Premium
Ser/ Thr residues at α3/β5 loop of Gαs are important in morphine‐induced adenylyl cyclase sensitization but not mitogen‐activated protein kinase phosphorylation
Author(s) -
Seyedabadi Mohammad,
Ostad Seyed Nasser,
Albert Paul R.,
Dehpour Ahmad R.,
Rahimian Reza,
GhaziKhansari Mahmoud,
Ghahremani Mohammad H.
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08459.x
Subject(s) - adenylyl cyclase , protein kinase a , phosphorylation , chemistry , sensitization , dephosphorylation , kinase , mapk/erk pathway , microbiology and biotechnology , receptor , opioid , gs alpha subunit , medicine , pharmacology , endocrinology , biology , biochemistry , immunology , phosphatase
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 μ m ) 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 μ m ) 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. Structured digital abstract• oprm1 physically interacts with Gsalphas by antibait coimmunoprecipitation ( View interaction ) • Gsalphas physically interacts with oprm1 by antitag coimmunoprecipitation ( View interaction )