Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

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 G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma 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 (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.

FIG. 1

Gα_i/o expression in BALB-D2S cells transfected with PTX-insensitive Gα_i/o mutants. Cell extracts (100 μg) of BALB-D2S cells (wild type) and BALB-D2S cells expressing Gα_o-PTX (BDo-14) (A), Gα_i1-PTX (BDi-11) (B), Gα_i2-PTX (BDi2-6, BDi2-22) (C), and Gα_i3-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-Gα_o (A), anti-Gα_i1-2 (B and C), and anti-Gα_i3 (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.

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 G_o-PTX; BDi2-6 and BDi2-22, cells expressing G_i2-PTX; BDi3-3 and BDi3-7, cells expressing G_i3-PTX.

FIG. 3

PTX blocks D2S-induced calcium mobilization in BALB-D2S cells expressing PTX-insensitive Gα_i/o-PTX mutants. BALB-D2S cells (A) and BALB-D2S cells expressing G_o-PTX (BDo-14) (B), G_i1-PTX (BDi1-11) (C), G_i2-PTX (BDi2-6) (D), and G_i3-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

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.

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).

FIG. 6

D2S-induced MAPK activation in BALB-D2S cells expressing PTX-insensitive Gα_i/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 G_o-PTX; BDi1-11, expressing G_i1-PTX; BDi2-22, expressing G_i2-PTX; BDi3-3, expressing G_i3-PTX.

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.

FIG. 8

D2S-induced DNA synthesis in BALB-D2S cells expressing a PTX-insensitive mutant of Gα_i/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 Gα_i/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 G_o-PTX; BDi1-11, expressing G_i1-PTX; BDi2-6 and BDi2-22, expressing G_i2-PTX; BDi3-3, expressing G_i3-PTX.

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).

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 Gα_i/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 G_o-PTX; BDi2-22, expressing G_i2-PTX; BDi3-3, expressing G_i3-PTX.