INTRODUCTION

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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 G and G subunits, which couple to various effectors in cell membrane (6, 38). The G_i and G_o proteins (collectively referred to as G_i/o proteins) couple negatively to adenylyl cyclase (AC), resulting in inhibition of cyclic AMP (cAMP) production. Pertussis toxin (PTX) selectively ADP-ribosylates the G_i/G_o subunits to block all actions of G_i/o proteins. The G 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 G_i/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 G_i/o proteins mediate enhancement of mitogen-activated kinase (MAPK) activity, DNA synthesis, and cell proliferation (1, 26, 43). Furthermore, a G_i-regulated role in mitosis has been identified in fibroblast cells (12). Moreover, expression of constitutively active mutants of G_i2 induces transformation in Rat-1 fibroblasts (18, 41), and mutationally activated G_i2 has been identified in human adrenal and ovarian tumors (35). In addition, the G 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 G_i/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 G subunits to D2S-mediated signaling was evaluated using PTX-insensitive mutants of G_i1, G_i2, G_i3, and G_o, in which the carboxyl-terminal ribosyl acceptor cysteine was changed to a nonaccepting serine. The CysSer 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 G subunits in D2S signaling has been investigated using the GRK-CT protein (carboxyl terminus of G protein-coupled receptor kinase) as a selective G scavenger (22). In BALB/c 3T3 cells transfected with D2S receptor, the D2S receptor utilized distinct G_i subunits to inhibit cAMP accumulation and specific G_i and G 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 G_i subunits but was blocked by inhibiting G function. These results indicate a strong G protein subunit specificity in D2S receptor-induced cell growth.

	MATERIALS AND METHODS

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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), [-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 G_o, G_i1, G_i2, and G_i3 were generously provided by Randall Reed, Johns Hopkins University, Baltimore, Md. The anti-G_o antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.); anti-G_i1-2 and anti-G_i3 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 G_i/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 G_i/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 G_i/o mutants were generated using rat cDNAs (20) encoding G_o, G_i1, G_i2, and G_i3 subunits as previously described (16). Briefly, the cysteine 351 codon (352 for G_i2), 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 _ex = 340/380 nm and _em = 510 nm. Calibration was done with 0.1% Triton X-100 and 20 mM Tris base to determine R_max and 10 mM EGTA (pH > 8) to obtain R_min, and the fluorescence ratio was converted to intracellular Ca²⁺ concentration ([Ca²⁺]_i) based on a K_d 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

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Expression of mutant G_i/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 (G_o-, G_i1-, G_i2-, and G_i3-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 G_o, G_i1 at apparently greater abundance than G_i2, and G_i3 at low levels, based on densitometric scanning. Comparing G_o and G_i1 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 G_i/o subunits (Fig. 1). Thus, approximately equal amounts of mutant and wild-type proteins were produced in the transfected cell lines.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Gi/o expression in BALB-D2S cells transfected with PTX-insensitive Gi/o mutants. Cell extracts (100 µg) of BALB-D2S cells (wild type) and BALB-D2S cells expressing Go-PTX (BDo-14) (A), Gi1-PTX (BDi-11) (B), Gi2-PTX (BDi2-6, BDi2-22) (C), and Gi3-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-Go (A), anti-Gi1-2 (B and C), and anti-Gi3 (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.

G_i2 and G_i3 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 G_i/o proteins.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   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.

View this table: [in this window] [in a new window]   TABLE 2.   Effect of D2S receptor on cAMP production in BDD cellsa

G_i/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 G_i/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 G subunit is involved. To test the role of G 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 G 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 G subunits than on particular G_i/o subunits as observed in Ltk cells (16).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   PTX blocks D2S-induced calcium mobilization in BALB-D2S cells expressing PTX-insensitive Gi/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.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   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 × 10⁷ and 6.3 × 10⁸ 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 G_i/o proteins (Fig. 5A).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   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).

G_i2 and G 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 G_o and G_i2 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 G_i/o proteins, only the G_i2-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 G_i2-PTX for p42 MAPK activation, but p44 MAPK activation was only partially recovered in two independent clones. Unlike clones expressing G_o-PTX or G_i2-PTX, in clones expressing G_i1-PTX or G_i3-PTX apomorphine failed to induce MAPK activation, suggesting that these subunits may antagonize or occlude D2S receptor signaling to MAPK.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   D2S-induced MAPK activation in BALB-D2S cells expressing PTX-insensitive Gi/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.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   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.

G_i2 and G 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 G_i/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 G mutants responded to apomorphine with a 30 to 50% increase in thymidine incorporation except G_i1-PTX-expressing clones, which were not examined further (Fig. 8B). However, after PTX pretreatment, only the G_i2-PTX clones (BDi2-6 and BDi2-22) retained D2S-induced increase in thymidine incorporation, indicating a important role for G_i2 protein in mediating this effect. D2S-induced thymidine incorporation was completely abolished in BDD cells, suggesting a major role for G 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 G_i2 and G subunits.

View larger version (28K): [in this window] [in a new window]   FIG. 8.   D2S-induced DNA synthesis in BALB-D2S cells expressing a PTX-insensitive mutant of Gi/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 Gi/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.

View larger version (29K): [in this window] [in a new window]   FIG. 9.   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).

G_i3 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 G_i/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 G_i/o subtypes, only the clone expressing G_i3-PTX displayed robust focus formation upon D2S receptor activation in the presence of PTX, indicating the involvement of G_i3 in this process (Fig. 10B). In the G_o-PTX clone, apomorphine-induced focus formation was almost completely blocked, suggesting that the G_o subunit may inhibit D2S-induced cell transformation. The G_i1-PTX-expressing clones displayed a constitutively transformed phenotype and were not responsive to dopamine agonists or PTX treatment (data not shown). The transfected G_i1-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 G_i3.

View larger version (26K): [in this window] [in a new window]   FIG. 10.   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 Gi/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

Top Abstract Introduction Materials and Methods Results Discussion References

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 G_i/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 G_i2 or G_i3 but not G_o, 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 G subunits as observed in other cell types. The importance of G_i2 in D2S-induced inhibition of forskolin-stimulated cAMP formation has been implicated in other cell types. Although G_i2 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 2-adrenergic receptor-induced MAPK activation is mediated by G_i 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 G_i/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 G subunits, as observed in Ltk cells (16). These results are consistent with the fact that G subunits of G_i/o proteins can activate phospholipase C-2 and -3 to initiate calcium mobilization (10). It has been estimated that the amount of G required to activate PLC-2 in vitro is 10-fold higher than the amount required for G_i-mediated activation of AC (7). It may be that multiple G_i/o subtypes, rather than a single subtype, must be activated to release sufficient G subunits to induce calcium mobilization in BALB-D2S cells. The crucial role of G 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 G 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 G mutants, G_i2 is crucial for D2S-induced activation of p42/44 MAPK (Fig. 6). In addition, blocking G signaling by ectopic expression of GRK-CT inhibited D2S-induced activation of endogenous p42/44 MAPK in BALB-D2S cells (Fig. 7). Mobilization of G subunits has been implicated in G_i/o-mediated activation of p42/44 MAPK in cells transfected with exogenous MAPK (15, 34). Activation of MAPK induced by G may be mediated by a common receptor tyrosine kinase pathway (32, 51) involving G-mediated activation of Src-like kinases to activate the Shc-Grb2-Sos pathway, leading to Ras-dependent MAPK activation. Thus, D2S-mediated activation of G_i2, which releases both G_i2 and G, specifically contributes to the activation of the MAPK pathway.

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 G_i3 but not the G_i2 and G_o mutants. Expression of the G_o-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 G_o. In contrast, constitutively active mutants of G_i2 and G_o 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.

Conclusion. These findings further indicate the precise signaling of the D2S receptor via specific G_i/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 G_i2- or G_i3-PTX clones), whereas MAPK activation was strongly correlated with D2S-induced DNA synthesis (in G_i2-PTX clones) but not with cellular transformation (G_i3-PTX clones). The G_i1 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 G_i subtypes are involved in these effects. Thus, the D2S receptor utilizes different G_i/o protein subunits to regulate a diversity of effector functions within the cell.