Endogenous RGS Protein Action Modulates µ-Opioid Signaling through Galpha o

EFFECTS ON ADENYLYL CYCLASE, EXTRACELLULAR SIGNAL-REGULATED KINASES, AND INTRACELLULAR CALCIUM PATHWAYS*

Mary J. ClarkDagger , Charlotte HarrisonDagger , Huailing ZhongDagger , Richard R. NeubigDagger §, and John R. TraynorDagger

From the Departments of Dagger  Pharmacology and § Internal Medicine/Hypertension, University of Michigan, Ann Arbor, Michigan 48109-0632

Received for publication, August 30, 2002, and in revised form, January 8, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the µ-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha o. We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha o that is either RGS-insensitive or RGS-sensitive. The µ-receptor agonist [D-Ala2,MePhe4,Gly5-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha o. In contrast, µ-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Opioid receptors are a typical seven-transmembrane domain receptor family that signal through inhibitory G proteins to a multitude of second messengers and cellular effectors, including adenylyl cyclase, voltage-operated calcium channels, and inwardly rectifying K+ channels (1); intracellular calcium stores (2); and the extracellular signal-regulated kinase (ERK)1 mitogen-activated protein kinase (MAPK) pathway (3, 4). There are three principal types of opioid receptors, µ, delta , and kappa , with ~60% homology. However, the µ-opioid receptor has generated the most interest as the principal site of action for clinical analgesics and abused opiate drugs. The µ-opioid receptor can couple to all members of the Galpha i/o family, with little selectivity for particular Galpha subunits (5). Selectivity of intracellular µ-opioid signaling would therefore appear to depend on cell-specific expression of G protein subunits coupled with the selectivity of G proteins to interact with particular effectors. However, it has been suggested that other factors besides G protein and effectors may contribute to signaling specificity (6-8).

Agonist activation of G protein-coupled receptors leads to exchange of GDP for GTP on Galpha and dissociation of the Galpha -GTP and Gbeta gamma subunits. Deactivation is brought about by the intrinsic GTPase activity of Galpha , causing GTP to be hydrolyzed to GDP and the subsequent reassociation of the Galpha -GDP and Gbeta gamma subunits. G protein signaling in this fashion is negatively controlled by a family of proteins known as RGS (regulators of G protein signaling) proteins (6). These proteins act as GTPase-activating proteins (GAPs) for Galpha and speed up the hydrolysis of the Galpha -bound GTP, thus reducing the steady-state levels of Galpha -GTP and inhibiting signaling. Therefore, it has been suggested that, as with other G protein-coupled receptors, RGS proteins act to inhibit µ-opioid signaling and may play a controlling role in the effectiveness of opioid receptor ligands. In support of this idea, overexpression of RGS2 shifts the concentration effect curve for morphine-stimulated pigment aggregation to the right to a small (2-fold) degree in cultured dermal melanophores from Xenopus laevis transiently expressing a murine µ-opioid receptor (9). Furthermore, a reduction in RGS9 levels in mice using antisense oligonucleotide leads to an increase in the anti-nociceptive potency of morphine (10). Although these changes are small, they are suggestive of a role for RGS proteins in opioid coupling efficiency.

An important question is whether RGS proteins alter the efficiency of all intracellular signaling pathways equally or whether there is a variable effect that would provide for selectivity. Selectivity for particular pathways may be obtained by several mechanisms. RGS-containing proteins have a wide variety of non-RGS domains (11-13) that, when RGS protein binds to Galpha , can link other proteins and signaling pathways to provide for diversity of signaling. In addition, the interaction of RGS protein with receptors may contribute to selectivity; for example, RGS12 binds to the carboxyl terminus of the interleukin-8 receptor (14), and inhibition of Ca2+ signaling in rat pancreatic acinar cells by RGS4 is selective for muscarinic receptors relative to bombesin and cholecystokinin receptors (15) possibly through interaction of the N-terminal domain of RGS4 with the receptors (16). Recently, Wang et al. (17) demonstrated, using ribozyme technology, that RGS3 is a negative modulator of m3 muscarinic receptor signaling, whereas RGS5 is a negative modulator of angiotensin type 1a receptor signaling through Gq/11.

In addition to RGS proteins selectively modulating the coupling of different receptors to a single effector, it is possible that RGS proteins could selectively modulate the coupling of a single receptor to different effectors. Indeed, we have recently proposed a "kinetic scaffolding" model for G protein signaling in which RGS proteins confer selectivity for signaling pathways by their ability to shorten the lifetime of Galpha -GTP (18). In this model, RGS protein accelerates hydrolysis of the Galpha -bound GTP, permitting recombination of Galpha -GDP and Gbeta gamma and recoupling of the heterotrimer and receptor and allowing rapid reactivation by agonist-bound receptors. This maintains active Galpha -GTP and Gbeta gamma proteins in the close vicinity of the receptor, but spillover of Galpha -GTP and Gbeta gamma to more distant effectors is prevented by the GAP activity of RGS. This effect can be mediated by the RGS domain alone and does not depend on other protein interaction modules.

Here we test the hypothesis that RGS proteins differentially regulate µ-opioid receptor coupling to signaling pathways, thus contributing to selectivity of receptor activation of second messenger pathways. Because 30 mammalian proteins with RGS activity have been identified to date (12, 13), the choice of which RGS protein to study is a difficult one. We have therefore made use of a point mutation in Galpha o (G184S) that is known to block interaction with all members of the RGS family without affecting GTPase activity (RGS-insensitive) (19), together with a mutation (C351G) that confers pertussis toxin (PTX) insensitivity (PTXi) (20). In this way, when the RGS- and PTX-resistant Galpha o mutant (Galpha oRGS/PTXi) is expressed in a cellular system, coupling to endogenously expressed G proteins can be inactivated by PTX treatment, and the system must then signal through the expressed Galpha o mutant (21).

Our findings demonstrate that the µ-opioid agonists DAMGO and morphine showed increased potency and/or efficacy of signaling to adenylyl cyclase in cells expressing RGS-insensitive Galpha o compared with those expressing RGS-sensitive Galpha o. Signaling through the MAPK pathway also showed an increased potency with the full agonist DAMGO, but not an increased maximal effect, although the maximal effect of the partial agonist morphine was significantly enhanced. In contrast, the ability of DAMGO or morphine to stimulate the release of calcium from intracellular stores was altered to a much lesser extent in cells expressing RGS-insensitive Galpha o compared with those expressing RGS-sensitive Galpha o. These results confirm that RGS proteins can modulate effector signaling by a single G protein and may play an important role in directing effector responses to µ-opioid receptor signaling.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [3H]DAMGO, [gamma -32P]GTP, and [35S]GTPgamma S were from PerkinElmer Life Sciences, and cAMP radioimmunoassay kits were from Diagnostic Products Corp. (Los Angeles, CA). Tissue culture medium, LipofectAMINE Plus reagent, Geneticin, Zeocin, fetal bovine serum, and trypsin were from Invitrogen. Morphine and naloxone were obtained through the Opioid Basic Research Center at the University of Michigan (Ann Arbor, MI), and DAMGO was obtained from Sigma. Trizma (Tris base), GDP, ATP, and other biochemicals were purchased from Sigma and were analytical grade. Anti-phospho-p44/42 MAPK (ERK1/2) antibody and anti-p44/42 MAPK (ERK1/2) antibody were from Cell Signaling Technology, Inc. (Beverly, MA); anti-Galpha o antibody (K-20) and secondary antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and SuperSignal West Pico chemiluminescent substrate was from Pierce. PTX-insensitive Galpha o (Galpha oPTXi, C351G) and RGS- and PTX-insensitive Galpha o (Galpha oRGS/PTXi, G184S/C351G) DNAs in the pCI vector were obtained from Dr. Stephen Ikeda (Guthrie Research Institute, Sayre, PA). GST fusion protein containing RGS8 (GST-RGS8) and His6-tagged Galpha o were prepared as previously described (22).

Expression of Galpha oPTXi or Galpha oRGS/PTXi in C6µ Cells and Cell Culture-- Galpha oPTXi or Galpha oRGS/PTXi DNA was excised from the plasmid vector pCI with NotI and NheI restriction enzymes and inserted into the Zeocin resistance vector pcDNA3.1zeo-. Plasmid DNA was transfected into C6 glioma cells stably expressing the rat µ-opioid receptor (C6µ cells) (23) using LipofectAMINE Plus reagent. Colonies were isolated from transfected cells grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum under 5% CO2 in the presence of 0.25 mg/ml Geneticin (to maintain expression of the µ-opioid receptor in a Geneticin-resistant plasmid) and 0.4 mg/ml Zeocin. Clones were maintained under the same conditions and typically subcultured at a ratio of 1:20 to 1:30, with partial replacement of the medium on Day 4 and the day before subculturing or harvesting at Day 7.

Membrane Preparation-- Cells were treated with or without PTX (100 ng/ml) overnight before collection. Cells were washed twice with ice-cold phosphate-buffered saline (0.9% NaCl, 0.61 mM Na2HPO4, and 0.38 mM KH2PO4, pH 7.4), detached from the plates by incubation in harvesting buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, and 0.68 mM EDTA) at room temperature, and pelleted by centrifugation at 200 × g for 3 min. The cell pellet was suspended in ice-cold 50 mM Tris-HCl buffer, pH 7.4, and homogenized with a Tissue Tearor (Biospec Products, Inc.) for 20 s at setting 4. The homogenate was centrifuged at 20,000 × g for 20 min at 4 °C, and the pellet was resuspended in 50 mM Tris-HCl, pH 7.4, with a Tissue Tearor for 10 s at setting 2, followed by recentrifugation. The final pellet was resuspended in 50 mM Tris-HCl, pH 7.4, to 0.5-1.0 mg/ml protein and frozen in aliquots at -80 °C. To determine protein concentration, membrane samples were dissolved with 1 N NaOH for 30 min at room temperature, neutralized with 1 M acetic acid, and assayed by the method of Bradford (42) using bovine serum albumin as the standard.

Determination of Galpha o Expression-- Membrane proteins (20 µg) or His6-Galpha o standards (10-40 ng) were separated by SDS-PAGE on 12% gels (Protogel, National Diagnostics, Inc., Atlanta, GA). Proteins were transferred to a nitrocellulose membrane (45 µm; Osmonics, Inc., Minnetonka, MN), probed with a 1:200 dilution of anti-Galpha o antibody, treated with horseradish peroxidase-conjugated goat anti-rabbit IgG, and visualized by enhanced chemiluminescence. Quantification was done using a Eastman Kodak Image Station 440.

Receptor Binding Assay-- Membranes (10-20 µg) were incubated in 50 mM Tris-HCl, pH 7.4, with 0.2-28 nM [3H]DAMGO with or without 50 µM naloxone (to determine nonspecific binding) in a total volume of 0.2 ml for 60 min in a shaking water bath at 25 °C. Samples were filtered through glass-fiber filters (No. 32; Schleicher & Schüll) mounted in a Brandel cell harvester and rinsed three times with ice-cold 50 mM Tris-HCl, pH 7.4. Radioactivity retained on the filters was counted by liquid scintillation counting in 4 ml of EcoLume scintillation mixture (ICN, Aurora, OH).

[35S]GTPgamma S Binding Assay-- Membranes (14-20 µg) were incubated for 60 min in a shaking water bath at 25 °C with 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 0.1 mM dithiothreitol (DTT; freshly prepared), 30 µM GDP, 0.1 nM [35S]GTPgamma S, and 0.01-10 µM DAMGO, 0.01-10 µM morphine, or dH2O. Samples were filtered through the glass-fiber filters mounted in a Brandel cell harvester and rinsed three times with ice-cold 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 100 mM NaCl. Radioactivity retained was determined as described above. For kinetic studies, membranes (~20 µg) were incubated for 10 min at 25 °C in 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 0.1 mM DTT, and 1 mM EDTA, pH 7.4, containing 30 µM GDP. DAMGO (10 µM) was added, and the mixture was further incubated for 10 min before the addition of 0.1 nM [35S]GTPgamma S to start the reaction. At various times (3 min to 2 h), bound and free radioactivities were separated and quantified as described above.

GTPase Assay-- Membranes (14-20 µg) were prewarmed for 5-20 min at 30 °C with 10 mM Tris, pH 7.6, 2 mM MgCl2, 20 mM NaCl, 0.2 mM EDTA, 0.1 mM DTT (freshly prepared), an ATP-regenerating system (0.2 mM ATP, 0.2 mM AppNHp, 50 units/ml creatine phosphokinase, and 5 mM phosphocreatine), and 0.01-10 µM DAMGO, 10 µM morphine, or dH2O with or without 1 µM GST-RGS8. The reaction was initiated by the addition of 0.1 µM [gamma -32P]GTP (prewarmed to 30 °C) to a final volume of 0.1 ml. The reaction was stopped after 15-120 s by the addition of ice-cold 15% charcoal with 20 mM phosphoric acid in 0.1% gelatin. After at least 30 min on ice, samples were centrifuged at 4000 × g for 20 min at 4 °C, and 0.3 ml was taken from the supernatant for liquid scintillation counting with 4 ml of EcoLume scintillation mixture. Blank values for each time point (without membranes) were subtracted from each value.

Inhibition of cAMP Accumulation-- Cells were plated to confluency in 24-well plates the day before the assay and treated overnight with 100 ng/ml PTX. To start the assay, the cells were rinsed with serum-free medium and then incubated with serum-free medium containing 30 µM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and 0.001-10 µM DAMGO, 0.01-10 µM morphine, or dH2O for 30 min at 37 °C. The reaction was stopped by replacing the medium with ice-cold 3% perchloric acid. After at least 30 min at 4 °C, 0.4 ml was removed from each sample, neutralized with 0.08 ml of 2.5 M KHCO3, vortexed, and centrifuged at 15,000 × g for 1 min. A radioimmunoassay kit was used to quantify accumulated cAMP in a 10-µl aliquot of the supernatant from each sample. Inhibition of cAMP formation was determined as a percentage of forskolin-stimulated cAMP accumulation in the absence of opioid agonist.

Stimulation of p44/42 MAPK Phosphorylation-- Cells were plated in six-well plates the day before the assay to reach 70-90% confluency on the day of the assay and treated overnight with 100 ng/ml PTX. The medium was replaced with serum-free medium for 2 h before the addition of 0.001-10 µM DAMGO, 10 µM morphine, or dH2O. The assay was stopped after 1-20 min by rinsing the cells twice with ice-cold phosphate-buffered saline and adding 0.1 ml of ice-cold SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromphenol blue). Samples were removed from the wells; sonicated for 10-15 s; boiled for 5 min; and then subjected (120 µg) to 12% SDS-PAGE, followed by transfer to 45-µm nitrocellulose membranes for Western blotting. The blot was probed with a 1:2000 dilution of anti-phospho-p44/42 MAPK (ERK1/2) antibody and visualized using horseradish peroxidase-conjugated anti-mouse IgG, followed by enhanced chemiluminescence detection and quantification using the Image Station 440. To assure equal loading, membranes were stripped and reblotted with a 1:1000 dilution of anti-p44/42 MAPK (ERK1/2) antibody to measure total ERK levels.

Release of Intracellular Calcium-- After overnight treatment with 100 ng/ml PTX and 5 µM forskolin, confluent cells were harvested with 10 mM HEPES-buffered 0.9% saline containing 0.05% EDTA, pH 7.4, and washed twice with and then resuspended in Krebs-HEPES buffer of the following composition: 143.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.7 mM glucose, and 10 mM HEPES, pH 7.4, with 10 M NaOH. Cell suspensions were loaded with 3 µM fura-2/acetoxymethyl ester for 30 min at 37 °C, washed, incubated at 20 °C for 20 min, and then rewashed. Intracellular calcium concentrations were measured in 1-ml volumes at 37 °C using a Shimazdu RF5000 spectrofluorophotometer at 340/380 nm excitation and 510 nm emission. In certain experiments, nominally Ca2+-free buffer containing 0.1 mM EGTA was used and was included in the final resuspension only. Data are presented as the Delta 340/380 nm ratio (mean ± S.E.).

Data Analysis-- Concentration-effect data from GTPase, [35S]GTPgamma S binding, adenylyl cyclase, MAPK phosphorylation, and [Ca2+]i assays were fitted to sigmoidal concentration-effect curves using GraphPAD Prism to determine EC50 values and maximal effects. Specific binding data were fitted to a one-site binding hyperbola using GraphPAD Prism to determine KD and Bmax values. Data are presented as means ± S.E. from at least three separate experiments and are compared using two-tailed Student's t test unless stated otherwise.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Expression of the Galpha oPTXi and Galpha oRGS/PTXi Mutants in C6µ Glioma Cells-- C6µ cells were stably transfected with Galpha oPTXi (C6µ-Galpha oPTXi cells) or Galpha oRGS/PTXi (C6µ-Galpha oRGS/PTXi cells). A C6µ-Galpha oPTXi clone (C1) and a C6µ-Galpha oRGS/PTXi clone (M1) chosen as expressing similar levels of Galpha o (Fig. 1A) were used for most studies. In membrane preparations, the binding affinity of [3H]DAMGO in Tris-HCl buffer was similar, with values of 2.0 ± 0.1 nM for C6µ-Galpha oPTXi cells (clone C1) and 3.5 ± 0.9 nM for C6µ-Galpha oRGS/PTXi cells (clone M1). The Bmax values for [3H]DAMGO were 7.6 ± 1.3 pmol/mg in C6µ-Galpha oPTXi cells (clone C1) and 14.4 ± 2.0 pmol/mg in C6µ-Galpha oRGS/PTXi cells (clone M1). PTX treatment (100 ng/ml overnight) of wild-type C6µ cells abolished opioid agonist-mediated signaling, as assessed by inhibition of cAMP accumulation, stimulation of [35S]GTPgamma S binding, stimulation of MAPK phosphorylation, and increases in [Ca2+]i through endogenous G proteins (data not shown). PTX treatment of C6µ cells expressing Galpha oPTXi or Galpha oRGS/PTXi allowed signaling through the transfected PTX-resistant Go proteins to be measured.


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Fig. 1.   Expression and activation of Galpha oPTXi or Galpha oRGS/PTXi in C6µ cells. A, membranes were prepared from wild-type (wt) C6µ cells or from C6µ cells stably transfected with Galpha oPTXi (PTXi) or Galpha oRGS/PTXi (RGSi) as described under "Experimental Procedures" and subjected to SDS-PAGE (20 µg of membranes or 20 ng of Galpha o standard (Go)). Proteins were transferred to a nitrocellulose membrane, incubated with anti-Galpha o antibody followed by horseradish peroxidase-conjugated anti-rabbit IgG, and visualized by chemiluminescence as described under "Experimental Procedures." Shown is a representative blot from three separate blots. B, membranes (14-20 µg) from PTX-treated C6µ-Galpha oPTXi (closed symbols) or C6µ-Galpha oRGS/PTXi (open symbols) cells were incubated for 60 min at 25 °C with 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 30 µM GDP, 0.1 nM [35S]GTPgamma S, and DAMGO (squares), morphine (circles), or dH2O. Data are derived from four assays, each carried out in duplicate, and are expressed as a percentage of basal binding. C, membranes (14-20 µg) from PTX-treated C6µ-Galpha oPTXi (black-square) or C6µ-Galpha oRGS/PTXi () cells were incubated for 10 min at 25 °C in 20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 0.1 mM DTT, and 1 mM EDTA, pH 7.4, containing 30 µM GDP, followed by a 10-min incubation with or without 10 µM DAMGO before the addition of 0.1 nM [35S]GTPgamma S to start the assay, which was allowed to proceed for 3-100 min. Samples were then harvested and counted as described under "Experimental Procedures." Data are presented as a percentage of the mean maximal DAMGO response from five assays, each performed in duplicate.

Stimulation of [35S]GTPgamma S Binding-- Basal levels of [35S]GTPgamma S binding were not different in membranes from PTX-treated Galpha oPTXi-expressing (0.026 ± 0.003 pmol/mg) and Galpha oRGS/PTXi-expressing (0.031 ± 0.002 pmol/mg) cells. The maximal stimulation of [35S]GTPgamma S binding produced by DAMGO over basal binding in membranes (Fig. 1B) was lower in C6µ-Galpha oRGS/PTXi membranes (157 ± 26% over control) versus C6µ-Galpha oPTXi membranes (250 ± 67% over control), but the difference was not statistically significant. A similar pattern was seen for maximal stimulation by morphine (C6µ-Galpha oRGS/PTXi membranes, 48 ± 7% over control; and C6µ-Galpha oPTXi membranes, 66 ± 17% over control). The potencies of the two µ-agonists for stimulation of [35S]GTPgamma S binding was also the same (DAMGO, EC50 = 296 ± 30 nM in C6µ-Galpha oPTXi cells and EC50 = 316 ± 30 nM in C6µ-Galpha oRGS/PTXi cells; and morphine, EC50 = 124 ± 24 nM in C6µ-Galpha oPTXi cells and EC50 = 159 ± 41 nM in C6µ-Galpha oRGS/PTXi cells). The rate of stimulation of [35S]GTPgamma S binding by 10 µM DAMGO (Fig. 1C) was the same in C6µ-Galpha oPTXi membranes (k = 0.17 ± 0.05 h-1) and C6µ-Galpha oRGS/PTXi membranes (k = 0.17 ± 0.09 h-1).

Stimulation of GTPase Activity-- The rates of basal GTP hydrolysis were 10.3 ± 0.3 and 13.2 ± 0.3 pmol/mg/min in membranes from PTX-treated C6µ-Galpha oPTXi and C6µ-Galpha oRGS/PTXi cells, respectively. DAMGO stimulated GTP hydrolysis in membranes from both PTX-treated C6µ-Galpha oPTXi and C6µ-Galpha oRGS/PTXi cells (Fig. 2A). The DAMGO stimulation of GTP hydrolysis in C6µ-Galpha oPTXi cells (5.70 ± 0.50 pmol/mg/min) was greater (p < 0.05) than in C6µ-Galpha oRGS/PTXi cells (2.64 ± 0.75 pmol/mg/min), consistent with reduced GTPase activity. Furthermore, the addition of 1 µM GST-RGS8 increased the DAMGO stimulation of GTP hydrolysis by a maximal concentration of DAMGO (1 µM) in C6µ-Galpha oPTXi (but not C6µ-Galpha oRGS/PTXi) membranes, indicating the effectiveness of the RGS-insensitive mutation in preventing the GAP activity of RGS8. GTPase stimulation in C6µ-Galpha oPTXi membranes by DAMGO at 2 min was concentration-dependent (Fig. 2B). GST-RGS8 increased the maximal stimulation over basal levels by DAMGO from 60 ± 7 to 151 ± 19% (p < 0.05), with a shift in the EC50 value from 34 ± 12 to 92 ± 31 nM, although this did not reach significance (p = 0.16). The maximal stimulation of GTP hydrolysis by 10 µM morphine at 2 min was 45 ± 5% in the C6µ-Galpha oPTXi membranes and increased significantly (p < 0.05) to 94 ± 17% in the presence of 1 µM GST-RGS8, although relative to DAMGO, morphine was significantly less efficacious (p < 0.05) in the presence of GST-RGS8 (61 ± 3%) than in its absence (75 ± 1%). RGS8 was chosen for these studies because it is structurally a simpler RGS protein and is known to be a GAP for Galpha o (22).


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Fig. 2.   A, stimulation of GTPase activity by 10 µM DAMGO in C6µ-Galpha oPTXi or C6µ-Galpha oRGS/PTXi cells in the presence or absence of RGS8. Membranes (14-20 µg) from PTX-treated C6µ-Galpha oPTXi (closed symbols) or C6µ-Galpha oRGS/PTXi (open symbols) cells were prewarmed for 5 min at 30 °C with 10 µM DAMGO in the absence (squares) or presence (triangles) of 1 µM RGS8 in a buffer system of 50 mM Tris, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, and an ATP-regenerating system. The reaction was initiated by the addition of prewarmed [32P]GTP to a final concentration of 0.1 µM and stopped at varying times between 15 and 120 s by the addition of ice-cold 15% charcoal in 20 mM H3PO4 plus 0.1% gelatin. Data are expressed as stimulation of Pi released by DAMGO after subtraction of basal release. Shown are the combined data from three assays. B, concentration-effect curve for stimulation of GTPase activity by DAMGO in C6µ-Galpha oPTXi cells in the presence and absence of RGS8. Membranes (14-20 µg) from PTX-treated C6µ-Galpha oPTXi cells were prewarmed for 5 min at 30 °C with varying concentrations of DAMGO in the absence (black-square) or presence (black-triangle) of 1 µM RGS8 in a buffer system of 50 mM Tris, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, and an ATP-regenerating system. The reaction was initiated by the addition of prewarmed [32P]GTP to a final concentration of 0.1 µM and stopped after 120 s by the addition of ice-cold 15% charcoal in 20 mM H3PO4 plus 0.1% gelatin. Data are given as pmol/Pi released/mg/min and are the combined data from three assays.

Inhibition of Adenylyl Cyclase-- Adenylyl cyclase activity was measured by the accumulation of cAMP stimulated by forskolin in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in PTX-treated whole cells. The level of accumulated forskolin-stimulated cAMP was the same in both the Galpha oPTXi- and Galpha oRGS/PTXi-expressing cells. The maximal inhibition of cAMP accumulation by DAMGO was significantly greater (p < 0.05) in C6µ-Galpha oRGS/PTXi cells (58 ± 5% inhibition) than in C6µ-Galpha oPTXi cells (35 ± 6% inhibition) (Fig. 3). More impressive was the fact that DAMGO was ~35-fold more potent (p < 0.05) in C6µ-Galpha oRGS/PTXi cells (EC50 = 12 ± 1 nM) than in C6µ-Galpha oPTXi cells (EC50 = 404 ± 112 nM). Morphine inhibition of forskolin-stimulated cAMP accumulation (Fig. 3) increased significantly (p < 0.01) from a maximum of 10 ± 5% in C6µ-Galpha oPTXi cells to 54 ± 7% in C6µ-Galpha oRGS/PTXi cells and showed an 8-fold increase in potency (C6µ-Galpha oRGS/PTXi cells, EC50 = 21.7 ± 11.2 nM; and C6µ-Galpha oPTXi cells, EC50 = 170 ± 53 nM), although this did not quite reach significance at the 0.05 level (p = 0.053). To ensure that the striking difference between the Galpha oPTXi- and Galpha oRGS/PTXi-expressing cells was not caused by differences in receptor and Galpha o expression levels, inhibition of cAMP accumulation was measured in two additional C6µ-Galpha oPTXi clones (C2 and C3) and an additional C6µ-Galpha oRGS/PTXi clone (M2). DAMGO and morphine were consistently more potent and gave higher maximal effects in the C6µ-Galpha oRGS/PTXi clones (Table I).


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Fig. 3.   Inhibition of cAMP accumulation. Cells were plated to confluency in 24-well plates the day before the assay and treated overnight with 100 ng/ml PTX. C6µ-Galpha oPTXi (closed symbols) or C6µ-Galpha oRGS/PTXi (open symbols) cells were rinsed with serum-free medium and then incubated with serum-free medium containing 30 µM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and DAMGO (squares), morphine (circles), or dH2O for 30 min at 37 °C. The reaction was stopped by replacing the medium with ice-cold 3% perchloric acid. After keeping the samples at 4 °C for at least 30 min, the samples were neutralized, and cAMP was quantified using a radioligand binding assay kit as described under "Experimental Procedures." Values are expressed as a percentage of the values with forskolin only (in the absence of ligand), which were the same for C6µ-Galpha oPTXi (6.6 ± 0.5 pmol/mg) and C6µ-Galpha oRGS/PTXi (6.8 ± 0.9 pmol/mg) cells. Shown are the combined data from three assays, each measured in duplicate.

                              
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Table I
Inhibition of cAMP accumulation in C6µ-Galpha oPTXi and C6µ-Galpha oRGS/PTXi cells by DAMGO and morphine after treatment with PTX

In wild-type C6µ, C6µ-Galpha oRGS/PTXi (clone M1), and C6µ-Galpha oPTXi (clone C1) cells not treated overnight with PTX, DAMGO and morphine robustly inhibited cAMP accumulation with similar EC50 values and maximal effects (Table II). DAMGO had somewhat increased potency in the Galpha oRGS/PTXi-expressing cells compared with the Galpha oPTXi-expressing cells and the wild-type C6µ cells. Morphine was more potent in the cells expressing Galpha oRGS/PTXi. When cells were treated with PTX overnight, the effect of DAMGO and morphine was completely lost in the C6µ cells expressing wild-type Galpha proteins; however, inhibition of cAMP accumulation was retained in the cells expressing Galpha oPTXi.

                              
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Table II
Inhibition of cAMP accumulation in wild-type C6µ, C6µ-Galpha oPTXi, and C6µ-Galpha oRGS/PTXi cells by DAMGO and morphine without PTX treatment

Stimulation of p44/42 MAPK Phosphorylation-- To determine whether MAPK regulation was also under the control of endogenous RGS proteins, stimulation of ERK phosphorylation by DAMGO (100 nM) was measured in PTX-treated C6µ cells expressing either Galpha oPTXi or Galpha oRGS/PTXi at intervals from 0 to 20 min (Fig. 4, A and B). Stimulation of phosphorylation by 100 nM DAMGO followed a similar time course in both cell clones, but the percent stimulation over basal levels was consistently higher in the C6µ-Galpha oRGS/PTXi cells. To determine whether the increased phosphorylation by 100 nM DAMGO in the Galpha oRGS/PTXi-expressing cells was due to a change in potency or a change in maximal effect, a concentration-effect curve for DAMGO was determined at the 5-min time point. DAMGO stimulated the phosphorylation of p44/42 MAPK (Fig. 5, A and B) with an 18-fold greater potency (p < 0.05) in C6µ-Galpha oRGS/PTXi cells (clone C1, EC50 = 48 ± 11 nM) than in C6µ-Galpha oPTXi cells (clone M1, EC50 = 839 ± 187 nM). Although the basal phosphorylation level was consistently lower in C6µ-Galpha oRGS/PTXi cells, the maximal percent increase over basal levels was similar (C6µ-Galpha oPTXi cells, 530 ± 115% stimulation; and C6µ-Galpha oRGS/PTXi cells, 590 ± 104% stimulation). Thus, the enhanced ERK phosphorylation seen in the initial time course study was due to a change in the EC50 for DAMGO and not in the maximal response. In contrast, even at 10 µM, morphine stimulated a low level of p44/42 MAPK phosphorylation in C6µ-Galpha oPTXi cells, representing just 14% of the stimulation seen with DAMGO. However, this concentration of morphine was significantly (p < 0.05) more efficacious in C6µ-Galpha oRGS/PTXi cells (527 ± 174% stimulation over basal levels) than in C6µ-Galpha oPTXi cells (68 ± 41% stimulation over basal levels) (Fig. 5C).


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Fig. 4.   Time dependence of DAMGO-stimulated p44/42 MAPK phosphorylation. C6µ-Galpha oPTXi (black-square) or C6µ-Galpha oRGS/PTXi () cells were treated overnight with 100 ng/ml PTX. The medium was replaced with serum-free medium for 2 h. The assay was started by the addition of 100 nM DAMGO and stopped after 0-20 min, and Western blotting was performed as described under "Experimental Procedures." Shown in A is a representative blot of phosphorylated and total MAPKs. Bands were quantitated as sum intensity (pixels) and plotted as a percentage of basal levels (without ligand). Shown in B are the combined data from three to four assays.


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Fig. 5.   Dose dependence of agonist-stimulated p44/42 MAPK phosphorylation. C6µ-Galpha oPTXi (black-square) or C6µ-Galpha oRGS/PTXi () cells were treated overnight with 100 ng/ml PTX. The medium was replaced with serum-free medium for 2 h. The assay started by the addition of 0-10 µM DAMGO (B) or 10 µM morphine (C) and stopped after 5 min, and Western blotting was performed as described under "Experimental Procedures." Shown in A is a representative blot of phosphorylated and total MAPKs. Bands were quantified as sum intensity (pixels) and plotted as a percentage of basal levels (without ligand). The basal sum intensity was lower in C6µ-Galpha oPTXi cells (157,000 ± 17,000 pixels) than in C6µ-Galpha oRGS/PTXi cells (67,000 ± 12,000 pixels). Shown in B are the combined data from three to four assays.

Increases in Intracellular Calcium-- To obtain a measurable increase in the intracellular calcium signal in response to opioid agonists, cells were grown for 24 h in the presence of 5 µM forskolin (24, 25). In these cells, DAMGO stimulated [Ca2+]i in PTX-treated C6µ-Galpha oRGS/PTXi (EC50 = 80 ± 34 nM) and C6µ-Galpha oPTXi (EC50 = 89 ± 17 nM) cells. There was no significant difference in the EC50 values or maximal stimulation (Fig. 6A). Compared with DAMGO, the relative maximal effect of morphine was somewhat higher in C6µ-Galpha oRGS/PTXi cells (71.9 ± 9.1%) than in C6µ-Galpha oPTXi cells (35.3 ± 14.5%) (Fig. 6B), but this did not reach statistical significance (p = 0.11, Wilcoxon matched pairs). At 1 µM DAMGO, the induced rise in [Ca2+]i was from intracellular stores because the increase was not significantly different (p > 0.05, Wilcoxon matched pairs) in the presence of extracellular Ca2+ (Delta 340/380 nm ratios of 0.11 ± 0.02 in C6µ-Galpha oRGS/PTXi cells and 0.06 ± 0.01 in C6µ-Galpha oPTXi cells) or in its absence (Delta 340/380 ratios of 0.07 ± 0.01 in C6µ-Galpha oRGS/PTXi cells and 0.07 ± 0.01 in C6µ-Galpha oPTXi cells).


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Fig. 6.   Agonist-stimulated increase in [Ca2+]i. [Ca2+]i was measured in the presence of 0-10 µM DAMGO (A) or 10 µM morphine (B) in fura-2-loaded whole cell suspensions from C6µ-Galpha oPTXi (black-square) and C6µ-Galpha oRGS/PTXi () cells treated for 24 h with forskolin and overnight with PTX as described under "Experimental Procedures." Morphine stimulation is expressed as a percentage of the 10 µM DAMGO response. Shown are the combined data from six to seven assays.

To confirm that the Galpha oPTXi- and Galpha oRGS/PTXi-expressing cells treated overnight with forskolin still showed different sensitivities to µ-opioid agonist inhibition of adenylyl cyclase, cells were examined for DAMGO inhibition of cAMP accumulation. Forskolin treatment increased the maximal effect and potency of DAMGO in the Galpha oPTXi-expressing cells. However, the differential response between the Galpha oPTXi- and Galpha oRGS/PTXi-expressing cells was retained. The degree of maximal inhibition of cAMP accumulation was increased in the presence of the RGS-insensitive Galpha o mutant (Galpha oPTXi, 68.5 ± 5.4%; and Galpha oRGS/PTXi, 83.8 ± 1.2%; p = 0.05), as was the enhanced potency of DAMGO (Galpha oRGS/PTXi, EC50 = 4.24 ± 1.44 nM; and Galpha oPTXi, EC50 = 157 ± 35 nM; p < 0.05).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have expressed Galpha o that is insensitive to RGS protein action in C6 cells expressing a µ-opioid receptor to demonstrate a role for endogenous RGS proteins in the control of opioid receptor signaling. Two main conclusions can be drawn from this study. First, endogenous RGS proteins reduce the effectiveness of Galpha o signaling to adenylyl cyclase and MAPK pathways, suggesting that endogenous RGS protein action has substantial regulatory effects on agonist potency and maximal response. Second, because only minor differences were seen between the RGS-insensitive Galpha o mutant compared with its RGS-sensitive counterpart in coupling to intracellular calcium release, but significant differences were measured with the adenylyl cyclase and MAPK pathways, we conclude that endogenous RGS proteins contribute to the control of effector selectivity of Galpha o signaling.

One of the most direct measures of receptor activation is stimulation of [35S]GTPgamma S binding seen upon the addition of agonist. The potency and maximal effect of DAMGO and morphine in stimulating [35S]GTPgamma S binding were similar in membranes from C6µ cells expressing Galpha oPTXi and Galpha oRGS/PTXi or, if anything, were greater for RGS-sensitive Galpha o. One would not expect the GAP activity of RGS proteins to play a role in the binding of the GTP analog to the Galpha o subunit because the assay is done in the presence of a large excess of GDP and the rate-limiting step is the dissociation of GDP from the Galpha subunit (26).

In contrast to agonist-stimulated [35S]GTPgamma S binding, DAMGO-stimulated GTPase activity in membranes from cells expressing Galpha oRGS/PTXi was significantly less than in membranes from cells expressing Galpha o/PTXi. This indicates that endogenous RGS protein GAP activity in C6µ cell membranes is significant, but this cannot function to stimulate GTP hydrolysis by the Galpha oRGS/PTXi mutant. Furthermore, DAMGO-stimulated GTP hydrolysis in membranes from the Galpha oPTXi-expressing cells was markedly enhanced in the presence of added RGS8, whereas no stimulation by exogenously added RGS8 was observed in membranes from the Galpha oRGS/PTXi-expressing cells, confirming the insensitivity of RGS-insensitive Galpha o. As a percentage of the maximal DAMGO stimulation, morphine produced a significantly smaller increase in GTPase activity in the presence of GST-RGS8 than in its absence, demonstrating that RGS protein is able to produce a greater enhancement of steady-state GTPase in the presence of a full agonist; this could relate to the greater rate of GDP release caused by the full agonist (26).

The potency and maximal effect of DAMGO and morphine in inhibiting adenylyl cyclase through activation of the transfected RGS-sensitive Galpha oPTXi mutant in PTX-treated cells were poor compared with both wild-type C6µ cells and cells expressing Galpha oPTXi before treatment with PTX. C6 cells do not endogenously express Galpha o, and Galpha i2 is the predominant Galpha subunit expressed (27). The robust inhibition of adenylyl cyclase in C6µ cells expressing exogenous Galpha o before PTX treatment confirms that endogenous Galpha i2 couples very efficiently to adenylyl cyclase. In contrast, it is probable that the transfected Galpha oPTXi mutant, although activated efficiently by the µ-opioid receptor, couples poorly to adenylyl cyclase such that this response can be dramatically improved by inhibition of RGS activity. In support of this, Galpha o does not couple well to adenylyl cyclase in NG108-15 cells or HEK293 cells compared with Galpha i2 (28, 29), although it is reported to have a role in this regard in SH-Galpha -interacting protein SY5Y cells (30). The inhibition of cAMP accumulation was very much improved in the Galpha oRGS/PTXi-expressing cells, giving a large increase in both the maximal inhibition and agonist potency in the different clones examined. It was also noticeable that, without PTX treatment, cells expressing the Galpha oRGS/PTXi mutant were more efficiently inhibited by morphine and DAMGO than cells expressing only Galpha oPTXi. In a series of experiments using the opposite approach, expression of RGS4 or Galpha -interacting protein in HEK293 cells reduced the level of somatostatin receptor-induced inhibition of cAMP accumulation (31), demonstrating the ability of RGS proteins to control the magnitude of inhibitory G protein signaling. Our data extend this by demonstrating a role for endogenous RGS proteins in this effect.

This effect of the RGS-insensitive Galpha o mutant on agonist-mediated inhibition of adenylyl cyclase was particularly marked for the partial µ-agonist morphine, which became almost (90%) as efficacious as the full agonist DAMGO in the C6µ-Galpha oRGS/PTXi cells. This indicates that RGS proteins may be more effective when the receptor/G protein/effector system is signaling at submaximal levels. In agreement with this, several authors have shown that, at high agonist concentrations, RGS proteins are less effective (32, 33). In addition, RGS5-mediated reduction in intracellular calcium release by the angiotensin type 1a receptor (Gq-linked) is less effective when receptors are expressed at high levels (34). Because the relative efficacy of an agonist is tissue-specific, it may be possible that differential expression of RGS proteins in tissues is one factor in determining agonist efficacy.

µ-Opioids strongly activate phosphorylation of p44/42 ERK in C6µ cells (3). This effect was retained in C6µ cells expressing either Galpha oRGS/PTXi or Galpha oPTXi after PTX treatment. However, similar to the µ-agonist effect on the cAMP system, µ-agonist activation of the MAPK pathway was increased in C6µ cells expressing the Galpha oRGS/PTXi mutant compared with the RGS-sensitive Galpha oPTXi mutant. Thus, when the µ-receptor was coupled through Galpha oRGS/PTXi, a >10-fold increase in DAMGO potency and an increase in the maximal effect of morphine were observed compared with coupling through Galpha oPTXi. These results are consistent with findings that 5-hydroxytryptamine 1beta receptor activation of ERK (p44/42 MAPK) is reduced by overexpression of RGS4 in neuroblastoma cells (32), as is stimulation of MAPK through interleukin-8 (35) and dopamine D2 (33) receptor activation. In contrast to the effects on adenylyl cyclase and MAPK, the µ-opioid-mediated increase in [Ca2+]i showed a much smaller increase in effect in the Galpha oRGS/PTXi-expressing cells such that the difference between these and the Galpha oPTXi-expressing cells did not quite reach significance. This differential effect causes a shift in the most potent response to the µ-opioid agonist DAMGO. Thus, in the presence of endogenous RGS activity, the rise in intracellular calcium is the most potent response (EC50 = 89 nM), and the inhibition of adenylyl cyclase is the weakest response (EC50 = 400 nM); but in the absence of endogenous RGS activity, the order is reversed, and the inhibition of adenylyl cyclase is the most potent response (EC50 = 12 nM), whereas the rise in intracellular calcium is the weakest response (EC50 = 80 nM).

The present data show that endogenous RGS proteins may differentially affect signaling by a single G protein depending on the effector pathway to which the G protein couples. Several mechanisms could account for this specificity. One is that the GAP activity of endogenous RGS proteins controls signaling by a kinetic scaffolding mechanism (18). The kinetic scaffolding model predicts that RGS action reduces depletion of local Galpha -GTP levels and so permits rapid recycling of G protein and rapid recoupling of the receptor and maintains local G protein activation. The adenylyl cyclase and MAPK pathways are poorly signaled to in the presence of RGS activity; but when this activity is blocked, as in cells expressing the RGS-insensitive Galpha o mutant, then signaling can occur because spatial control is lost, allowing spillover of Galpha -GTP and Gbeta gamma subunits to more distant effectors. In contrast, coupling to intracellular calcium stores is more similar in cells expressing Galpha oPTXi and Galpha oRGS/PTXi. Thus, for the kinetic scaffolding model to account for this effect, the G proteins involved in coupling to this pathway must be organized closely with receptor and effector such that they show a reduced RGS-dependent effect.

The differential effect of RGS on the three pathways examined is consistent with this theory, but does not provide direct proof. Other mechanisms may explain the findings. The increased opioid effect at adenylyl cyclase and MAPK may simply be due to an increased lifetime of Galpha -GTP in the absence of RGS protein GAP activity; but if so, then the question arises as to why the intracellular calcium signal is not enhanced to a similar extent. There may be differential localization or compartmentalization of effectors within the cell (36) such that the intracellular calcium signaling complex is protected from RGS action. One possibility is that RGS proteins provide a protein scaffold that allows signaling to the intracellular calcium pathway; but in the absence of this restraining scaffold, other pathways become available to the Galpha -GTP or Gbeta gamma subunits. Certainly, C6 cells contain message for a variety of RGS proteins (RGS2, RGS3, RGS8, RGS10, RGS12, and RGS14) (37), several of which have regions outside of the RGS box that could be involved in protein-protein interactions. Alternatively, RGS proteins may not have a controlling function in modulation of the intracellular calcium signal; the rate of GTP hydrolysis and the lifetime of Galpha -GTP may not be the rate-limiting step in this signaling pathway. The increase in [Ca2+]i response is transient in nature. Opioid stimulation of intracellular concentration is thought to be mediated through Gbeta gamma subunit activation of phospholipase Cbeta 1 to break down phosphatidylinositol 4,5-bisphosphate and to provide inositol 1,4,5-trisphosphate, which binds to the inositol 1,4,5-triphosphate receptor on the intracellular store, causing an increase in [Ca2+]i (23). The nature of the intracellular calcium signal may be controlled by other factors such as inositol 1,4,5-triphosphate receptor desensitization and the fullness of the Ca2+ store; indeed, there may not be a direct temporal relationship between inositol 1,4,5-triphosphate and Ca2+ signaling (38).

In summary, we have shown, in a transfected C6 cell line, that RGS proteins differentially regulate µ-opioid receptor-mediated signaling to different effectors through Galpha o, consistent with a kinetic scaffolding mechanism. Coupling to adenylyl cyclase and the MAPK pathway appears to be efficiently limited by endogenous RGS proteins, whereas coupling to intracellular calcium stores is less susceptible to RGS protein action. Because the potency and maximal effect of agonist are altered, it is possible that differential expression of RGS proteins in tissues plays a role in tissue-specific differences in agonist selectivity and efficiency. Finally, because cAMP (39) and MAPK (40, 41) have been implicated in contributing to the tolerance associated with long-term opioid administration, the effect of endogenous RGS proteins on these cellular adaptations merits further investigation.

    ACKNOWLEDGEMENTS

We thank Dr. Huda Akil for the µ-opioid receptor DNA and Dr. Stephen Ikeda for the RGS- and PTX-insensitive Galpha o mutants.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DA 04087 (to J. R. T.) and GM 39561 (to R. R. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Tel.: 734-647-7479; Fax: 734-763-4450; E-mail: jtraynor@umich.edu.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M208885200

    ABBREVIATIONS

The abbreviations used are: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GAP, GTPase-activating protein; PTX, pertussis toxin; DAMGO, [D-Ala2,MePhe4,Gly5-ol]enkephalin; [35S]GTPgamma S, guanosine 5'-O-(3-[35S]thiotriphosphate); GST, glutathione S-transferase; DTT, dithiothreitol; dH2O, distilled H2O; AppNHp, adenyl-5'-yl imidodiphosphate; [Ca2+]i, intracellular calcium concentration.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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