부산대학교 도서관

The signaling switch of β2-adrenergic and μ1-opioid receptors from stimulatory G-protein (Gαs) to inhibitory G-protein (Gαi) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1/2 activation. Post-translational modifications, including dephosphorylation of Gαs, enhance opioid receptor coupling to Gαs. In the present study, we substituted the Ser/Thr residues of Gαs at the α3/β5 and α4/β6 loops aiming to study the role of Gαs 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 μ m) in Gαs-transfected S49 cyc− cells but not in nontransfected cells. However, there was no significant difference between the Gαs-wild-type (wt) and mutants. Morphine (10 μ m) inhibited AC activity more efficiently in cyc− compared to Gαs-wt introduced cells ( P < 0.05); however, we did not find a notable difference between Gαs-wt and mutants. Interestingly, Gαs-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); μ1-opioid receptor interacted with Gαs, 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 α3/β5, 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 Gαs-transfected cells; mutations of α3/β5 and α4/β6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that Gαs interacts with the μ1-opioid receptor, and the Ser/Thr mutation to Ala at the α3/β5 loop of Gαs enhances morphine-induced AC sensitization. In addition, Gαs was required for the rapid phosphorylation of ERK1/2 by isoproterenol but not morphine. Structured digital abstract [ABSTRACT FROM AUTHOR]