Ser/Thr residues at a3/beta 5 loop of Gas are important in morphine-induced adenylyl cyclase sensitization but not mitogen-activated protein kinase phosphorylation
The signaling switch of beta 2-adrenergic and mu 1-opioid receptors from stimulatory G-protein (Gas) to inhibitory G-protein (Gai) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1/2 activation. Post-translational modifications, including dephosphorylation of Gas, enhance opioid receptor coupling to Gas. In the present study, we substituted the Ser/Thr residues of Gas at the a3/beta 5 and a4/beta 6 loops aiming to study the role of Gas lacking Ser/Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC50 = 22.8 +/- 3.4 mu m) in Gas-transfected S49 cyc- cells but not in nontransfected cells. However, there was no significant difference between the Gas-wild-type (wt) and mutants. Morphine (10 mu m) inhibited AC activity more efficiently in cyc- compared to Gas-wt introduced cells (P < 0.05); however, we did not find a notable difference between Gas-wt and mutants. Interestingly, Gas-wt transfected cells showed more sensitization with respect to AC after chronic morphine compared to nontransfected cells (101 +/- 12% versus 34 +/- 6%; P < 0.001); mu 1-opioid receptor interacted with Gas, and both co-immunoprecipitated after chronic morphine exposure. Furthermore, mutation of T270A and S272A (P < 0.01), as well as T270A, S272A and S261A (P < 0.05), in a3/beta 5, resulted in a higher level of AC supersensitization. ERK1/2 phosphorylation was rapidly induced by isoproterenol (by 9.5 +/- 2.4-fold) and morphine (22 +/- 2.2-fold) in Gas-transfected cells; mutations of a3/beta 5 and a4/beta 6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that Gas interacts with the mu 1-opioid receptor, and the Ser/Thr mutation to Ala at the a3/beta 5 loop of Gas enhances morphine-induced AC sensitization. In addition, Gas was required for the rapid phosphorylation of ERK1/2 by isoproterenol but not morphine.