Dopamine D-2 receptor-induced heterologous sensitization of adenylyl cyclase requires G alpha(s): Characterization G alpha(s)-insensitive mutants of adenylyl cyclase V

Whereas acute stimulation of G alpha (i/o)-coupled receptors inhibits the a ctivity 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 G alpha (s)-coupled receptors. G alpha (s)-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization re quires Ga.-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D-2 L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimula ted cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D-2 receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three G alpha (s)-insensitive mutants (F379L, R1021Q, and F109 3S) in HEK293 cells stably expressing the D-2L receptor demonstrated that a ll three mutants had little or no responsiveness to beta -adrenergic recept or-mediated activation of Ga. but that the mutants retained sensitivity to forskolin and to D-2L receptor-mediated inhibition. Transiently expressed a denylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpiro le. In contrast, the G alpha (s)-insensitive mutants displayed no sensitiza tion of forskolin-stimulated cyclic AMP accumulation, indicating that respo nsiveness to G alpha (s) is required for the expression of heterologous sen sitization.