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

Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, New York

Received for publication March 19, 2007.

Accepted for publication June 15, 2007.

	Abstract

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The recent biochemical demonstration of the association of the µ-opioid receptor (MOR) with G_s that increases after long-term 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.

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, G₁₁ 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 immune-complexed pp60c-src enhances rates of -adrenergic receptor-mediated binding of guanosine 5'-O-(2-[³⁵S]thio)triphosphate (GTPS) as well as receptor-stimulated steady-state rate of GTP hydrolysis by G_s (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 ₁₁₂ dimer regulates its activity in an effector-specific fashion (Yasuda et al., 1998). Threonine-phosphorylated G 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 G_s 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 opioid-tolerant tissue. Furthermore, our data suggest that increased association of G_s (G_s) with protein phosphatase 2A (PP2A) is functionally linked to the long-term morphine treatment-induced reduction in G_s phosphorylation.

	Materials and Methods

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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 µg/ml G-418 (Geneticin; Mediatech, Herndon, VA). The neuroblastoma x 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 µ-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.

Morphine Treatment Cell Culture. Cells were plated (4 x 10⁶ cells/150 mm dishes) and grown at 37°C in a humidified atmosphere of 90% air/10% CO₂ for MOR-CHO and 95% air/5% CO₂ for F11 cells. Two days later, at 90 to 95% confluence, cells were treated with vehicle or morphine (1 µM) for 48 h. Media containing morphine or vehicle was replenished every 24 h.

Spinal Cord. Experiments employed male Sprague-Dawley rats (250–300 g; Charles River Laboratories, Kingston, NY) that were maintained in an approved controlled environment on a 12-h light/dark cycle. Food and water were available ad libitum. Studies were carried out in accordance with the Guide for the Care and Use of laboratory Animals as adopted and promulgated by the National Institutes of Health. All experimental procedures were reviewed and approved by the Animal Care and Use Committee of SUNY Downstate Medical Center.

Morphine pellets were supplied by the National Institute on Drug Abuse, Bethesda, MD). Morphine tolerance was induced by subcutaneous implantation of one morphine pellets on day 1, two pellets on day 3, and three pellets on day 5 (each containing 75 mg of morphine base) (Bhargava and Villar, 1991) under sodium pentobarbital anesthesia (40 mg/kg, i.p., Anpro Pharmaceutical, Arcadia, CA). On the day 7 after pellet implantation, rats were decapitated, spinal cords were quickly expelled and washed extensively in Krebs buffer (4°C), and membranes were prepared.

³²P_i Labeling of MOR-CHO Cells On the day of harvest, cells were incubated for 2 h in phosphate- and serum-free DMEM at 37°C under normal culture conditions. Later, MOR-CHO cells were washed once with 10 ml of phosphate- and serum-free media and incubated with 10 ml of the same media containing [³²P]orthophosphate (100 µCi/ml; PerkinElmer Life and Analytical Sciences, Waltham, MA) for additional 2 h at 37°C under 90% air/10% CO₂.

Membrane Preparation and Immunoprecipitation Cells were washed thoroughly (twice, 15 ml each) with ice-cold phosphate-buffered saline, pH 7.3, and harvested directly in 20 mM HEPES, pH 7.4, containing 10% sucrose, 5 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol (DTT), 10 mM sodium pyrophosphate, 10 mM NaF; protease inhibitors 1 mM benzamidine, 0.2 mg/ml bacitracin, and 2 mg/l aprotinin; 3.2 mg/l each of trypsin inhibitor from soybean and leupeptin, 20 mg/l each of N-tosyl-L-phenyl-alanine chloromethyl ketone, Na-p-tosyl-L-lysine chloromethyl ketone, and phenylmethylsulfonyl fluoride, and complete cocktail inhibitor tablet/50 ml. Calyculin A, a protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) inhibitor was added after ³²P labeling before the onset of membrane preparation. Cells were homogenized and centrifuged at 1000g, 4°C for 10 min. Spinal tissue was homogenized in HEPES buffer of identical composition. Supernatants obtained from the low-speed spin were subjected to a high-speed spin at 30,000g for 40 min at 4°C.

Membrane fractions obtained were re-suspended in HEPES buffer, pH 7.4, containing 1 mM each EDTA, EGTA, and DTT, 10 mM sodium pyrophosphate, and the same protease and phosphatase inhibitors as mentioned above. Membranes were either stored at –80°C in aliquots or processed further. For immunoprecipitation, membranes were solubilized in the same buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10% glycerol, agitated for 60 min at 4°C and centrifuged (14,000g for 20 min at 4°C). Clear supernatants were used for Protein Assay (Bradford) and immunoprecipitation. G_s was immunoprecipitated from solubilized membrane using a rabbit anti-G_s (bovine) polyclonal affinity purified antibody generated against the C terminus of the G_s subunit (aa 385–394; US Biologicals, Swampscott, MA; 1 µl/100 µg protein). Prewashed Protein A-agarose (50 µl; Roche Molecular Biologicals, Indianapolis, IN) was used for immunoprecipitation overnight at 4°C. The beads were washed in 20 mM HEPES buffer, pH 7.4, containing 1 mM each DTT and EDTA, 150 mM NaCl, 0.05% Nonidet P-40, and the same protease inhibitors as mentioned above. Immunoprecipitates were eluted by heating samples in 30 µl of sample buffer (15 min at 85°C). Samples separated on 4–12% gradient Bis-Tris gels (Invitrogen) were electro-transferred onto nitrocellulose membranes and used for Western analyses or were exposed to Phosphorimager screens that were scanned in Phosphorimager Storm 860 (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). ³²P_i incorporated into phosphorylated samples was determined using densitometric analysis (Imagequant; GE Healthcare).

Western Analysis MOR protein was visualized using a 1:5000 dilution of a rabbit polyclonal antibody (affinity-purified) generated against the C-terminal 50 aa of MOR (generously provided by Dr. Thomas Cote, Uniformed Services University of the Health Sciences, Bethesda, MD). G_s, PP2A, and phosphothreonine proteins were visualized by using a 1:10,000 dilution of a polyclonal anti-G_s antibody (generously provided by Dr. J. Hildebrandt, Medical University of South Carolina, Charleston, SC), a 1:2000 dilution of an anti-PP2A monoclonal antibody (Upstate/Millipore, Charlottesville, VA), and a 1:1000 dilution of a polyclonal anti-phosphothreonine antibody (Invitrogen), respectively. The secondary antibody used was either a peroxidase-labeled donkey anti-rabbit or a peroxidase-labeled sheep anti-mouse antibody from GE Healthcare. Antibody-substrate complex was visualized using a Supersignal West Dura Chemiluminescence detection kit (Pierce, Rockford, IL). Specificity of Western signals was demonstrated via their diminution/elimination after incubation with antibodies while in the presence of a 3- to 5-fold excess of their respective blocking peptides. Specificity of G_s immunoprecipitation was demonstrated via the diminution of MOR Western signal when G_s immunoprecipitation was performed in the presence of 10 µg of the G_s immunogen (aa 385–394 blocking peptide; Calbiochem, San Diego, CA). Sample pairs, obtained from opioidnaive and long-term morphine-treated MOR-CHO cultures were processed, electrophoresed, and blotted in parallel, after which they were exposed concomitantly to GeneGnome (CCD camera; Syngene, Frederick, MD) or to a Kodak X-Omat film (Denville Scientific, Metuchen, NJ). Intensity of signal was quantified using Syngene software in GeneGnome or NIH imaging software from films.

Stoichiometry of G_s Phosphorylation Stoichiometry of ³²P incorporation into G_s by protein kinase C (PKC) was assessed after incubation of 160 ng of recombinant purified G_s (rG_s; purified from Escherichia coli; generously provided by Dr. W-J Tang, University of Chicago, Chicago, IL) with catalytic subunits of PKC (PKCcat; purified from bovine brain; 20 mU/reaction; obtained from Calbiochem) and [-³²P]ATP (2.5 µCi/reaction; obtained from PerkinElmer Life and Analytical Sciences). The phosphorylation reaction was carried out in a 50-µl reaction mixture containing 20 mM HEPES, pH 7.6, 10 mM MgCl₂, 1 mM CaCl₂, 0.25% bovine serum albumin (BSA), 1 mM DTT, 40 mg/liter leupeptin, 40 mg/liter phenylmethylsulfonyl fluoride, and the phosphatase inhibitors 0.1 mM sodium vanadate, 0.5 µM okadaic acid, 25 nM calyculin A, and 100 µM ATP. The reaction was initiated by addition of PKCcat (as recommended by the manufacturer, Calbiochem, San Diego, CA), and incubated at 30°C for 2 h. The reaction was terminated by heating samples at 85°C for 8 min. Phosphorylated G_s was resolved by gel electrophoresis (4–12% Bis-Tris gel), visualized by autoradiography using PhosphorImager analysis, and quantified by liquid scintillation spectroscopy. To assess whether G_s exists as a phosphoprotein, the stoichiometry of G_s phosphorylation was compared with that obtained using rG_s that had been phosphatase-treated (see below).

In Vitro Dephosphorylation of Purified G_s and Immunoprecipitation Purified recombinant G_s was dephosphorylated using commercially available PP1 and PP2A. Phosphatase reaction was initiated using 2 units each of purified PP1 and PP2A (from PP1/PP2A Toolbox kit; Upstate/Millipore) with 1 µg rG_s in phosphatase reaction buffer (Upstate) at 30°C for 1h. Calyculin A (25 nM) was added to terminate the activity of the phosphatases. To assess the effect of dephosphorylating rG_s on its association with MOR, phosphatase-treated and untreated rG_s was incubated with solubilized membranes from opioid-naive MOR-CHO cells, and the content of MOR in G_s IP obtained from each sample was determined and compared.

Preparation of AC2 Transfected MOR-CHO Cell Membranes and Determination of AC Activity Forty-eight hours after transient transfection of AC2 in MOR-CHO cells, membranes were prepared as described above. Membrane pellets were resuspended in the homogenizing buffer without sucrose and stored in aliquots at –80°C for future use. G_s stimulation of AC activity was determined in AC2-MOR-CHO membranes in the presence of rG_s with or without dephosphorylation as described previously (Chakrabarti et al., 1998a). In brief, after the termination of dephosphorylation (or mock) reaction, rG_s was activated by incubation (1 h at 30°C) with 100 µM GTPS in 50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA, 1 mM DTT, 5 mM MgSO₄, and 1.25 mg/ml bovine serum albumin as described previously (Tang and Gilman, 1991). The activated rG_s-GTPS was separated from free GTPS using gel filtration (Sephadex G-25 spin columns). AC activity was determined by measuring the synthesis of [-³²P]cAMP from [-³²P]ATP (MP Biomedicals, Irvine, CA). Assays were initiated by the addition of the reaction mixture (50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl₂, 20 mM creatine phosphate, 10 units/sample creatine phosphokinase, 0.1 µM ATP, 10 µM GTP, 20 mM NaCl, 1 mM DTT, 1 mM EGTA, 0.125 µM rolipram, 0.1% bovine serum albumin, and [-³²P]ATP; 1 µCi/sample) to cell membranes (5 µg) with prior incubation (30°C, 15 min) with activated rG_s. Reactions (30°C, 15 min) were terminated by the addition of 10 µl of 2.2 N HCl (4°C). Thereafter, [³²P]cAMP generated was separated by neutral alumina column chromatography as described previously (Alvarez and Daniels, 1990) and quantified using liquid scintillation spectroscopy.

Protein Phosphatase 2A Assay PP2A activity was measured by immunoprecipitation phosphatase Assay kit per the manufacturer's instructions (Upstate/Millipore,). PP2A was first immunoprecipitated from membranes as described above. Afterward the enzyme was used on phosphopeptide substrate to release phosphate molecules, which were measured colorimetrically and estimated from standard phosphate curves. It is important to note that all buffers and chemicals used for this procedure should be free of any contaminating phosphates.

Statistical Analysis Significance of differences in the magnitude of autoradiographic and Western signals was assessed using paired two-tailed Student's t test. A repeated-measures ANOVA using a general linear mixed model was used to assess the effect of G_s dephosphorylation on its ability to stimulate AC activity.

	Results

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G_s Was Endogenously Expressed As a Phosphoprotein. G_s was immunoprecipitated from ³²P_i-metabolically labeled opioid-naive MOR-CHO cell membranes and subjected to sequential autoradiographic and Western analyses. Three radiolabeled bands of 45, 48, and 52 kDa were observed (Fig. 1A, lane 1). It is noteworthy that when the same sample was subjected to sequential autoradiographic and Western analyses, two of the three radiolabeled bands (45- and 48-kDa signals) coincided with signals observed in Westerns blotted with anti-G_s antibodies (Fig. 1A, compare lanes 1 and 2 versus lanes 4/5 and 6/7). These observations in combination with the abolition/reduction of the Western signal when blotting was performed in the presence of a 3- to 5-fold excess of G_s blocking peptide (Fig. 1A, lanes 8 and 9 indicates the ³²P-radiolabeled bands to be G_s or its splice variants. The 52-kDa radiolabeled band (comparable in molecular mass to G_s "long"), which was much weaker than the 45- and 48-kDa signals, did not have a corresponding Western signal, suggesting that its protein content is extremely low, below the detection limits of the Western analysis employed.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Gs was dephosphorylated after long-term morphine treatment. A, lanes 1, 2, and 3, autoradiographic analysis of Gs IP obtained, in parallel, from opioid-naive and long-term morphine-treated MOR-CHO cells in the absence and presence of calyculin A, respectively. MOR-CHO cells were labeled with 32Pi, and membranes were prepared as described under Materials and Methods.Gs immunoprecipitation was initiated using 400 µgof solubilized, radiolabeled MOR-CHO cell membranes and anti-Gs antibodies (4 µl/400 µg). The involvement of PP1/PP2A in the long-term morphine treatment-induced decrement in Gs phosphorylation is underscored by its abolition by calyculin A (lane 3). The Gs identity of the 48- and 45-kDa radiolabeled bands was validated by demonstrating their coincidence with Western signals when the nitrocellulose membranes used for autoradiographic analyses was probed with anti-Gs antibodies (lanes 4 and 5) or cell membranes obtained from MOR-CHO were subjected to Western blots using anti-Gs antibodies (lanes 6 and 7). The absence of a Gs Western signal that corresponds to the 52-kDa radiolabeled band most likely reflects its low protein content. Specificity of the Gs IP and Western is indicated by the absence of signal when the immunoprecipitation (lane 8) or Western analysis (lane 9) is performed in the presence of 3- to 5-fold excess antigen. B, phosphothreonine Western analysis of Gs IP from spinal cord membranes of opioid naive (lane 1) and long-term morphine treatment treated rats (lane 2). In lanes 3 and 4, the same immunoblot was reprobed with anti-Gs antibodies after stripping off anti-phosphothreonine antibodies. Gs phosphorylation decreased after long-term morphine treatment, which cannot be attributed to a decrement in the Gs content of the Gs IP. Results represent replicates of three observations.

G_s Phosphorylation Decreased after Long-Term Morphine Treatment. G_s IP was obtained in parallel from membranes of opioid-naive and long-term morphine-treated MOR-CHO cells that had been metabolically labeled with ³²P_i. Sequential autoradiographic and Western analyses of G_s IP obtained from membranes of long-term morphine-treated cells revealed the phosphorylation of 45-, 48-, and 52-kDa molecular mass forms of G_s, as was observed in G_s IP obtained from membranes of opioid-naive MOR-CHO (Fig. 1A, lanes 1 and 2). However, densitometric analyses of radiolabeled bands revealed that long-term morphine treatment decreased ³²P incorporation into the predominant 45-kDa band by 47 ± 11% (Fig. 1A, lane 2 versus 1; n = 3; p < 0.05). ³²P incorporation into the 48- and 52-kDa minor molecular mass forms of G_s was similarly decreased (40 and 59%, respectively; Fig. 1A, lane 2 versus 1; n = 2). It is noteworthy that sequential G_s Western analyses revealed that the efficiency of G_s immunoprecipitation was not altered by long-term morphine treatment (Fig. 1A, compare lanes 5 and 4). Thus, long-term morphine treatment results in the net decrease in G_s phosphorylation. It is noteworthy that the decrement in G_s phosphorylation after long-term morphine treatment is obliterated by the addition of calyculin A (25 nM) during the last 30 min of the ³²P radiolabeling period (Fig. 1A, lane 3 versus 2; n = 3) suggesting the preeminent importance of PP1/PP2A to the tolerant-associated decrement in G_s phosphorylation.

The effect of chronic systemic morphine on the phosphorylation of G_s that had been immunoprecipitated from spinal tissue was assessed via Western analyses using anti-phosphothreonine antibodies (Fig. 1B, lanes 1 and 2). One major band of 45 kDa was observed, the G_s identity of which was confirmed by stripping and reprobing with anti-G_s antibodies (Fig. 1B, lanes 3 and 4). It should be noted that although G_s Western analysis revealed the expected 45- and 48-kDa G_s species (Fig. 1B, lanes 3 and 4), only the 45-kDa molecular mass species appeared to be threonine-phosphorylated. Long-term systemic morphine administration in rats diminished spinal G_s phosphorylation (27 ± 5.8%; Fig. 1B, lane 2 versus 1; n = 3; p < 0.05). This indicates that reduction of G_s phosphorylation after long-term morphine treatment exposure generalizes to complex integrated neuronal systems and is not a cell culture phenomenon. The reduced magnitude of the long-term morphine treatment-induced decrement in G_s phosphorylation in spinal cord versus MOR-CHO (27 versus 47%, respectively) could suggest a reduction in phosphorylation at sites in G_s in addition to threonine.

Long-Term Morphine Treatment Enhanced Association of PP2A with G_s. A central role of PP2A in the decrement in G_s phosphorylation after long-term morphine treatment is suggested by 1) the observation that PP2A pretreatment of rG_s results in an increment in its in vitro phosphorylation that was 80% of that observed when using PP2A/PP1, and 2) the ability of calyculin A to abolish the decrement in G_s phosphorylation after long-term morphine treatment. Thus, we explored the putative physiological relevance of PP2A to the in vivo phosphorylation state of G_s and its modulation by long-term morphine treatment. This was investigated by assessing their association in vivo by quantifying the presence of PP2A in IP obtained with anti-G_s antibodies. PP2A Western analysis of G_s IP obtained from spinal cord, F11, and MOR-CHO cells, without or after long-term morphine treatment, revealed a single band of 36 kDa (Fig. 2). After long-term morphine treatment, there was a significant increase in the content of PP2A in G_s IP obtained from all three sources (spinal cord, 109%; F11 cells, 57%; and MOR-CHO, 118%). It is noteworthy that the long-term morphine treatment-induced increased coimmunoprecipitation of PP2A with G_s occurred in the absence of any detectable increase in the membrane content of PP2A (Fig. 2, top, lanes 7 and 8,) or G_s (Fig. 2, bottom). Thus, long-term morphine treatment results in a net increment in the interaction between PP2A and G_s.

View larger version (18K): [in this window] [in a new window]   Fig. 2. PP2A coimmunoprecipitated with Gs and their association increased after long-term morphine treatment. Gs IP was performed as mentioned under Materials and Methods. Top, Western blot of PP2A in Gs IP, obtained in parallel from membranes of opioid-naive and long-term morphine-treated rat spinal cord, F11, and MOR-CHO cells, respectively (lanes 1 through 6). Lanes 7 and 8 represent PP2A Western of membranes obtained from naive and long-term morphine-treated MOR-CHO cells (5 µg, respectively). Chronic morphine induced an increase in the PP2A that coimmunoprecipitated with Gs (lanes 1–6). Bottom, Gs Western analysis of the same blot after stripping and reprobing with anti-Gs antibodies. The long-term morphine treatment-induced increment in coimmunoprecipitation of PP2A with Gs occurred in the absence of increased content of Gs.

View larger version (10K): [in this window] [in a new window]   Fig. 3. PP2A activity increased in MOR-CHO cells after long-term morphine treatment. A, bar diagram denotes increased PP2A activity measured in PP2A IP from long-term morphine treated versus untreated MOR-CHO cell membranes (bar 2 versus 1). Details of PP2A activity assay are mentioned under Materials and Methods. In brief, anti-PP2A monoclonal antibody was used to IP PP2A, the activity of which was quantified using a commercially available phosphopeptide substrate. Phosphates liberated were measured colorimetrically (650 nm) using Malachite green. Results are expressed as percentage of control (picomoles of phosphate per sample). *, p < 0.05 for long-term morphine treatment versus naive; n = 4. B, Western analysis illustrating that the increased activity of PP2A after long-term morphine treatment is not accompanied by an increase in the MOR-CHO membrane content of PP2A or the efficiency of PP2A immunoprecipitation.

View larger version (9K): [in this window] [in a new window]   Fig. 4. In vitro dephosphorylation of Gs enhanced its association with MOR. Western analysis of Gs IP obtained from MOR-CHO membranes after incubation with purified recombinant Gs that had been either dephosphorylated in vitro using protein PP1 and 2A or mock-dephosphorylated (PP+ and PP–, respectively). The content of MOR in the Gs IP was quantified by Western analyses using anti-MOR antibodies. The Gs Western blot of the Gs IP, shown below the break in the gel, illustrates that the augmented coimmunoprecipitation of MOR with Gs after its pretreatment with protein phosphatases did not result from differential loading. Bar graphs shown below illustrate that the content of MOR (80 kDa) in Gs IP was substantially increased after incubation of MOR-CHO membranes with in vitro dephosphorylated versus mock-dephosphorylated rGs. * p < 0.05; n = 3.

Effect of G_s Dephosphorylation on Its Ability to Stimulate AC2 Activity. Because AC phosphorylation state has been shown to be a critical determinant of the stimulatory responsiveness of some AC isoforms to G_s (Jacobowitz and Iyengar, 1994; Watson et al., 1994), we determined whether the phosphorylation state of G_s would similarly influence its ability to stimulate AC. rG_s was either dephosphorylated with PP2A or mock-dephosphorylated, after which their ability to stimulate AC2 was determined and compared. As shown in Table 1, in vitro dephosphorylated rG_s was more potent than mock-dephosphorylated rG_s in stimulating cAMP production by AC2. The increment in cAMP production by 20, 40, and 60 nM dephosphorylated rG_s, although modest in magnitude, reached significance (p < 0.05).

View this table: [in this window] [in a new window]   TABLE 1 Effect of Gs dephosphorylation on its ability to stimulate AC2 activity cAMP formation represents the differences in activity of AC2 when stimulated by mock (vehicle-treated) dephosphorylated versus dephosphorylated (via PP2A + PP1) rGs. Analysis of variance and Tukey's post hoc test were used to assess significance of difference from zero for each indicated change in picomoles of cAMP formed.

	Discussion

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This study demonstrates that the G_s subunit of G proteins exists as a phosphorylated protein. Data supporting this conclusion consists of 1) coincidence of radiolabeled and Western signals after sequential autoradiographic and G_s Western analysis of the same sample that been metabolically labeled with ³²P_i, 2) -fold increase in stoichiometry of rG_s phosphorylation after phosphatase treatment, and 3) demonstration that (spinal cord) G_s is immunoreactive with anti-phosphothreonine antibodies. The observed ³²P metabolic labeling of G_s and its in vitro phosphorylation by PKCcat is consonant with the presence of multiple phosphorylation sites scattered throughout G_s (e.g., seven serine, eight threonine, and three tyrosine; predicted by NetPhos 2.0; Net-Phosk 1.0 predictions include 10 PKC/protein kinase A sites of >0.5 probability, of which three have a probability of >0.70).

The second salient finding is that long-term morphine treatment decreases the stoichiometry of G_s phosphorylation concomitant with its increased association with PP2A. This inference of their causal association is validated by the demonstration that the long-term morphine treatment-induced decrement in G_s phosphorylation is abolished by short-term (30 min) pretreatment with calyculin A, an inhibitor of PP1/PP2A (data not shown). The third notable finding is that in vitro dephosphorylation of G_s increases its association with MOR. It is noteworthy that augmented phosphorylation of G_s (on tyrosine by pp60c-src) potentiates signaling via the -adrenergic receptor (Hausdorff et al., 1992), which normally predominantly couples to G_s to stimulate AC, whereas a diminution in G_s phosphorylation augments its association with MOR, which normally predominantly couples to G_i/G_o to inhibit AC activity. More extensive analysis will be required to assess if inverse consequences of G_s phosphorylation generalizes to other "stimulatory" versus "inhibitory" receptors.

It is noteworthy that demonstration of findings in MOR-CHO and F11 cells as well as spinal cord indicate 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 strongly suggest that the phosphorylation state of G_s is a critical determinant of its interaction with MOR, which is regulated (decreased) by long-term morphine treatment. We previously demonstrated that long-term morphine treatment increases the association of MOR with G_s (Chakrabarti et al., 2005a). The present results identify decreased G_s phosphorylation as a mechanism central to this change in MOR-G_s protein coupling.

Regulation of protein phosphorylation has long been recognized to be a key mechanism underlying opioid tolerance formation. Heretofore, most of the attention has been focused on augmented protein phosphorylation, predominantly via enhanced activity of protein kinase A/PKC pathways (Guitart and Nestler, 1989; Nestler, 1992; Zhang et al., 1996). More recently, we have demonstrated the causal association of tolerance formation and increased phosphorylation of multiple signaling proteins. These include the G subunit of G proteins, phospholipase C3 and AC (Chakrabarti et al., 1998b, 2001; Chakrabarti and Gintzler, 2003a,b). A reduction in phosphorylation of phospholipase C1 (Chakrabarti and Gintzler, 2003b) and mitogen-activated protein kinase (Schulz and Hollt, 1998) has been demonstrated after long-term morphine treatment and its withdrawal. However, so far, with a few notable exceptions, opioid tolerance has been associated mostly with enhancement in phosphorylation of multiple signaling protein(s). The current demonstration that long-term morphine treatment decreases phosphorylation of G_s that in turn augments its association with MOR represents a novel dimension of adaptation to long-term morphine treatment.

Increased interaction of MOR with G_s after long-term morphine treatment would act in parallel with and complement signaling consequences of adaptations to long-term morphine treatment we previously identified [i.e., increased availability of G (Chakrabarti et al., 2001), increased phosphorylation of G (Chakrabarti et al., 2001; Chakrabarti and Gintzler, 2003a), augmented AC isoform-specific synthesis and phosphorylation (Chakrabarti et al., 1998a,8b; Rivera and Gintzler, 1998)]. These converge to shift MOR-coupled signaling from predominantly G_i-inhibitory to G AC stimulatory that would mitigate the persistent opioid inhibition of AC(s) via the opioid receptor-coupled generation of G_i (for review, see Gintzler and Chakrabarti, 2006).

The tolerance-associated emergence of MOR-coupled G stimulatory AC signaling does not require a shift in G protein coupling. Nevertheless, it is well known that the presence of activated G_s is also essential for a substantial component of G stimulation of AC (Tang and Gilman, 1991). In vivo, activated G_s could be generated via the ongoing activation of a multitude of G_s-coupled receptors, independent of opioid receptor function. Nonetheless, the present report underscores that direct coupling of MOR to G_s represents an additional source of activated G_s; concomitant opioid receptor signaling via G_s as well as G_i/G_o would result in the coordinate generation of activated G_s (from G_s) and G (from G_s as well as G_i//G_o). Thus, increased MOR-coupled generation of activated G_s during morphine tolerance would be functionally coordinated with the previously described concomitant emergence of opioid receptor-coupled AC stimulatory G signaling. In addition, enhanced MOR-G_s coupling (the effects of which would be amplified by the modestly more AC stimulatory activity of dephosphorylated G_s; see Table 1) represent a parallel pathway for shifting opioid receptor signaling from predominantly inhibitory to stimulatory; direct stimulation of AC by MOR-coupled generation of activated G_s would be additive with that resulting from the action of G, and would thus further contribute to the neutralization of the predominant inhibitory G_i/G_o-coupled opioid receptor signaling (i.e., opioid tolerance formation).

Coimmunoprecipitation of proteins demonstrates their presence in stable high-affinity complexes. However, difficulties in distinguishing between a naturally occurring complex versus those that may form in vitro during lysate preparation and incubation can confound interpretation of results. In the present study, this concern is mitigated by the observation that the phosphorylation state of the G_s that coprecipitates with increasing amounts of PP2A diminishes after long-term morphine treatment and can be blocked by calyculin A. This strongly suggests an increased functional association between G_s and PP2A at the time of ³²P labeling, preceding lysate incubation. A priori, one would not expect a phosphatase to remain associated with its substrate after catalysis of its dephosphorylation. The coimmunoprecipitation of PP2A and dephosphorylated G_s demonstrated in the present study can most easily be explained by postulating the presence of a third as-yet-unidentified protein/lipid that serves as an anchor for both PP2A and G_s.

The observed increased association of PP2A with G_s after long-term morphine treatment most likely results from the translocation of one or both because their membrane content does not change after this treatment. We previously demonstrated that a macromolecular signaling complex containing PKC, G, and AC underlies, in part, the previously reported shift from predominantly G_i-inhibitory to G-stimulatory AC signaling (Chakrabarti et al., 2005b) and that long-term morphine treatment induces the concomitant phosphorylation of G protein-coupled receptor kinase 2/3, -arrestin, and G, signaling molecules that exist in a multimolecular complex, with attendant modulation of their association (Chakrabarti et al., 2001). The present study underscores the contribution of the concomitant modulation of multiple membrane microsignaling domains by long-term morphine treatment to opioid tolerance formation and that PP2A should be considered as a scaffolding molecule that facilitates the interaction between MOR and G_s, in addition to its enzymatic function.

It is important to note that tolerant-producing mechanisms demonstrated in this study are not mutually exclusive. Numerous adaptations to long-term morphine treatment have been observed and postulated to be causally associated with opioid tolerance. These include opioid receptor down-regulation/internalization (Chavkin and Goldstein, 1984; Chakrabarti et al., 1995; Cox and Crowder, 2004), MOR G protein uncoupling (Sim et al., 1996), and AC superactivation. Altered association/activity of regulators of G-protein signaling (Zachariou et al., 2003; Xu et al., 2004; Garzon et al., 2005; Xie and Palmer, 2005) has also been suggested to be a tolerance-producing adaptation. In general, studies designed to assess the relative contribution to opioid tolerance of the many proposed tolerant mechanisms are woefully lacking. Future studies will be required to parse the relative importance to opioid tolerance of the adaptations demonstrated in this study, the mechanisms suggested by the literature, and how they might intersect. This process should be facilitated by understanding tolerance within the context of physiological plasticity and the realization that opioid tolerance is the result of the combined effect of the loss of specific opioid receptor-coupled signaling sequelae as well as the emergence of novel signaling strategies.

	Footnotes

 
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.107.036145.

ABBREVIATIONS: MOR, µ-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; GTPS, guanosine 5'-O-(2-[³⁵S]thio)triphosphate; P_i, inorganic phosphate.

Address correspondence to: Dr. Alan Gintzler, Box 8, Department of Biochemistry, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203. E-mail: alan.gintzler{at}downstate.edu

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