Phosphorylation of G s Influences Its Association with the -Opioid Receptor and Is Modulated by Long-Term Morphine Exposure

The recent biochemical demonstration of the association of the -opioid receptor (MOR) with G s that increases after longterm morphine treatment (Mol Brain Res 135:217–224, 2005) provides a new imperative for studying MOR-G s interactions and the mechanisms that modulate it. A persisting challenge is to elucidate those neurochemical parameters modulated by long-term morphine treatment that facilitate MOR-G s association. This study demonstrates that 1) G s exists as a phosphoprotein, 2) the stoichiometry of G s phosphorylation decreases after long-term morphine treatment, and 3) in vitro dephosphorylation of G s increases its association with MOR. Furthermore, our data suggest that increased association of G s with protein phosphatase 2A is functionally linked to the long-term morphine treatment-induced reduction in G s phosphorylation. These findings are observed in MOR-Chinese hamster ovary and F11 cells as well as spinal cord, indicating that they are not idiosyncratic to the particular cell line used or a “culture ” phenomenon and generalize to complex neural tissue. Taken together, these results indicate that the phosphorylation state of G s is a critical determinant of its interaction with MOR. Long-term morphine treatment decreases G s phosphorylation, which is a key mechanism underlying the previously demonstrated increased association of MOR and G s in opioid tolerant tissue. The coupling of -opioid receptors (MOR) to Gs has long been controversial. The ability of MOR to signal via Gs, while persistently suggested by pharmacological experiments (Xu et al., 1989; Shen and Crain, 1990; Gintzler and Xu, 1991; Cruciani et al., 1993; Wang and Gintzler, 1997; Szucs et al., 2004), has met with considerable skepticism and has not been incorporated into commonly accepted models of shortand long-term opioid actions. This has largely resulted from the inability to demonstrate the physical association of MOR and G s in vivo. Our recent report that MOR is present in G s immunoprecipitate (IP), which increases after long-term morphine treatment (Chakrabarti et al., 2005a), provides a new imperative for studying MOR-G s interactions and mechanisms that modulate it. A remaining challenge is to identify the parameter(s) modulated by long-term morphine treatment and causally linked to the observed increased MOR-G s association during the tolerant condition. Many biochemical parameters of receptors and G proteins could influence their functional interactions, of which phosphorylation has received much attention. Phosphorylation of G protein subunits has been shown to play a major role in adaptive changes in receptor signaling, altering their signaling patterns. Phosphorylation of G i suppresses the hormonal inhibition of adenylyl cyclase (AC) in human platelet membranes (Katada et al., 1985) and -opioid receptor mediated inhibition of AC activity in NG108-15 cells (Strassheim and Malbon, 1994). Recently, G 11 protein phosphorylation has been demonstrated to contribute to diminishing 5-HT2A receptor signaling (Shi et al., 2007). In addition, tyrosine phosphorylation of purified recombinant G s by immunecomplexed pp60c-src enhances rates of -adrenergic receptor-mediated binding of guanosine 5 -O-(2-[S]thio)triphosphate (GTP S) as well as receptor-stimulated steady-state rate of GTP hydrolysis by Gs (Hausdorff et al., 1992). Phosphorylation of G protein and subunits has also been shown to be an important parameter of G protein signaling via G . Protein kinase C phosphorylation of 12 in the 1 12 dimer regulates its activity in an effector-specific fashion (Yasuda et al., 1998). Threonine-phosphorylated G Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.036145. ABBREVIATIONS: MOR, -opioid receptor; IP, immunoprecipitate; AC, adenylyl cyclase; CHO, Chinese hamster ovary; MOR-CHO, Chinese hamster ovary cells stably transfected with MOR; PP2A, protein phosphatase 2A; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; aa, amino acids; rG s, recombinant purified G s; PKC, protein kinase C; GTP S, guanosine 5 -O-(2-[S]thio)triphosphate; Pi, inorganic phosphate. 0026-895X/07/7203-753–760$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 3 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 36145/3247037 Mol Pharmacol 72:753–760, 2007 Printed in U.S.A. 753 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from has been demonstrated in spinal cord (Chakrabarti and Gintzler, 2003a), and histidine-phosphorylated G has been demonstrated in membranes of bovine retinae (Wieland et al., 1991), liver, and brain and in human placental tissue (Nurnberg et al., 1996). It is noteworthy that long-term morphine treatment augments phosphorylation of G in guinea pig longitudinal muscle myenteric plexus tissue (Chakrabarti et al., 2001), rat spinal cord (Chakrabarti and Gintzler, 2003a) and Chinese hamster ovary (CHO) cells stably transfected with MOR (MOR-CHO) (Chakrabarti et al., 2005b). Phosphorylation of G has notable consequences on G signaling. It decreases the association of G with G protein receptor kinase (Chakrabarti et al., 2001) (which increases its availability for interaction with effectors, e.g., AC) and increases its potency to stimulate AC2 activity (Chakrabarti and Gintzler, 2003a). The relevance of G protein subunit phosphorylation to G protein receptor-coupled signaling suggests that G s phosphorylation could be a regulatory parameter that is modulated by long-term morphine treatment and a determinant of the association of Gs with MOR. Accordingly, we investigated whether or not G s exists as a phosphoprotein, the modulation of its phosphorylation state by long-term morphine treatment, and the relevance of G s phosphorylation to its association with MOR. The results reveal that long-term morphine treatment decreases the phosphorylation of G s and that this is causally associated with the previously reported increased interaction of G s with MOR in opioidtolerant tissue. Furthermore, our data suggest that increased association of G s (Gs) with protein phosphatase 2A (PP2A) is functionally linked to the long-term morphine treatment-induced reduction in G s phosphorylation. Materials and Methods Cell Culture and Transfection MOR-CHO were maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose with L-glutamine (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Nova-Tech Inc., Grand Island, NE), 100 units/ml penicillin/streptomycin, and 100 g/ml G-418 (Geneticin; Mediatech, Herndon, VA). The neuroblastoma dorsal root ganglia neuron hybrid F11 cell line was generously provided by Dr. Richard Ledeen (University of Medicine and Dentistry of New Jersey, Newark, NJ). These cells endogenously express -opioid receptors and manifest tolerance and dependence in response to long-term opioid treatment (Wu et al., 1995). Monolayer cultures of F11 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. To investigate whether dephosphorylated versus phosphorylated G s changes its stimulation of AC activity, MOR-CHO cells were transiently transfected with AC2 cDNA (AC2-pRC/CMV) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction.