Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: Changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation

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 in creased AC activity (especially evident on removal of the inhibitory agonis t), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also lead s to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a ti me-dependent reversal of the cholate insolubility. With Chinese hamster ova ry and COS cells transfected with several G(i/o)-coupled receptors (i.e., m u- and kappa-opioid, and m(4)-muscarinic), we observed that although no ove rall change occurred in total content of G(alpha i)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% ch olate to solubilize G(beta gamma) as well as G(alpha i). This solubility sh ift is exclusively observed with G(alpha i), and was not seen with G(alpha s). The disappearance and reappearance of G(alpha i) and G(beta gamma) subu nits from and to the detergent-soluble fractions occur with similar time co urses as observed for the onset and disappearance of AC superactivation. La stly, pertussis toxin, which blocks acute and chronic agonist-induced AC in hibition and superactivation, also blocks the shift in detergent solubility . These results suggest a correlation between the solubility shift of the h eterotrimeric G(i) protein and the generation of AC superactivation.