Dopamine D₂ receptor-induced heterologous sensitization of adenylyl cyclase requires Gα_s: Characterization of Gα_s-insensitive mutants of adenylyl cyclase V

Whereas acute stimulation of Gα_i/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Gα_s-coupled receptors. Gα_s-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Gα_s-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D_2L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D₂ receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Gα_s-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D_2L receptor demonstrated that all three mutants had little or no responsiveness to β-adrenergic receptor-mediated activation of Gα_s but that the mutants retained sensitivity to forskolin and to D_2L receptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Gα_s-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Gα_s is required for the expression of heterologous sensitization.