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. 2000 Mar;20(5):1497–1506. doi: 10.1128/mcb.20.5.1497-1506.2000

Distinct Roles for Gαi2 and Gβγ in Signaling to DNA Synthesis and Gαi3 in Cellular Transformation by Dopamine D2S Receptor Activation in BALB/c 3T3 Cells

Mohammad H Ghahremani 1, Christine Forget 2, Paul R Albert 2,*
PMCID: PMC85319  PMID: 10669727

Abstract

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates Gi and Go proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Gαi/o subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Gαi2 and Gαi3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Gαi2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Gαi3 was required for formation of transformed foci. Transfection of toxin-resistant Gαi1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Gαo inhibited dopamine-induced transformation. The role of Gβγ subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gβγ activity. Mobilization of Gβγ subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Gαi2 and Gβγ) and oncogenic transformation (Gαi3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.

Growth signaling of a large family of receptors is mediated by heterotrimeric guanine nucleotide binding proteins (G proteins). Activation of G protein-coupled receptors results in dissociation of Gα and Gβγ subunits, which couple to various effectors in cell membrane (6, 38). The Gi and Go proteins (collectively referred to as Gi/o proteins) couple negatively to adenylyl cyclase (AC), resulting in inhibition of cyclic AMP (cAMP) production. Pertussis toxin (PTX) selectively ADP-ribosylates the Gαi/Gαo subunits to block all actions of Gi/o proteins. The Gβγ subunits are mobilized upon G protein activation and couple to a variety of cell-specific effectors (10, 38). G protein-coupled receptors appear to utilize receptor-specific combinations of subunits to initiate distinct responses (17). Signaling through PTX-sensitive Gi/o proteins can enhance or inhibit cell growth and transformation (36, 42). In mesenchymal cells, such as BALB/c 3T3 cells, several receptors that couple to Gi/o proteins mediate enhancement of mitogen-activated kinase (MAPK) activity, DNA synthesis, and cell proliferation (1, 26, 43). Furthermore, a Gαi-regulated role in mitosis has been identified in fibroblast cells (12). Moreover, expression of constitutively active mutants of Gαi2 induces transformation in Rat-1 fibroblasts (18, 41), and mutationally activated Gαi2 has been identified in human adrenal and ovarian tumors (35). In addition, the Gβγ subunits also contribute to MAPK activation and DNA synthesis in transfected cell lines (34, 51).

The dopamine D2 receptor short form (D2S receptor) couples to PTX-sensitive Gi/o proteins to mediate inhibitory or stimulatory cellular responses, depending on the cell type (2, 9, 37). In lactotroph and neuronal cells, the D2S receptor activates potassium channels to hyperpolarize the cell membrane, inhibits L-type calcium channels, and prevents AC activation, actions that together mediate inhibition of (i) hormone secretion and gene transcription and (ii) cell proliferation (4, 14, 29, 47, 50). In contrast, when expressed in cells of mesenchymal origin, the D2 receptor displays a stimulatory phenotype, mediating stimulation of phospholipase C activity to induce calcium mobilization and activating the MAPK cascade leading to enhanced gene transcription and cell proliferation (24, 28, 33, 50). These findings suggest that the D2 receptor mediates opposite actions on cell growth depending on the repertoire of cell-specific effectors that is expressed.

To address the G protein signaling specificity of the D2S receptor in cell growth, nontransformed BALB/c 3T3 fibroblast cells were transfected stably with D2S receptor cDNA. The contribution of specific Gα subunits to D2S-mediated signaling was evaluated using PTX-insensitive mutants of Gαi1, Gαi2, Gαi3, and Gαo, in which the carboxyl-terminal ribosyl acceptor cysteine was changed to a nonaccepting serine. The Cys→Ser mutation is a structurally conservative change, and the mutant G proteins remain functional following PTX pretreatment (8, 16, 19, 48). This approach has the advantage over pharmacological or dominant negative inhibitors of signaling components since mitogen-activated pathways are not inhibited indiscriminately, allowing a selective characterization of D2S receptor-mediated actions. The role of Gβγ subunits in D2S signaling has been investigated using the GRK-CT protein (carboxyl terminus of G protein-coupled receptor kinase) as a selective Gβγ scavenger (22). In BALB/c 3T3 cells transfected with D2S receptor, the D2S receptor utilized distinct Gαi subunits to inhibit cAMP accumulation and specific Gαi and Gβγ subunits to enhance MAPK activation and DNA synthesis and to mediate cellular transformation. In contrast, calcium mobilization induced by the D2S receptor was not reconstituted with Gαi subunits but was blocked by inhibiting Gβγ function. These results indicate a strong G protein subunit specificity in D2S receptor-induced cell growth.

MATERIALS AND METHODS

Materials.

Apomorphine, dopamine, EGTA, forskolin, 3-isobutyl-1-methylxanthine, and PTX were from Sigma (St. Louis, Mo.). Fura-2 AM was purchased from Molecular Probes (Eugene, Oreg.), and hygromycin B was purchased from Calbiochem. [125I]succinyl cAMP (2,200 Ci/mmol) and polyvinylidene difluoride (PVDF) membranes were from New England Nuclear Corp. (Boston, Mass.); [3H]thymidine (76.0 Ci/mmol), [3H]spiperone (125 Ci/mmol), [α-32P]dCTP (3,000 Ci/mmol), and enhanced chemiluminescence Western blot detection kits were from Amersham Corp. (Arlington Heights, Ill.). Sera, media, and Geneticin (G418) were obtained from Gibco/BRL. Plasmid pY3 was obtained from the American Type Culture Collection (Manassas, Va.). Endonucleases and DNA polymerase were purchased from New England Biolabs (NEB; Mississauga, Ontario, Canada). The cDNAs encoding wild-type rat Gαo, Gαi1, Gαi2, and Gαi3 were generously provided by Randall Reed, Johns Hopkins University, Baltimore, Md. The anti-Gαo antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.); anti-Gαi1-2 and anti-Gαi3 were obtained from Calbiochem (San Diego, Calif.); anti-RGS-His6 was from Qiagen (Santa Clarita, Calif.); and anti-phospho-p42/44 MAPK antibody (T202/Y204) was from NEB.

Cell culture and transfection.

BALB/c 3T3 cells and derivative clones were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). For transfection, BALB/c 3T3 cells plated at 50% confluence were cotransfected with 20 μg of rat D2S-pZEM and 2 μg of pY3, using calcium phosphate coprecipitation (46). The transfected cells were cultured in DMEM–10% FBS containing hygromycin B (400 μg/ml) for 2 to 3 weeks (3). Antibiotic-resistant clones were subjected to Northern blot analysis and subsequent clonal expansion (generating the BALB-D2S clone). The receptor number in the BALB-D2S clone was quantified by saturation binding analysis using [3H]spiperone. Based on receptor binding results, BALB-D2S cells express 143.6 ± 35.9 fmol of D2S receptor/mg. The mutant Gαi/o subunit constructs (Go-PTX, Gi1-PTX, Gi2-PTX, and Gi3-PTX) and His-GRK-CT were transfected individually (30 μg) into BALB-D2S (clone 11), and the cells were cultured in medium containing G418 (700 μg/ml) for 2 to 3 weeks. Antibiotic-resistant clones of each transfection were picked (24 clones/transfection) and tested for expression of the corresponding Gαi/o proteins, using Northern blot and Western blot analyses. A minimal amount of serum (1%) was used in DNA synthesis and MAPK activity measurements since the D2-induced signaling was lost in serum-free medium.

Plasmid construction.

PTX-insensitive Gαi/o mutants were generated using rat cDNAs (20) encoding Gαo, Gαi1, Gαi2, and Gαi3 subunits as previously described (16). Briefly, the cysteine 351 codon (352 for Gαi2), TGT, was mutated to TCT in order to encode serine, and the mutation was confirmed by Sanger dideoxynucleotide sequencing. The mutant cDNAs were then subcloned into the EcoRI site of the pcDNA3 mammalian expression vector (Invitrogen). The carboxyl-terminal domain of OK-GRK2 cDNA (27) starting from Thr493 was isolated and used for the construct GRK-CT (16). An RGS-His6 tag was incorporated at the N terminus of GRK-CT, and the His-GRK-CT fragment was cloned in pcDNA3. The structure of the His-GRK-CT construct was confirmed by DNA sequencing.

Western blot analysis.

Cells (107/10-cm-diameter plate) were harvested and resuspended in 200 μl of RIPA-L buffer (10 mM Tris [pH 8], 1.5 mM MgCl2, 5 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.1% sodium lauryl sulfate, 0.5% sodium deoxycholate, 5 μg of leupeptin/ml) on ice. The cell lysate was passed through a 25-gauge needle three times to shear genomic DNA and incubated on ice. After 30 min, the lysate was centrifuged (10,000 × g, 10 min, 4°C), and the supernatant was recovered and assayed for protein content by the bicinchoninic acid protein assay kit (Pierce). Lysates (100 μg/lane) were electrophoresed on sodium lauryl sulfate-containing 12% polyacrylamide gels at 100 V and 40 mA for 1 h and blotted onto PVDF membranes for 1 h at 250 mA and 4°C. Blots were blocked overnight in 5% nonfat dry milk in TBS-T (10 mM Tris, 150 mM NaCl [pH 8.0], 0.05% Tween 20) at 4°C. The blots were then incubated at room temperature in TBS-T for 1 h with primary antibody followed by 30 min of incubation with horseradish peroxidase-conjugated secondary antibody; the peroxidase product was developed using the enhanced chemiluminescence Western blot protocol.

cAMP measurement.

Equal numbers of cells were plated in six-well plates and grown to 70 to 80% confluence. After being rinsed with HBBS buffer (118 mM NaCl, 4.6 mM KCl, 1.0 mM CaCl2, 10 mM d-glucose, 20 mM HEPES [pH 7.2]), the cells were incubated with or without experimental compounds in of HBBS–100 μM 3-isobutyl-1-methylxanthine (1 ml/well) at 37°C. After 20 min, the media were recovered and stored at −20°C. Samples were analyzed by specific radioimmunoassay to detect cAMP (4). Percent inhibition was calculated as 100 − [100(D − C)/(S − C)], where D is cAMP in apomorphine-treated cells, C is cAMP in control or nontreated cells (basal cAMP), and S is stimulated cAMP in forskolin-treated cells.

Measurement of calcium mobilization.

Cells were grown to 80% confluence, harvested with trypsin-EDTA, resuspended in 1 ml of HBBS with 2 μM Fura-2 AM, and incubated at 37°C for 45 min with shaking (100 rpm). The cells were washed twice with HBBS, resuspended in 2 ml of HBBS, and subjected to fluorometric measurement. The fluorescence ratio (R) of Fura-2 was monitored in a Perkin-Elmer LS-50 spectrofluorometer at λex = 340/380 nm and λem = 510 nm. Calibration was done with 0.1% Triton X-100 and 20 mM Tris base to determine Rmax and 10 mM EGTA (pH > 8) to obtain Rmin, and the fluorescence ratio was converted to intracellular Ca2+ concentration ([Ca2+]i) based on a Kd of 227 nM for the Fura-2–calcium complex (4). Experimental compounds were added directly to cuvettes from 100-fold-concentrated solutions at times indicated in the figures. Because of fluorescent interference of the Fura-2 signal by apomorphine autofluorescence, dopamine was used in these experiments.

Measurement of MAPK activity.

Equal numbers of cells (3 × 105 cells/well) were plated in six-well dishes. At 80% confluence, the cells were serum starved for 24 to 36 h in DMEM–0.2% FBS, and the assay was performed in the presence of indicated drugs for 10 min at 37°C. The cells were lysed in 100 μl of sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue). Samples were heated (100°C, 5 min) and centrifuged. Supernatants (15 μl) were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted on PVDF membranes, and subjected to Western blot analysis. Active MAPK was detected using (1:1,000) anti-phospho-p42/44 MAPK antibody (NEB). The corresponding band for p42 MAPK and p44 MAPK (collectively referred to as p42/44 MAPK) was normalized to the actin band on each lane, and the normalized ratio was used for further analysis. PTX treatment was attained by incubation of the cells in 10 ng of PTX/ml for 4 h. In this study, PTX treatment reduced MAPK activity of serum-free and low-serum medium conditions. However, the PTX sensitivity was not altered in any of the clones compared to BALB-D2S cells.

Measurement of DNA synthesis.

Cells were plated in 24-well dishes at a density of 104 cells/well and were serum starved upon confluence in DMEM–0.2% FBS for 36 h. The cells were incubated in low-serum (1%) medium with experimental drugs for 16 h, followed by addition of 1 μCi of [3H]thymidine/ml for 6 h. After drug treatment, wells were washed with phosphate-buffered saline, 1 ml of 10% trichloroacetic acid was added to each well, and the dishes were incubated for 30 min at 4°C. The precipitated materials were washed with ice-cold 10% trichloroacetic acid, resuspended in 1 ml of NaOH (1 M)-SDS (1%), and counted in 5 ml of scintillation cocktail (1). PTX treatment was performed by incubation of the cells in 10 ng of PTX/ml for 4 h prior to drug treatment. In PTX-pretreated conditions, thymidine incorporation was attenuated compared to nontreated cells. However, the PTX sensitivity of DNA synthesis was not altered in any of the clones compared to BALB-D2S cells.

Focus formation.

The cells were plated in six-well dishes at a density of 3 × 105 cells/well and grown to confluence in DMEM–5% FBS. Every 2 days, fresh medium containing the appropriate drugs was added for 10 to 14 days. The cells were then stained with methylene blue (1). Briefly, the cells were fixed in 3.7% formaldehyde in phosphate-buffered saline for 5 min, incubated in 0.02% methylene blue in 50% methanol for 10 min, rinsed twice with distilled water, and air dried. Focus formation was analyzed by counting foci in plates using MCID imaging system (Imaging Research Inc., St. Catherines, Ontario, Canada).

RESULTS

Expression of mutant Gαi/o subtypes in BALB-D2S cells.

In nontransfected BALB/c 3T3 cells, dopamine D2 receptors were not detected by Northern blot analysis or binding assays, nor did we observe responses to dopamine agonists (data not shown). For consistency, apomorphine was used as an agonist in most experiments (except calcium assays; see Materials and Methods) since, unlike dopamine, it is stable in long-term culture in serum-containing medium. To define D2S receptor growth signaling pathways, a clone of BALB/c 3T3 cells stably transfected with rat D2S receptor cDNA plasmid (BALB-D2S) was selected for D2S receptor expression. The BALB-D2S cells (referred to as wild-type cells) were then transfected separately with each PTX-insensitive G protein mutant (Gαo-, Gαi1-, Gαi2-, and Gαi3-PTX). Cell extracts from clones and the wild-type cells were used to analyze the level of expression of the G proteins by Western blot analysis (Fig. 1). BALB-D2S cells expressed Gαo, Gαi1 at apparently greater abundance than Gαi2, and Gαi3 at low levels, based on densitometric scanning. Comparing Gαo and Gαi1 protein expression in each transfectant to the level in BALB-D2S cells indicates that the transfectant cell lines expressed approximately twofold more than the most abundant endogenous Gαi/o subunits (Fig. 1). Thus, approximately equal amounts of mutant and wild-type proteins were produced in the transfected cell lines.

FIG. 1.

FIG. 1

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Gαi/o expression in BALB-D2S cells transfected with PTX-insensitive Gαi/o mutants. Cell extracts (100 μg) of BALB-D2S cells (wild type) and BALB-D2S cells expressing Gαo-PTX (BDo-14) (A), Gαi1-PTX (BDi-11) (B), Gαi2-PTX (BDi2-6, BDi2-22) (C), and Gαi3-PTX (BDi3-3 and BDi3-7) (D) were subjected to Western blot analysis as described in Materials and Methods. The blots were probed with anti-Gαo (A), anti-Gαi1-2 (B and C), and anti-Gαi3 (D) antibodies. Densitometric analysis indicated the following data for different clones (fold increase compare to wild type): BDo-14, 1.8; BDi-11, 2.0; BDi2-6, 2.1, BDi2-22, 1.8; BDi3-3, 2.9; and BDi3-7, 3.5.

Gαi2 and Gαi3 mediate D2S receptor inhibition of forskolin-stimulated cAMP production.

In BALB-D2S cells, activation of the D2S receptor did not affect the basal cAMP production (data not shown). Stimulation of the cells with forskolin (10 μM) increased cAMP levels eightfold (4.2 ± 0.27 [mean ± standard error of the mean {SEM}] versus 0.51 ± 0.04 pmol/ml) above the basal level. Activation of the D2S receptor by apomorphine (1 μM) inhibited forskolin-induced cAMP production by 87.1% ± 2.5%, an effect which was reversed by pretreatment with PTX (27.4% ± 7.2%), indicating the role of Gi/o proteins.

The G protein specificity of D2S-induced inhibition was examined using BALB-D2S clones stably expressing mutant Gαi/o subtypes. In all clones, apomorphine inhibited forskolin-induced cAMP production to a comparable extent as in BALB-D2S cells (Fig. 2). In multiple experiments, apomorphine-induced inhibition of forskolin-stimulated cAMP level was not blocked by PTX treatment in clones expressing Gi2-PTX (BDi2-6 and BDi2-22) or Gi3-PTX (BDi3-3 and BDi3-7), whereas in the other clones a significant blockade of apomorphine effect was observed (Fig. 2; Table 1). The Gi1-PTX clones gave inconsistent responses to apomorphine in the absence of PTX and hence could not be analyzed further (data not shown). As described below, the expression of Gi1-PTX appears to have direct actions in BALB/c 3T3 cells that are not evident in transformed Ltk− cells (16). Furthermore, in BALB-D2S cells expressing GRK-CT (BDD−), apomorphine inhibition of forskolin-induced cAMP production was comparable to that in BALB-D2S cells (Table 2), suggesting a minor role for Gβγ subunits in this process. These results indicate that Gαi2 and Gαi3 subtypes can mediate D2S-induced inhibition of forskolin-stimulated cAMP production in BALB-D2S cells.

FIG. 2.

FIG. 2

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Apomorphine inhibition of forskolin-induced cAMP accumulation in BALB-D2S cells. Cells were incubated for 20 min with no drugs, forskolin (10 μM), apomorphine (1 μM), or both forskolin and apomorphine, with or without PTX pretreatment (50 ng/ml, 4 to 6 h); percent inhibition of apomorphine action from two independent experiments was calculated as described in Materials and Methods. The data are expressed as mean ± SEM and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest. In all clones, basal and forskolin-induced cAMP levels were not significantly different from levels in nontransfected BALB-D2S cells. BALB-D2S cells, parent cell line; BDo-14, BALB-D2S cells expressing Go-PTX; BDi2-6 and BDi2-22, cells expressing Gi2-PTX; BDi3-3 and BDi3-7, cells expressing Gi3-PTX.

TABLE 1.

Percent inhibition of forskolin-induced cAMP accumulation in BALB-D2S cells not treated or treated with a low PTX concentrationa

Cell type % Inhibition
−PTX +PTX
BALB-D2S 84.3 ± 6.8 38.6 ± 4.2*
BDo-14 73.8 ± 12.2 27.8 ± 13.4*
BDi2-22 79.2 ± 2.1 63.5 ± 7.1 (NS)
BDi3-3 66.3 ± 5.7 56.5 ± 5.8 (NS)
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a

Cells were incubated for 20 min with no drugs, forskolin (10 μM), apomorphine (1 μM), or both apomorphine and forskolin, with or without PTX pretreatment (10 ng/ml, 4 to 6 h). Percent inhibition of apomorphine action was calculated as described in Materials and Methods. The data are expressed as mean ± SEM of four independent experiments (n = 4) and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest (*, P < 0.05; (NS), not significant; PTX treatment compared to no PTX treatment). In all clones, basal and forskolin-induced cAMP levels were not significantly different from levels in nontransfected BALB-D2S cells. BALB-D2S cells, parent cell line; BDo-14, BALB-D2S cells expressing Go-PTX; BDi2-22, cells expressing Gi2-PTX; BDi3-3, cells expressing Gi3-PTX. 

TABLE 2.

Effect of D2S receptor on cAMP production in BDD− cellsa

Cell type Forskolin (fold increase) Apomorphine (% inhibition)
BALB-D2S 7.9 ± 0.8 80.0 ± 9.5
BDD− 6.6 ± 0.2 68.2 ± 9.2
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a

Forskolin fold increase was calculated as amount over the basal cAMP level. Percent inhibition of forskolin-induced cAMP production by apomorphin was calculated as described in Materials and Methods. The data represent the mean ± SEM of two to four independent experiments. 

Gi/o protein subtypes involved in D2S receptor-induced calcium mobilization.

In BALB-D2S cells but not nontransfected BALB/c-3T3 cells, dopamine (10 μM) induced an immediate threefold increase in [Ca2+]i which was blocked completely by PTX pretreatment, indicating signaling via Gi/o proteins (Fig. 3A). In each of the clones expressing mutant G proteins, dopamine induced a 2- to 2.5-fold increase in [Ca+2]i which was also blocked by PTX treatment (Fig. 3B to E). Thus, none of the mutant G proteins rescued the D2S-mediated calcium response, indicating that no single Gα subunit is involved. To test the role of Gβγ subunits, BALB-D2S cells were stably transfected with a plasmid encoding His6-tagged GRK-CT, which lacks the kinase domain of GRK and is known to bind and specifically inactivate free Gβγ subunits (22). As shown in Fig. 4, dopamine-induced [Ca+2]i was reduced by 80% in a clone expressing GRK-CT (BDD−) compared to BALB-D2S cells (Fig. 4). In another clone expressing a lower level (20%) of GRK-CT, the dopamine-induced increase in [Ca+2]i was reduced by only 35% (data not shown). These results indicate that D2S-induced increase in [Ca+2]i is more dependent on Gβγ subunits than on particular Gαi/o subunits as observed in Ltk− cells (16).

FIG. 3.

FIG. 3

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PTX blocks D2S-induced calcium mobilization in BALB-D2S cells expressing PTX-insensitive Gαi/o-PTX mutants. BALB-D2S cells (A) and BALB-D2S cells expressing Go-PTX (BDo-14) (B), Gi1-PTX (BDi1-11) (C), Gi2-PTX (BDi2-6) (D), and Gi3-PTX (BDi3-7) (E) mutant G proteins were treated without (solid line) or with (dashed line) PTX pretreatment (10 ng/ml, 4 to 6 h), and the change in [Ca2+]i in response to dopamine (10 μM) or ATP (10 μM) (as an indicator of cell responsiveness) was measured. Arrows indicate the addition of dopamine or ATP.

FIG. 4.

FIG. 4

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Inhibition of calcium mobilization in BALB-D2S cells expressing GRK-CT protein. Change in [Ca2+]i was measured in BALB-D2S cells (wild type [wt]) and BALB-D2S cells stably transfected with His-GRK-CT protein (BDD−). Arrows indicate the addition of dopamine (10 μM) or ATP (10 μM). (Inset) Western blot analysis of BALB-D2S and BDD− cells. Cell extracts (100 μg/lane) from BALB-D2S and BDD− cells were subjected to SDS-PAGE, and recombinant protein was detected using an anti-RGS-His6 (Qiagen) antibody. The arrow indicates the 24-kDa recombinant GRK-CT protein.

D2S receptor induces p42/44 MAPK activation in BALB/c cells.

Activation of MAPK was detected by Western blotting using an antibody specific for dually phosphorylated MAPK (active form). In BALB/c-D2S but not nontransfected BALB/c 3T3 cells, activation of D2S receptor by apomorphine increased p42/44 MAPK phosphorylation by 42.3% ± 0.2% and 66.9% ± 0.3%, respectively (Fig. 5A). Apomorphine also augmented serum-induced phospho-MAPK level by 50 to 80%. Comparable apomorphine-induced MAPK activation was observed in the presence of 1% FBS (data not shown), which was included in the assay for DNA synthesis. Apomorphine-induced MAPK phosphorylation reached a maximum level within 5 min and remained elevated for at least 30 min (data not shown). Apomorphine activated MAPK in a concentration-dependent fashion, with 50% effective concentrations of 2.7 × 10−7 and 6.3 × 10−8 M for activation of p42 MAPK and p44 MAPK, respectively (Fig. 5B). Apomorphine-induced enhancement of MAPK phosphorylation was blocked by PTX treatment, indicating mediation by Gi/o proteins (Fig. 5A).

FIG. 5.

FIG. 5

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MAPK activation by apomorphine in BALB-D2S cells. (A) BALB-D2S cells were treated with or without PTX (10 ng/ml, 4 h) and incubated for 10 min with no drugs (Control), 1 μM apomorphine in serum-free medium (Apo), minimal-serum (1%) medium (FBS), or apomorphine in minimal-serum medium (FBS/Apo). Then the cell lysate was prepared and subjected to SDS-PAGE as described in Material and Methods. The corresponding bands for p42/44 MAPK were detected using anti-phospho-p42/44 MAPK on Western blots. The numbers indicate the densitometric analysis of the corresponding bands for each lane (fold increase compared to the control level, set at 1.0). (B) Dose-response curve of apomorphine-induced MAPK activation in BALB-D2S cells. Cells were treated with different concentrations of apomorphine for 10 min, and MAPK activation was measured by Western blotting. The data obtained for p42 MAPK (solid line)- and p44 MAPK (dashed line)-specific bands were plotted as percent increase over the basal level (n = 3). The 50% effective concentration for apomorphine effect on p42/44 MAPK (EC50) was calculated using nonlinear regression with variable slope on Prism software (GraphPad).

Gαi2 and Gβγ involvement in D2S-induced p42/44 MAPK activation.

The G protein specificity of D2S-induced MAPK activation was examined. BALB-D2S clones expressing PTX-insensitive Gαo and Gαi2 mutants displayed 1.42 ± 0.15- and 1.79 ± 0.16-fold increases in p42 MAPK activation and 1.41 ± 0.22- and 1.46 ± 0.23-fold increases in p44 MAPK activation over the basal level, respectively (Fig. 6). After PTX treatment to inhibit endogenous Gi/o proteins, only the Gαi2-PTX-expressing clone mediated p42/44 MAPK activation, suggesting an important role in D2S-induced activation of MAPK (Fig. 6). Apomorphine-induced enhancement was fully rescued by Gαi2-PTX for p42 MAPK activation, but p44 MAPK activation was only partially recovered in two independent clones. Unlike clones expressing Go-PTX or Gi2-PTX, in clones expressing Gi1-PTX or Gi3-PTX apomorphine failed to induce MAPK activation, suggesting that these subunits may antagonize or occlude D2S receptor signaling to MAPK.

FIG. 6.

FIG. 6

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D2S-induced MAPK activation in BALB-D2S cells expressing PTX-insensitive Gαi/o mutants. Cells were pretreated with or without PTX (10 ng/ml, 4 h) and incubated for 10 min at 37°C with 1 μM apomorphine. D2S-induced MAPK activation was calculated as fold increase over the basal level based on densitometric analysis of phospho-p42/44 MAPK bands. The data are expressed as mean ± SEM (n = 3) and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest (*, P < 0.05 compared to BALB-D2S cells). The dashed line indicates the basal ratio of MAPK (set at 1.0). BALB-D2S cells, parent cell line; BDo-14, BALB-D2S cells expressing Go-PTX; BDi1-11, expressing Gi1-PTX; BDi2-22, expressing Gi2-PTX; BDi3-3, expressing Gi3-PTX.

To examine the role of Gβγ subunits of Gi/o proteins, D2S-induced MAPK activation was tested in the BDD− clone (Fig. 7). In BDD− cells, the D2S receptor-induced activation of MAPK was altered to a 25 to 50% reduction of the basal level of phospho-p42/44 MAPK upon apomorphine addition. These results indicate a major role for Gβγ in apomorphine-induced MAPK activation. In the absence of Gβγ activation, D2S receptor activation inhibits MAPK activity, perhaps utilizing Gαi1 or Gαi3 subunits. Based on these results, Gαi2 and Gβγ both play an important roles in D2S-induced activation of p42/44 MAPK in BALB-D2S cells.

FIG. 7.

FIG. 7

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D2S-induced MAPK activation in BALB-D2S cells expressing GRK-CT protein. MAPK activation was measured in BALB-D2S cells (wild type) and BALB-D2S cells stably transfected with His-GRK-CT protein (BDD−). Cells were treated with or without 1 μM apomorphine for 10 min at 37°C. D2S-induced MAPK activation was calculated as percent basal level, and data are expressed as mean ± SEM (n = 2). The dashed line indicates the basal (control) level of MAPK in each cell line, set at 100%. The Western blots at the top shows detection of active p44/42 in BDD− cells with anti-phospho-MAPK antibody. The cell lysate was prepared from BDD− cell incubated in serum-free medium (Control), 1 μM apomorphine (Apo), minimal-serum medium (FBS1%), or 1 μM apomorphine in minimal serum medium (FBS/Apo) and subjected to SDS-PAGE as described in Materials and Methods.

Gαi2 and Gβγ mediate D2S-induced DNA synthesis.

As an indicator of DNA synthesis, [3H]thymidine incorporation into acid-precipitable material was measured. A minimal amount of FBS (1%) was required to preserve D2 responsiveness in BALB-D2S cells over the 16-h time course. No response to apomorphine was observed in nontransfected BALB/c 3T3 cells. In BALB-D2S cells, activation of the D2S receptor by apomorphine augmented thymidine incorporation by 60% (58.7% ± 16.0%), which was blocked by PTX pretreatment, indicating the role of Gi/o protein in mediating D2S receptor action (Fig. 8A). PTX pretreatment attenuated thymidine incorporation in BALB-D2S cells given no drug treatment. However, the PTX sensitivity of DNA synthesis in control conditions was not altered in any of the clones compared to BALB-D2S cells (data not shown). All BALB-D2S clones stably expressing individual PTX-resistant Gα mutants responded to apomorphine with a 30 to 50% increase in thymidine incorporation except Gi1-PTX-expressing clones, which were not examined further (Fig. 8B). However, after PTX pretreatment, only the Gi2-PTX clones (BDi2-6 and BDi2-22) retained D2S-induced increase in thymidine incorporation, indicating a important role for Gαi2 protein in mediating this effect. D2S-induced thymidine incorporation was completely abolished in BDD− cells, suggesting a major role for Gβγ in mediating this action (Fig. 9). In another clone expressing a lower level of GRK-CT, the D2S effect was partially (30%) blocked (data not shown). These results demonstrate that D2S receptor activation induces DNA synthesis in BALB-D2S cells and that this effect is mediated through both Gαi2 and Gβγ subunits.

FIG. 8.

FIG. 8

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D2S-induced DNA synthesis in BALB-D2S cells expressing a PTX-insensitive mutant of Gαi/o. (A) BALB-D2S cells were pretreated with or without PTX (10 ng/ml, 4 h) in the absence (Control) or presence of 1 μM apomorphine (Apo), and thymidine incorporation was measured as described in Materials and Methods. The data represent the mean ± SEM of three independent experiments (n = 3) and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest (*, P < 0.05 compared to control). (B) Apomorphine-induced increase in DNA synthesis in BALB-D2S cells expressing mutant Gαi/o with or without PTX pretreatment. Percent increase in DNA synthesis was calculated as 100(D − C)/(S − C), where D is [3H]thymidine incorporation in apomorphine-treated cells, C is the basal level of [3H]thymidine incorporation in serum-free medium, and S is [3H]thymidine incorporation in minimal serum with no drug treatment. The data are expressed as mean ± SEM of three independent experiments and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest (*, P < 0.05, PTX-treated compared to no PTX treatment; n/s, not significant). BALB-D2S cells, parent cell line; BDo-14, BALB-D2S cells expressing Go-PTX; BDi1-11, expressing Gi1-PTX; BDi2-6 and BDi2-22, expressing Gi2-PTX; BDi3-3, expressing Gi3-PTX.

FIG. 9.

FIG. 9

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D2S-induced DNA synthesis in BALB-D2S cells expressing GRK-CT protein. DNA synthesis was measured in BALB-D2S cells and BALB-D2S cells stably transfected with His-GRK-CT protein (BDD−). Cells were incubated with or without 1 μM apomorphine in minimal-serum medium, and thymidine incorporation was determined. [3H]thymidine incorporation for each condition was measured in triplicate, and the data are expressed as mean ± SEM of two independent experiments (n = 2).

Gαi3 mediates D2S-induced focus formation in BALB-D2S cells.

The role of D2S receptor signaling in cellular transformation was examined. In BALB-D2S cells but not nontransfected BALB/c 3T3 cells (not shown), persistent activation of D2S receptor by apomorphine induced cellular transformation that was manifest as an increase in focus formation (Fig. 10A), implicating an oncogenic role for the D2S receptor in these nontransformed cells. The extent of apomorphine-induced focus formation was comparable to that for thrombin and was completely blocked with PTX treatment, indicating the role of Gi/o proteins (Fig. 10A). By contrast, the low rate of spontaneous transformation of BALB-D2S cells was not affected by PTX treatment. In BALB-D2S cells stably expressing mutant Gαi/o subtypes, only the clone expressing Gi3-PTX displayed robust focus formation upon D2S receptor activation in the presence of PTX, indicating the involvement of Gαi3 in this process (Fig. 10B). In the Go-PTX clone, apomorphine-induced focus formation was almost completely blocked, suggesting that the Gαo subunit may inhibit D2S-induced cell transformation. The Gi1-PTX-expressing clones displayed a constitutively transformed phenotype and were not responsive to dopamine agonists or PTX treatment (data not shown). The transfected Gi1-PTX protein may couple to other receptors that are activated by serum components (e.g., insulin-like or fibroblast growth factors [30, 39]) to induce cellular transformation. Thus, unlike other actions of the D2S receptor, cellular transformation appears to be selectively mediated by Gi3.

FIG. 10.

FIG. 10

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Apomorphine induces focus formation in BALB-D2S cells. (A) BALB-D2S cells were treated with apomorphine (1 μM), thrombin (1 U/ml), or apomorphine (1 μM) and PTX (1 ng/ml, added every 2 days) (Apo/PTX). The foci were counted as indicated in Materials and Methods. The data represent the results from four independent experiments (n = 4). (B) Apomorphine stimulation of focus formation in BALB-D2S cells expressing PTX-insensitive Gαi/o mutants. The results are presented as fold increase over the basal the for each clone. The data are expressed as mean ± SEM of at least three independent experiments and were analyzed by repeated-measures analysis of variance with Bonferroni multiple comparison posttest (∗, P < 0.01, PTX treated compared to no PTX treatment; n/s, not significant). BALB-D2S cells, parent cell line; BDo-14, BALB-D2S cells expressing Go-PTX; BDi2-22, expressing Gi2-PTX; BDi3-3, expressing Gi3-PTX.

DISCUSSION

The D2S receptor has been characterized as growth inhibitory in the pituitary (21, 45) but has been found to stimulate proliferation of various mesenchymal cells, including BALB/c 3T3 cells as shown here. PTX-insensitive G protein mutants were used to distinguish and compare the G protein specificities of signaling pathways (inhibition of AC, calcium mobilization, and MAPK activation) and cell proliferative actions (DNA synthesis and foci formation) of the D2S receptor in BALB/c-D2S cells.

D2S-mediated inhibition of AC.

Inhibitory regulation of AC appears to be a ubiquitous pathway of Gi/o-coupled receptors, including the D2S receptor (6, 10, 17). In BALB-D2S cells, inhibition of forskolin-stimulated cAMP accumulation by D2S receptor activation was rescued by PTX-insensitive Gαi2 or Gαi3 but not Gαo, consistent with previous studies utilizing antisense or PTX-insensitive G proteins. Transfection of GRK-CT, which inhibited dopamine-induced calcium mobilization, did not alter inhibition of cAMP levels, indicating that the latter action of the D2S receptor does not require mobilization of Gβγ subunits as observed in other cell types. The importance of Gαi2 in D2S-induced inhibition of forskolin-stimulated cAMP formation has been implicated in other cell types. Although Gαi2 is also implicated in D2S-mediated MAPK activation and DNA synthesis in BALB-D2S cells, the lack of the effect of D2S receptor activation on basal cAMP levels suggests that this pathway is not involved in D2S-induced actions on cell growth. This finding is consistent with the report by Alblas et al., who found that α2-adrenergic receptor-induced MAPK activation is mediated by Gi proteins and is independent of AC inhibition by the same receptor (5).

D2S-induced calcium mobilization.

One signaling pathway that is initiated by the D2S receptor in fibroblast cells, but not in pituitary cells, is a PTX-sensitive stimulation of [Ca2+]i (1, 4, 16). Hence, this pathway could be involved in D2S-mediated stimulation of cell growth that occurs only in fibroblast cells. None of the PTX-insensitive G protein mutants rescued dopamine-induced calcium mobilization after PTX treatment, suggesting that Gαi/o subunits play a minor or secondary role in this pathway. On the other hand, expression of GRK-CT in BALB-D2S cells inhibited D2S-induced calcium mobilization, indicating a predominant role for Gβγ subunits, as observed in Ltk− cells (16). These results are consistent with the fact that Gβγ subunits of Gi/o proteins can activate phospholipase C-β2 and -β3 to initiate calcium mobilization (10). It has been estimated that the amount of Gβγ required to activate PLC-β2 in vitro is 10-fold higher than the amount required for Gαi-mediated activation of AC (7). It may be that multiple Gi/o subtypes, rather than a single subtype, must be activated to release sufficient Gβγ subunits to induce calcium mobilization in BALB-D2S cells. The crucial role of Gβγ subunits in D2S-induced calcium mobilization and DNA synthesis suggests that calcium mobilization may contribute in part to D2S-induced cell proliferation. Calcium mobilization leads to activation of calcium-calmodulin-dependent proteins kinases, and diacylglycerol (a product of the phospholipase C reaction) activates protein kinase C, which could potentially activate MAPK or enhance cell proliferation. However, roles of particular Gα subunits in D2S-induced MAPK activation, DNA synthesis, and focus formation suggest that pathways other than calcium mobilization are more important for D2S-induced cell growth.

D2S-induced MAPK activation and DNA synthesis.

In BALB-D2S cells, the D2S receptor induced a rapid activation of p42/44 MAPK (Fig. 5), as observed previously in mesenchymal cells (53) and C6 glioma cells (33). Our results demonstrate that in PTX-insensitive Gα mutants, Gαi2 is crucial for D2S-induced activation of p42/44 MAPK (Fig. 6). In addition, blocking Gβγ signaling by ectopic expression of GRK-CT inhibited D2S-induced activation of endogenous p42/44 MAPK in BALB-D2S cells (Fig. 7). Mobilization of Gβγ subunits has been implicated in Gi/o-mediated activation of p42/44 MAPK in cells transfected with exogenous MAPK (15, 34). Activation of MAPK induced by Gβγ may be mediated by a common receptor tyrosine kinase pathway (32, 51) involving Gβγ-mediated activation of Src-like kinases to activate the Shc-Grb2-Sos pathway, leading to Ras-dependent MAPK activation. Thus, D2S-mediated activation of Gi2, which releases both Gαi2 and Gβγ, specifically contributes to the activation of the MAPK pathway.

Activation of the D2S receptor transfected in BALB/c 3T3 augmented DNA synthesis, as observed for other Gi/o-coupled receptors (1, 26, 43). The D2S-induced stimulation of DNA synthesis was rescued solely by the PTX-insensitive Gαi2 subtype (Fig. 8). LaMorte et al. microinjected anti-Gαi2 antibody to demonstrate that Gαi2 mediates PTX-sensitive thrombin-induced DNA synthesis in BALB/c 3T3 cells (25). This suggests that Gi2 is crucial for stimulation of DNA synthesis by endogenous receptors in addition to the D2S receptor. The expression of GRK-CT in BALB-D2S cells revealed that Gβγ subunits also have a major role in D2S-induced thymidine incorporation in these cells (Fig. 9). Thus, as observed for MAPK activation, both Gαi2 and Gβγ participate in the DNA synthesis induced by D2S receptor activation.

Activation of MAPK has been implicated in a variety of receptor-mediated signaling pathways that mediate cell growth and proliferation (32, 44). Luo et al. have reported that by blocking p42/44 MAPK activation pharmacologically, D2-induced DNA synthesis was blocked in C6 glioma cells (33). Actions of MAPK on cell proliferation and differentiation are postulated to require persistent activation of MAPK (11, 42, 49), which may occur only in the presence of serum. Thus, although other signaling pathways may participate, the shared specificity of D2S-induced MAPK activation and DNA synthesis for Gαi2 and Gβγ suggests that MAPK activation plays an important role in the regulation of DNA synthesis in BALB-D2S cells.

D2S-induced cellular transformation.

In BALB-D2S cells, continuous activation of D2S receptor by apomorphine induced PTX-sensitive focus formation, which was rescued by the PTX-insensitive mutant Gαi3 but not the Gαi2 and Gαo mutants. Expression of the Go-PTX mutant inhibited apomorphine-induced transformation, consistent with a lack of oncogenic function for this G protein. Thus, the antiproliferative action of the D2S receptor (e.g., in pituitary cells [37]) may involve Gαo. In contrast, constitutively active mutants of Gαi2 and Gαo induce transformation when transfected in Rat-1 or NIH 3T3 cells (23, 41). This discrepancy may reflect differences in cell types or between receptor-mediated and mutational activation G proteins.

The specificity of the transformation response for Gαi3 is surprising given that Gi2, but not Gi3, is implicated in D2S-induced MAPK activation and DNA synthesis. This suggests a dissociation between induction of DNA synthesis and transformation. The identity of the signaling pathway induced by Gαi3 that could mediate transformation is not known. Gi3 has been specifically implicated in several processes, including vesicle trafficking (exocytosis and autophagic endocytosis) and activation of potassium channels (40, 52). Alterations in the internalization of cell surface adhesion molecules could initiate the loss of contact inhibition that characterizes focus formation and oncogenic transformation. Alternately, Gi3 may couple to tyrosine phosphatase activation, which is implicated in control of cell proliferation via PTX-sensitive G proteins (54). For example, the tyrosine phosphatase SHP-1 associates specifically with Gi3 and somatostatin receptor to mediate Gi3-selective actions on cell growth (31).

Conclusion.

These findings further indicate the precise signaling of the D2S receptor via specific Gαi/o proteins to control cell proliferation. Each of the clones expressing PTX-insensitive G proteins displayed distinct patterns of D2S-induced actions that were insensitive to PTX treatment. In particular, acute inhibition of basal cAMP and stimulation of calcium mobilization were dispensable for D2S-induced growth modulation (in Gi2- or Gi3-PTX clones), whereas MAPK activation was strongly correlated with D2S-induced DNA synthesis (in Gi2-PTX clones) but not with cellular transformation (Gi3-PTX clones). The Gαi1 clones grew abnormally (13), precluding analysis of D2S-mediated MAPK, DNA synthesis, and transformation. Our results indicate that the MAPK pathway is linked to DNA synthesis but may be separate from cellular transformation since different Gαi subtypes are involved in these effects. Thus, the D2S receptor utilizes different Gi/o protein subunits to regulate a diversity of effector functions within the cell.

ACKNOWLEDGMENTS

We acknowledge the helpful editorial comments of H. Jafar-Nejad.

This research was supported by the National Cancer Institute, Canada, and the Ontario Mental Health Foundation. M.H.G. was supported by the Iranian Ministry of Health and the Schizophrenia Society of Canada; P.R.A. holds the Novartis/MRC Michael Smith Chair in Neurosciences.

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