Abstract

Prolonged G_i/o protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G_i-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G_i/o-coupled receptors (i.e., μ- and κ-opioid, and m₄-muscarinic), we observed that although no overall change occurred in total content of G_αi- and β₁-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G_βγ as well as G_αi. This solubility shift is exclusively observed with G_αi, and was not seen with G_αs. The disappearance and reappearance of G_αi and G_βγ subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G_i protein and the generation of AC superactivation.