INTRODUCTION

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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  subunit leading to exchange of GDP for GTP in the protein's nucleotide binding site. Binding of GTP induces a conformational change in the  subunit, simultaneously activating the protein and markedly decreasing its affinity for the dimer (1). Both the GTP-bound form of the  subunit and the released 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  subunit have been studied extensively. All  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 subunit (1, 3, 4, 8). Both changes induce formation of the stable, heterotrimeric form of the G protein. Interestingly, the GTPase activity of many  subunits can be increased by a class of proteins termed RGS molecules (9, 10) and by certain effectors such as PLC- (11).

Although the activity of the  subunit is regulated by multiple mechanisms, regulation of the activity of the dimer is not well characterized. Recently, the ₁₂ subunit has been shown to be a substrate for protein kinase C (12, 13), suggesting that dimers containing this  subunit may be subject to regulation by phosphorylation. The ₁₂ subunit is widely expressed (12-15) and, given the extensive role of the subunit in cell signaling (6, 16), its phosphorylation may have important consequences. To examine the effects of ₁₂ subunit phosphorylation on its activity, we purified recombinant ₁₁₂ dimers from baculovirus-infected Sf9 insect cells, phosphorylated them with PKC  and ₁, and tested their activity in three assays of function. We examined the ability of phosphorylated dimers to support coupling of the G_i1  subunit to the A1 adenosine receptor and to activate two effectors, type II adenylyl cyclase or phospholipase C-. Phosphorylation of the ₁₁₂ subunit had no effect on its ability to activate PLC-, 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 signal to increase cyclic AMP levels and favors activation of other effectors.

	EXPERIMENTAL PROCEDURES

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Construction of Recombinant Baculoviruses for the  and  Subunits-- Full-length clones encoding the human ₁₀ and ₁₂ proteins were identified in the EST data base and obtained from Research Genetics, Inc (₁₂, GenBank^TM N42722; ₁₀, GenBank^TM U31383). To minimize the length of the construct 5' from the ATG start codon, the end of the ₁₂ cDNA was modified using the polymerase chain reaction (PCR). For the 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 ₁₂ coding sequence excised from pCNTR with SmaI and XbaI, and ligated into these sites in the baculovirus transfer vector, pVL1393. The native ₁₀ cDNA was excised from the pT7T3D plasmid with EcoRI, further digested with BanII and Asp700 and subcloned into the pCNTR shuttle vector. The 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 ₁₀ protein was modified to have a protein kinase C phosphorylation site by mutagenesis of the ₁₀ cDNA using PCR. The primers used were: (sense primer: 5'-GGATCCATGTCCTCCAAGGCTAGC-3'; antisense primer: 5'-CACTTTGTGCTTGAAGGAATTCC-3'). This modification introduced a Gly⁴  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 ₁ subunit, the polymerase chain reaction was used to add XbaI and BamHI restriction sites to the 5' and 3' ends of the 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 ₁ coding region (17). The _1HF coding region was excised from pDT with HindIII and BamHI and subcloned into the pCNTR shuttle vector. The _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  and  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 G_i1 and G_s  subunits and the A1 adenosine receptor have been described (20, 21).

Expression and Purification of Recombinant G Protein  and Subunits-- G protein  and subunits were overexpressed by infecting suspension cultures of Sf9 insect cells with recombinant baculoviruses (22, 23). The G_i1  subunit was purified to homogeneity as described (22). The recombinant dimers were extracted from Sf9 cells and purified using DEAE chromatography and an  subunit affinity column (23).

Phosphorylation of Purified Subunits by PKC-- The purified _1HF12 subunit was incubated for 30 min at 30 °C in 50 mM Tris, pH 7.5, 1 mM -mercaptoethanol, 10 mM MgCl₂, 0.4 mM CaCl₂, 40-100 µM ATP, recombinant PKC  or , 40 µg/ml phosphatidylserine, and 0.8 µg/ml diolein. Usually, 25 µg of dimer was incubated with 0.74 unit of PKC  to achieve stoichiometric phosphorylation of the  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 subunits to Tricine/SDS-PAGE (24). The resolved  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 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 -mercaptoethanol, 0.6% (w/v) CHAPS, and 5 mM imidazole to remove PKC. The 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 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 dimer. Controls were also performed to determine whether the 30 °C incubation with PKC and subsequent re-purification reduced the activity of the dimer in the functional assays. In these experiments, a mock-phosphorylation reaction was performed in the absence of PKC, the dimer re-purified and its activity compared with that of dimers subjected only to the -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  and 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 G_i1  subunit, 0-10 nM 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- 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 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 preparations in each assay tube was kept below 0.01% (w/v) to eliminate effects of detergent on PLC- activity (29). The reaction was begun by addition of 10 ng of recombinant, turkey erythrocyte PLC- 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 G_s  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 GTPS-activated G_s  subunit (32) and varying concentrations of 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  and 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  and 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  3 Prime, Inc. (Boulder, CO). All other reagents were of the highest purity available.

	RESULTS

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Stoichiometry of Phosphorylation of the ₁₂ and the _10G4K Subunits-- Recent experiments have demonstrated that the bovine 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 ₁₂ subunit is widely expressed (12-15), its phosphorylation may have important consequences. To examine the effects of ₁₂ subunit phosphorylation on its activity, we purified recombinant ₁₁₂ dimers from Sf9 insect cells, phosphorylated them with PKC  and , and tested their activity in assays of function. The human ₁₂ subunit was rapidly phosphorylated by PKC  to a stoichiometry of about 1 mol/mol as shown in Fig. 1, A and B. Protein kinase C ₁ was less effective than PKC ; the stoichiometry only reached 0.5 mol/mol after 60 min of incubation. Addition of a hexahistidine-FLAG affinity tag to the ₁ subunit (_1HF) facilitated removal of PKC from the reaction mixture prior to assessing function. Thus, the phosphorylated _1HF12 dimer in the reaction mix can be applied to a Ni²⁺-NTA-agarose column and pure 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 dimer was free of residual kinase activity (see "Experimental Procedures"). The right side of the figure shows that only the ₁₂ subunit in the dimer is phosphorylated. Addition of the hexahistidine-FLAG affinity tag to the N terminus of the ₁ subunit did not affect the association of the  and  subunits or purification of the dimer on an -subunit affinity column (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of 1HF12 by protein kinase C. A, purified 1HF12 dimer was incubated with protein kinase C  for the indicated times. The autoradiograph shows the time course of the phosphorylation of the 12 subunit by PKC  (upper panel) and PKC 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 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 1HF12 on the Ni2+-NTA-agarose column. The right panel shows that only the 12 subunit is phosphorylated. Experiments are representative of 10 similar experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of the mutated 1HF10 dimer by protein kinase C. A, comparison of the N-terminal amino acid sequences of the bovine and human 12 subunits, the human 10 subunit, and the Gly4  Lys mutant 10 subunit (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 10 subunit is underlined. B, autoradiograph showing the phosphorylation of the 10G4K and 12 subunits by PKC . The native 1 and 10 proteins were not phosphorylated. C, section from a silver-stained Tricine/SDS-PAGE gel showing the mobilities of the four  subunits. Experiments are representative of five similar experiments.

Effect of Phosphorylation of the  Subunit on Receptor Coupling-- Having established the ability of protein kinase C to phosphorylate the two recombinant  subunits, we examined the effects of phosphorylation on the function of the dimer. The subunits play several important roles in the signaling mechanisms used by receptors to activate effectors. In combination with the 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  subunit for GDP (7, 8); and when released from the  subunit, they activate effectors such as type II adenylyl cyclase and phospholipase C- (6, 7). We first examined the effect of  subunit phosphorylation on the ability of the 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 G_i  and subunits into these membranes establishes high affinity agonist binding and provides a sensitive assay for receptor- interactions (21). Fig. 3 shows that the dimers used in this study, ₁₁₂, ₁₁₀, _110G4K, and _1HF12, 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 ₁₂ dimer (26). All dimers tested support coupling with a K_act of 0.5-1.0 nM (see Fig. 3 legend). Thus, the newly discovered ₁₀ and ₁₂ subunits are able to couple very effectively to the G_i1  subunit and the A1 adenosine receptor when dimerized with the ₁ subunit. Importantly, neither the hexahistidine-FLAG tag added to the N terminus of the ₁ subunit nor the G4K mutation made in the N terminus of the ₁₀ subunit affect the ability of these recombinant dimers to induce the high affinity conformation of the A1 adenosine receptor.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Ability of native and modified 112 and 110 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  subunit, the indicated concentrations of the defined dimers, and high affinity 125I-aminobenzyladenosine binding measured as described under "Experimental Procedures." The ratio of receptor:: 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.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Comparison of the ability of phosphorylated and unphosphorylated 1HF12 and 1HF10 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  subunit and the indicated concentrations of unphosphorylated (closed circles) or phosphorylated (open circles) 1HF12 dimers. Formation of the high affinity, agonist binding state of the receptor was measured as described under "Experimental Procedures." Phosphorylation of the 1HF12 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) 1HF10G4K dimers. Phosphorylation of the 1HF10G4K 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  Subunit Phosphorylation on Effector Activity-- The data in Fig. 5A show the ability of the native and modified dimers used in this study to activate PLC- in an in vitro assay using [³H]PIP₂ incorporated into phospholipid vesicles as substrate. Note that ₁₁₂, ₁₁₀, _1HF10G4K, and _1HF12 are equally as effective as the ₁₂ dimer. All four forms of the protein stimulated the release of [³H]inositol 1,4,5-trisphosphate with a K_act 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 _1HF12 dimers to activate PLC-. The phosphorylated or unphosphorylated forms of the _1HF10G4K 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 activity in the PLC assay.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Comparison of the ability of phosphorylated and unphosphorylated dimers to activate phospholipase C-. A, the ability of native and modified 112 and 110 dimers to activate phospholipase C- as compared with the effect of 12. The indicated concentrations of dimers were reconstituted with recombinant, turkey phospholipase C- 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) 1HF12 dimers to activate phospholipase C-. 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.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Comparison of the ability of phosphorylated and unphosphorylated 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 dimers as described under "Experimental Procedures." B, the effect of phosphorylation on the ability of 1HF12 and 1HF10G4K to stimulate type II adenylyl cyclase. The dimers were phosphorylated by protein kinase C  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) 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."

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Stimulation of type II adenylyl cyclase by 1HF12 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 dimers on cyclic AMP production as described under "Experimental Procedures." The 1HF12 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 1 for 15 min at 30 °C; 0.5 mol of PO4/mol (open circles), the dimer was incubated with PKC  for 2 min at 30 °C; 1.0 mol of PO4/mol (open triangles), the dimer was incubated with PKC  for 30 min at 30 °C. The 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.

	DISCUSSION

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Identification of the diversity in the family of G protein  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 ₁ subunit, the newly discovered ₁₂ and ₁₀ subunits are equal in potency and efficacy to the well studied ₁₂ dimer. Both the ₁₁₂ and ₁₁₀ dimers were fully effective in the receptor coupling assay using the G_i1  subunit and the A1 adenosine receptor and able to maximally activate type II adenylyl cyclase and PLC-. As these  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 ₁₁₂ 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 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 _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 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 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  subunit can reduce the ability of the 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 activity only involved sequestration by either G protein  subunits or phosducin (3, 6, 7). Because the GDP bound form of the  subunit has a higher affinity for the subunit, an important mechanism for regulating the activity of the subunit is the return of the active, GTP-bound  subunit to its basal state (1, 3, 4, 7). Indeed, overexpression of  subunits is an effective means of decreasing the activity of subunits in cultured cells (46, 47). Phosducin also has a nanomolar affinity for the subunit (48) and, whereas its role in cell function is still emerging, it appears to inhibit the dimer by sequestration of the protein following dissociation of the : heterotrimer (49). Because the dimer is needed for coupling the  subunit to receptors (21, 38, 39), the continued activation of  subunits is decreased. Accordingly, overexpression of phosducin in cultured cells can inhibit the ability of released to activate PLC- or type II adenylyl cyclase (50). Interestingly, phosphorylation of Ser⁷³ in phosducin via the cyclic AMP-dependent protein kinase decreases its affinity for the 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 ₁₂ subunit with protein kinase C can inhibit the activity of the dimer toward certain effectors offers new paradigms for understanding the regulation of this important signal.

The observation that phosphorylation of the ₁₂ subunit on Ser¹ inhibits the ability of the dimer to stimulate type II adenylyl cyclase suggests that the N-terminal region of the  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 ₂ protein can inhibit the ability of ₁₂ 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  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  subunit, and the receptor (4, 5, 26, 52, 53), and the central region of the molecule is important for specific interaction with the  subunits (54, 55). The importance of these interaction sites is clearly supported by the x-ray structures of the heterotrimer, which indicate a stretch of 15 amino acids beginning at Arg³⁰ of ₁ that interact with the  subunit and likely contacts between the C-terminal domain of the  subunit and the membrane- 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  subunits through the first helical region (Val²⁶ in 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- molecule that interact with the dimer have been examined using a number methods. Multiple lines of evidence indicate that the dimer binds near the Y domain in the catalytic core of PLC- (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 dimer (59). Experiments using small peptides indicate a potential binding domain in the 10 amino acids between Glu⁵⁷⁴ and Lys⁵⁸³ (60). The domains in the  subunit that interact with effectors appear to be similar to those responsible for binding the  subunit (7). However, the domains in the  subunits responsible for interaction with PLC- have not been clearly identified. The finding that the ability of the subunit to stimulate PLC- is not affected by phosphorylation suggests that the central or C-terminal regions in the protein are more likely to interact with PLC- than the N-terminal domain. Alternatively, the negative charge introduced by phosphorylation of the protein may not inhibit binding of the dimer to PLC-.

It is especially interesting that phosphorylation of the ₁₂ subunit increases the affinity of the receptor- interaction, since the structure of the heterotrimer shows no interaction between the N terminus of the  subunit and the