Selective Activation of Gαoby D2LDopamine Receptors in NS20Y Neuroblastoma Cells

D2Ldopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2Ldopamine 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 D2Lreceptors with the selective D2agonist 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-D2Lcells. 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 (Gαi1*, Gαi2*, Gαi3*, and Gαo*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPγS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Gα coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein α subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2Lcells with HSV-Gαo* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Gαi1*, HSV-Gαi2*, or HSV-Gαi3* failed to rescue either response. In summary, the current study provides strong evidence that the D2Ldopamine receptor couples to Gαoin neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2receptor activation on adenylate cyclase activity.