Abstract

Short-term activation of Gα_i/o-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Gα_i/o-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by Gα_s or forskolin. Several indirect observations have suggested the involvement of increased Gα_s-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for Gα_s has not been well established. In the present report, we used two genetic approaches to further examine the role of Gα_s in heterologous sensitization of Ca²⁺-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed Gα_s-insensitive mutants of AC1 (F293L and Y973S) that retained sensitivity to Ca²⁺ and forskolin activation. Persistent (2 h) activation of the D₂ dopamine receptor resulted in a significant augmentation of basal or Ca²⁺- and forskolin-stimulated AC1 activity; however, sensitization of Gα_s-insensitive mutants of AC1 was markedly reduced compared with wild-type AC1. In the second strategy, we examined the requirement of an intact receptor-Gα_s signaling pathway for the expression of sensitization using dominant-negative Gα_s mutants (α3β5 G226A/A366S or α3β5 G226A/E268A/A366S) to disrupt D₁ dopamine receptor activation of recombinant AC1. D₁ dopamine receptor-Gα_s signaling was attenuated in the presence of α3β5 G226A/A366S or α3β5 G226A/E268A/A366S, but D₂ agonist-induced sensitization of Ca²⁺-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves Gα_s-adenylyl cyclase interactions.