SELECTIVE ACTIVATION OF G-ALPHA(O) BY D-2L DOPAMINE-RECEPTORS IN NS20Y NEUROBLASTOMA

D-2L dopamine receptor activation results in rapid inhibition and dela yed heterologous sensitization of adenylate cyclase in several host ce ll types. The D-2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neur onal cell environment and to identify the particular G-proteins involv ed. Acute activation of D-2L receptors with the selective D-2 agonist quinpirole inhibited forskolin-stimulated cAMP ccumulation, whereas pr olonged incubation (2 hr) with quinpirole resulted in heterologous sen sitization (more than twofold) of forskolin-stimulated cAMP accumulati on in NS20Y-D-2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitizatio n, we used viral-mediated gene delivery to assess the ability of genet ically engineered PTX-resistant G-proteins (G alpha(i1)(star), G alpha (i2)(star), G alpha(i3)(star), and G alpha(0)(star)) to rescue both re sponses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibi tion of GTP gamma S-stimulated adenylate cyclase, respectively. To ass ess the specificity of D-2L-G alpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-res istant G-protein alpha subunits and treated with PTX, and quinpirole-i nduced responses were measured. Infection of NS20Y-D-2L cells with HSV -G alpha(o)(star) rescued both inhibition and sensitization in PTX-tre ated cells, whereas infection with HSV-G alpha(i1)(star), HSV-G alpha( i2)(star), or HSV-G alpha(i3)(star) failed to rescue either response. In summary, the current study provides strong evidence that the D-2L d opamine receptor couples to G alpha(o) in neuronal cells, and that thi s coupling is responsible for both the acute and subacute effects of D -2 receptor activation on adenylate cyclase activity.