Selective activation of Galphao by D2L dopamine receptors in NS20Y neuroblastoma cells

Abstract

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.

Fig. 1.

Specificity of D_2L receptor-mediated inhibition of cAMP accumulation. cAMP accumulation was stimulated by forskolin (10 μm) in NS20Y-D_2L cells in the absence or presence of quinpirole (1 μm) or dopamine (1 μm) for 15 min. As indicated in the figure, some experiments were performed in the presence of 1 μmspiperone (+ Spip) or 1 μm SCH23390 (+ SCH). Data shown are the mean ± SE of three to four independent experiments, each assayed in duplicate. *p < 0.01 compared with vehicle-treated cells (Dunnett’s post-repeated measures ANOVA).

Fig. 2.

Potency of quinpirole for inhibition and sensitization of forskolin-stimulated cAMP accumulation. Each point is the average of duplicate determinations, expressed as a percentage of forskolin-stimulated activity in the absence of quinpirole (•,Inhibition) or after 2 hr pretreatment with 10 μm quinpirole (○, Sensitization). Dose–response curves for quinpirole inhibition of cAMP accumulation were determined in NS20Y-D_2L cells stimulated with 10 μm forskolin and increasing concentrations of quinpirole for 15 min. For sensitization, NS20Y-D_2L cells were treated with increasing concentrations of quinpirole for 2 hr and washed, and cAMP accumulation was stimulated with 10 μm forskolin. The experiments shown are representative of three independent experiments. In the inhibition curve shown, the maximal inhibition was 88% and the IC₅₀ value was 3.3 nm. Forskolin stimulation in the absence of quinpirole was 135 pmol/well. The EC₅₀ value for sensitization was 18 nm; forskolin-stimulated cAMP accumulation was 130 pmol/well in vehicle-treated cells and 320 pmol/well in cells treated with 10 μm quinpirole.

Fig. 3.

D₂ agonist-induced heterologous sensitization. NS20Y-D_2L cells were treated with vehicle, quinpirole (1 μm), or dopamine (1 μm) for 2 hr at 37°C. Where indicated, some agonist treatments were completed in the presence of 1 μm spiperone (+ Spip) or after overnight treatment with 50 ng/ml pertussis toxin (+ PTX). Cells were extensively washed, and cAMP accumulation was stimulated with forskolin (10 μm) for 15 min. Data shown are the mean ± SE of four to six independent experiments, each assayed in duplicate. *p < 0.01 compared with vehicle-treated cells (Dunnett’s post-repeated measures ANOVA).

Fig. 4.

Expression of HSV-Gα_i1*, -Gα_i2*, -Gα_i3*, and -Gα_o* in NS20Y-D_2L cell membranes. Lane 1 of each gel was loaded with 50 μg of control NS20Y-D_2L cell membranes from an enriched membrane preparation (EP) as described in Materials and Methods. Lanes 2 and 3were loaded with 10 μg of cell membranes from a standard membrane preparation (StdP) of control and HSV-Gα_i1*, -Gα_i2*-, -Gα_i3*-, or -Gα_o*-infected cells (18 hr), and Western analysis was completed using corresponding α subunit specific antibodies. The data shown are from a single experiment representative of three independent experiments.

Fig. 5.

Gα subunit specificity for inhibition of cAMP accumulation in NS20Y-D_2L cells. Quinpirole-induced inhibition of forskolin-stimulated cAMP accumulation was examined in cells infected with PTX-resistant HSV-Gα subunits. NS20Y-D_2L cells were untreated (Control), infected with HSV-LacZ, or infected with individual PTX-resistant Gα subunits for 18 hr. cAMP accumulation was stimulated with forskolin (10 μm) in the presence or absence of quinpirole (10 μm) for 15 min. Where indicated, cAMP accumulation was assessed after 6 hr treatment with 100 ng/ml pertussis toxin (PTX). Data shown are the mean ± SE of four or more independent experiments, assayed in duplicate. **p < 0.01, *p < 0.05 compared with forskolin alone (Student’s t test).

Fig. 6.

Gα subunit specificity for heterologous sensitization in NS20Y-D_2L cells. Quinpirole-induced sensitization of forskolin-stimulated cAMP accumulation was examined in cells infected with PTX-resistant HSV-Gα subunits. NS20Y-D_2L cells were untreated (Control), infected with HSV-LacZ, or infected with individual PTX-resistant Gα subunits for 18 hr. NS20Y-D_2L cells were incubated in the presence or absence of quinpirole (10 μm) for 2 hr and extensively washed, and cAMP accumulation was stimulated with forskolin (10 μm) for 15 min. Some experiments were completed after pertussis toxin (PTX) treatment. Cells were initially treated with PTX (500 ng/ml) for 2 hr, followed by a 2 hr incubation with quinpirole in the presence of PTX (250 ng/ml). Data shown are the mean ± SE of five to seven independent experiments, assayed in duplicate. **p < 0.01 compared with matched vehicle-treated cells (Student’s t test).