Using molecular tools to dissect the role of Galphas in sensitization of AC1.

Short-term activation of Galpha(i/o)-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Galpha(i/o)-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by Galpha(s) or forskolin. Several indirect observations have suggested the involvement of increased Galpha(s)-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for Galpha(s) has not been well established. In the present report, we used two genetic approaches to further examine the role of Galpha(s) in heterologous sensitization of Ca(2+)-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed Galpha(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 Ca(2+)- and forskolin-stimulated AC1 activity; however, sensitization of Galpha(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-Galpha(s) signaling pathway for the expression of sensitization using dominant-negative Galpha(s) mutants (alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S) to disrupt D1 dopamine receptor activation of recombinant AC1. D1 dopamine receptor-Galpha(s) signaling was attenuated in the presence of alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S, but D2 agonist-induced sensitization of Ca(2+)-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves Galpha(s)-adenylyl cyclase interactions.