title: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Seyedabadi
authors.forename: Seyed Nasser
authors.initials: SN
authors.lastname: Ostad
authors.forename: Paul R
authors.initials: PR
authors.lastname: Albert
authors.forename: Ahmad R
authors.initials: AR
authors.lastname: Dehpour
authors.forename: Reza
authors.initials: R
authors.lastname: Rahimian
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Ghazi-Khansari
authors.forename: Mohammad H
authors.initials: MH
authors.lastname: Ghahremani
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009020:Morphine / Q000494:pharmacology*; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D000318:Adrenergic beta-Agonists; D000701:Analgesics, Opioid; C404943:Oprm protein, mouse; D018343:Receptors, Adrenergic, beta-2; D017450:Receptors, Opioid, mu; D013912:Threonine; D012694:Serine; D009020:Morphine; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D007545:Isoproterenol