Received for publication March 8, 2004.
Revised September 3, 2004.
Accepted for publication September 10, 2004.

Using molecular tools to dissect the role of G_s in sensitization of AC1

Abstract

Acute activation of G_i/o-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of G_i/o-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity following subsequent activation by G_s or forskolin. Several indirect observations have suggested the involvement of increased G_s-adenylyl cyclase interactions in the expression of sensitization, however, evidence supporting a direct role for G_s has not been well established. In the present report, we have used two genetic approaches to further examine the role of G_s in heterologous sensitization of Ca²⁺-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed G_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 G_s-insensitive mutants of AC1 was markedly reduced compared to wild-type AC1. In the second strategy, we examined the requirement of an intact receptor-G_s signaling pathway for the expression of sensitization using dominant negative G_s mutants (35 G226A/A366S or 35 G226A/E268A/A366S) to disrupt D₁ dopamine receptor activation of recombinant AC1. D₁ dopamine receptor-G_s signaling was attenuated in the presence of 35 G226A/A366S or 35 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 G_s-adenylyl cyclase interactions.

Key words: Dopamine, Gi family, Gs family, Adenylyl cyclases, cAMP, G protein regulation, Drug tolerance/dependence