Ser/ Thr residues at α3/β5 loop of Gαs are important in morphine-induced adenylyl cyclase sensitization but not mitogen-activated protein kinase phosphorylation

Abstract

The signaling switch of β2-adrenergic and μ(1) -opioid receptors from stimulatory G-protein (G(αs) ) to inhibitory G-protein (G(αi) ) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1/2 activation. Post-translational modifications, including dephosphorylation of G(αs) , enhance opioid receptor coupling to G(αs) . In the present study, we substituted the Ser/Thr residues of G(αs) at the α3/β5 and α4/β6 loops aiming to study the role of G(αs) lacking Ser/Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC(50) = 22.8 ± 3.4 μm) in G(αs) -transfected S49 cyc- cells but not in nontransfected cells. However, there was no significant difference between the G(αs) -wild-type (wt) and mutants. Morphine (10 μm) inhibited AC activity more efficiently in cyc- compared to G(αs) -wt introduced cells (P < 0.05); however, we did not find a notable difference between G(αs) -wt and mutants. Interestingly, G(αs) -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 G(αs) , 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 G(αs) -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 G(αs) interacts with the μ1-opioid receptor, and the Ser/Thr mutation to Ala at the α3/β5 loop of G(αs) enhances morphine-induced AC sensitization. In addition, G(αs) was required for the rapid phosphorylation of ERK1/2 by isoproterenol but not morphine.

Fig. 1

Expression of Gαs, ADRB2, OPRM1 and sequence alignment of Gαs. S49 cyc– cell lysates were prepared and (A) G_αs, (B) OPRM1 and (C) ADRB2 protein expression was analyzed by western blotting; PC12 cell lysates were used as a positive control. (D) Expression of Flag tagged-G_αs in cyc– cells was studied at different times after transient transfection. (E) Protein sequence of the short isoform of G_αs with the point mutations underlined.

Fig. 2

cAMP accumulation in response to Isoproterenol. S49 cyc– cells transfected with G_αs-wt were exposed to different doses of isoproterenol for 45 min (acute) and 24 h (chronic), and then the cAMP concentration was assayed with a competitive ELISA method (A; n = 5). The effects of (B) acute and (C) chronic isoproterenol (10 μM) on the cAMP concentration were studied in cyc– cells transfected with indicated constructs. The data are presented as the ratio to control. *Compared to cyc–; mean ± SD; n = 7.

Fig. 3

cAMP accumulation in response to morphine. S49 cyc– cells transfected with G_αs-wt were exposed to different doses of morphine for 45 min (acute) and 24 h (chronic), and then the cAMP concentration was assayed with a competitive ELISA method (A; n = 5). The effects of (B) acute and (C) chronic morphine (10 μM) on cAMP concentration were studied in cyc– cells transfected with indicated constructs. The data are normalized to Fsk. *Compared to cyc–. #Compared to G_αs-wt; mean ± SD; n = 7.

Fig. 4

Co-immunoprecipitation of G_αs with OPRM1. S49 cyc–, G_αs-wt and mutant transfected cells were treated with morphine (10 μM) for 24 h and pull-down experiments with (A) Flag or (B) OPRM1 antibodies were conducted, and western blotting was used for the detection of protein bands. The ratio of receptor to G-protein (and vice versa) are presented in (C) (mean ± SD; n = 2).

Fig. 5

Time course of isoproterenol-induced MAPK activation. S49 cyc–, G_αs-wt and mutant transfected cells were serum starved for 24 h and then treated with isoproterenol (10 μM), cell lysates were prepared at the indicated times, and phosphorylation of ERK1 ⁄ 2 was studied by western blotting; blots were stripped and reprobed for ERK1 ⁄ 2 (A) and β-actin (not shown). (B) The pERK1 ⁄ 2 band intensities were normalized to corresponding ERK1 ⁄ 2 and β-actin, and are presented as the mean ± SD of the ratio to time 0 (t₀) (n = 2).

Fig. 6

Time course of morphine-induced MAPK activation. S49 cyc–, G_αs-wt and mutant transfected cells were serum starved for 24 h and then treated with morphine (10 μM), cell lysates were prepared at the indicated times, and phosphorylation of ERK1 ⁄ 2 was studied by western blotting; blots were stripped and reprobed for ERK1 ⁄ 2 (A) and β-actin (not shown). (B) The pERK1 ⁄ 2 band intensities were then normalized to corresponding ERK1 ⁄ 2 and β-actin, and are presented as the mean ± SD of the ratio to time 0 (t₀) (n = 2).