino acid identity varies from 15 to 85%. Thus, the diversity of this domain may be important for specificity in effector signaling.

The domains in the PLC-beta molecule that interact with the beta gamma dimer have been examined using a number methods. Multiple lines of evidence indicate that the beta gamma dimer binds near the Y domain in the catalytic core of PLC-beta (58). Refinement of this location using overexpression of glutathione S-transferase fusion proteins containing small regions of the molecule suggests that the amino acids between Leu580 and Val641 are involved in binding the beta gamma dimer (59). Experiments using small peptides indicate a potential beta gamma binding domain in the 10 amino acids between Glu574 and Lys583 (60). The domains in the beta  subunit that interact with effectors appear to be similar to those responsible for binding the alpha  subunit (7). However, the domains in the gamma  subunits responsible for interaction with PLC-beta have not been clearly identified. The finding that the ability of the beta gamma subunit to stimulate PLC-beta is not affected by phosphorylation suggests that the central or C-terminal regions in the protein are more likely to interact with PLC-beta than the N-terminal domain. Alternatively, the negative charge introduced by phosphorylation of the protein may not inhibit binding of the dimer to PLC-beta .

It is especially interesting that phosphorylation of the gamma 12 subunit increases the affinity of the receptor-alpha beta gamma interaction, since the structure of the heterotrimer shows no interaction between the N terminus of the gamma  subunit and the alpha  subunit (56, 57). In addition, the N-terminal region of the gamma  subunit is predicted to be some distance from the receptor and the membrane (57). A similar situation occurs with phosducin, where phosphorylation changes its affinity for the beta gamma dimer (49, 51), yet the phosphorylation site is not directly in contact with either the beta  or gamma  subunit (61). One possible explanation of this result is that phosphorylation may cause an indirect effect on receptor-heterotrimer interactions by an induced conformational change in the beta gamma dimer. However, a definitive answer should emerge from direct structural analysis of the receptor-alpha beta gamma interaction.

The finding that phosphorylation of beta 1gamma 12 markedly decreases its ability to stimulate type II adenylyl cyclase and focuses the beta gamma signal toward other effectors is likely to be a broadly important regulatory mechanism. The gamma 12 subunit is widely expressed and has been demonstrated to be phosphorylated by PKC in intact cells following receptor activation (12, 13). The type II adenylyl cyclase is expressed at high levels in the brain and the type IV adenylyl cyclase, with nearly identical regulatory properties, is widely expressed in peripheral tissues such as lung, heart, kidney, and liver (62). Thus, the phosphorylation of the gamma 12 subunit has the potential to affect the interplay of the Ca2+ and cyclic AMP signaling networks in most cells. As one example, in vascular smooth muscle cells where phosphorylation of the gamma 12 subunit occurs following application of the contractile agonists vasopressin or angiotensin II (12), this mechanism may augment the ability of Ca2+ to cause contraction by blunting a rise in cyclic AMP, which relaxes smooth muscle (63). A similar mechanism could be used in neural networks to amplify the effects of a Ca2+ signal and blunt those of cyclic AMP.

The fact that only the gamma 12 subunit has a protein kinase C phosphorylation site in its N terminus is intriguing. Whether other gamma  subunits can be phosphorylated is an important issue. However, the gamma 12 subunit is highly expressed in all regions of the brain (14) and in most peripheral tissues (12). Thus, gamma 12 may be the gamma  subunit in the beta gamma dimers used in many important signaling systems. Our findings that the beta 1gamma 12 dimer is equal in activity to the better studied beta 1gamma 2 dimer support this conclusion. However, the gamma 12 subunit may also be used by cells for undiscovered or specialized signaling roles where phosphorylation is critical to its activity. In this regard, in Swiss 3T3 and C6 cells, dimers containing gamma 12 appear to be localized to actin stress fibers, whereas those containing the gamma 5 subunit are found in focal adhesions. Moreover, the beta gamma 12 dimers appear to bind much more tightly to purified actin filaments than do dimers containing the gamma 5 subunit (15). These findings suggest that the cytoskeleton may be an important site for gamma 12 function and may lead to discovery of new roles for the beta gamma dimer in cell signaling.

The observation that phosphorylation can change the effect of the beta gamma dimer on certain effectors may have consequences for many signaling systems not studied in this report. The beta gamma dimer is emerging as an important regulatory signal for a large number of effectors including: K+ and Ca2+ channels (64), the beta  adrenergic receptor kinase (65), phosphatidylinositol 3-kinase (66-68), mitogen-activated protein kinase (16), and novel kinases such as p21-activated protein kinase (69). It will be important to determine whether phosphorylation alters the activity of the beta 1gamma 12 dimer toward any of these signaling molecules.

    ACKNOWLEDGEMENTS

We thank Dr. Joel M. Linden for the 125I-N6-(aminobenzyl)adenosine, the pDT vector, and help with statistical analysis; Dr. Ravi Iyengar for the baculovirus encoding type II adenylyl cyclase; and Dr. T. K. Harden for the turkey phospholipase C-beta . We also acknowledge Rimma Khazan for technical assistance, the University of Virginia Biomolecular Research Facility for DNA sequencing, and the Diabetes Core Facility for [32PO4]ATP and the cAMP assays.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants PO1-CA 40042 and RO1-DK-19952.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.

Dagger Supported by a fellowship from the Virginia Affiliate of the American Heart Association. Present address: Dept. of Medicine, University of Tokyo, Tokyo 112-8688, Japan.

§ To whom correspondence should be addressed: Box 448, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5618; Fax: 804-982-3878; E-mail: jcg8w{at}virginia.edu.

The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; Sf9 cells, Spondoptera frugiperda cells (ATCC number CRL 1711)CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonateGENAPOL® C-100, polyoxylethylene(10)dodecyl etherPKC, protein kinase CPMA, phorbol 12-myristate 13-acetatePCR, polymerase chain reactionPLC, phospholipase CPAGE, polyacrylamide gel electrophoresisTricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineNTA, nitrilotriacetic acidGTPgamma S, guanosine 5'-O-(thiotriphosphate).

2 H. Y. and J. C. G., unpublished experiments.

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Abstract
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Procedures
Results
Discussion
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