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CELLULAR AND MOLECULAR
-Opioid Receptors
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Munich, Germany
Received October 17, 2003; accepted January 20, 2004.
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Abstract |
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-opioid receptors (DORs) expressed in human embryonic kidney (HEK)293 cells. Receptor activation by etorphine turned out to transiently phosphorylate ERK/MAP kinases and bring about DOR internalization within 20 min. In contrast, prolonged exposure of HEK293 cells to morphine excited persistent phosphorylation of ERK/MAP kinases, and those cells failed to internalize the opioid receptor. When ERK/MAP kinase phosphorylation was blocked by 2'-Amino-3'-methoxyflavone (PD98059), morphine gained the ability to strongly induce DOR endocytosis. The importance of activated MAP kinases for DOR internalization is further demonstrated by glutamate and paclitaxel because these substances induce phosphorylation of ERK1/2 and concomitantly prevent DOR sequestration by etorphine. In addition, receptor internalization by morphine was facilitated by inhibition of protein kinase C and opioid-mediated transactivation of epidermal growth factor receptor (EGFR), both activating ERK/MAP kinases by opioids. The mechanism affording DOR internalization by PD98059 may relate to arrestin, which uncouples GPCRs and thus triggers receptor internalization. Arrestin considerably translocates toward the cell membrane upon DOR activation by morphine in presence of the MAP kinase blocker, but it fails in the absence of PD98059. We conclude that ERK/MAP kinase activity prevents opioid receptor desensitization and sequestration by blocking arrestin 2 interaction with activated DORs.
). One such mechanism reflects the internalization of activated receptors that is preceded by their phosphorylation brought about by G protein-coupled receptor kinases (GRKs). Subsequently, cytosolic arrestin (Bünemann and Hosey, 1999) uncouples the receptor from associated G proteins. This mechanism accounts for desensitization of receptors and eventually their internalization (Goodman et al., 1996).
The disappearance of opioid receptors from the cell surface diminishes signal transmission and thus contributes to the development of opioid tolerance (Williams et al., 2001). However, whether opioid receptors undergo internalization depends on the ligand used for their activation. Exposure of receptors to etorphine or certain enkephalin derivatives rapidly triggers internalization of -(DOR; Trapaidze et al., 1996) and µ-opioid receptors (MOR; Sternini et al., 1996). In contrast, morphine almost fails to desensitize and sequester activated opioid receptors (Keith et al., 1996; Sternini et al., 1996). This feature of morphine implicates a sustained receptor signaling in the presence of this drug (Williams et al., 2001). That is, morphine uses the full capacity of existing opioid receptors, and this may not change during the course of chronic drug exposure. Therefore, the high-dependence liability of morphine reported for isolated cells (Ammer and Schulz, 1997) and animals (Narita et al., 2001) is likely to relate to the failure of morphine to diminish opioidergic signaling by its inability to bring about opioid receptor internalization. Consequently, the phenomenon of morphine tolerance underlies adaptations within the receptor-associated signal transduction pathway (Williams et al., 2001).
Organized interaction of several cellular components (Bünemann and Hosey, 1999) and especially the binding of adaptor proteins, e.g., arrestins (Ferguson et al., 1996), to activated receptors regulates signaling and triggers their internalization. This process is linked to the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), representing isoforms of the mitogen-activated protein (MAP) kinase family (Johnson and Lapadat, 2002). Primarily known for their kinase activity to nuclear transcription factors, these enzymes also phosphorylate cytosolic proteins participating in the process of receptor internalization. This mechanism is known to apply to GRK2, which loses its activity when phosphorylated by ERKs (Pitcher et al., 1999). In contrast, inhibition of ERK-mediated phosphorylation potentiates GRK2 activity, and concomitantly enhances receptor internalization (Pitcher et al., 1999). Phosphorylation of arrestin 2 by MAP kinases impairs binding to clathrin, preventing internalization of 
2-adrenergic receptors (Lin et al., 1999). GTPase activity of dynamin that is required for pinching-off internalizing vesicles from plasma membrane is also inhibited by phosphorylation of dynamin through ERK (Earnest et al., 1996).
Although the opioid alkaloids etorphine and morphine activate both MORs and DORs (Zhang et al., 1999), only etorphine, but not morphine, induces opioid receptor sequestration. The failure of morphine to internalize opioid receptors may be explained by its reduced ability to bring about receptor phosphorylation by GRKs and arrestin binding (Zhang et al., 1998, 1999). Because GRKs and arrestins are regulated by MAP kinases, we examined whether distinct differences of morphine and etorphine on ERK1/2 activation accounts for differences of DOR internalization. Therefore, endocytosis of DORs and ERK/MAP kinase activation were studied using human embryonic kidney (HEK)293 cells. We demonstrate here that continuous activation of DORs by etorphine triggers in parallel their internalization and transient ERK/MAP kinase activation. In contrast, prolonged morphine exposure caused a sustained ERK stimulation but failed to internalize DORs. Blockade of morphine-mediated ERK activation by 2'-amino-3'-methoxyflavone (PD98059) enabled the opiate to strongly induce DOR internalization. Etorphine, which is well known to effectively internalize opioid receptors, fails to sequester DORs in presence of the ERK1/2 activator glutamate and paclitaxel. We further report here that ERK/MAP kinases control DOR internalization by impairing the interaction of arrestin 2 with DOR.
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Materials and Methods |
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-opioid receptor cDNA (Yasuda et al., 1993) inserted into pcDNA3.1 (Invitrogen BV, Groningen, The Netherlands) using the calcium phosphate method. Cells permanently expressing the -receptor were grown in DMEM supplemented with 10% fetal calf serum (complete medium), glutamine and penicillin/streptomycin (Schulz et al., 2002). For confocal studies, HEK293 cells were transfected to permanently express the mouse DOR fused to enhanced green fluorescence protein (DOR-EGFP; Schulz et al., 2002) and grown on coverslips in complete medium (60% confluence).
Cell Treatment. HEK293/ cells were challenged with etorphine (National Institute on Drug Abuse, Bethesda, MD) and morphine (Merck, Mannheim, Germany), respectively, for up to 1 h at 37°C. To investigate the effect of MAP kinase on DOR internalization, cells were treated with either the MAP kinase blocker PD98059 (10 µM, 15 min; Merck-Eurolab, Darmstadt, Germany) or MAP kinase activator glutamate (5 mM, 12 h; Sigma Chemie, Deisenhofen, Germany) and paclitaxel (1 µM, 24 h; Sigma Chemie), respectively, before opioid exposure. To inhibit receptor internalization, cells were challenged with concanavalin A (con A; 250 µg/ml; Merck-Eurolab) 30 min before and during etorphine (100 nM) exposure and with sucrose (0.4 M) for 2 h followed by opiate incubation for 60 min. Cell pretreatment with tyrphostin (AG1478; 5 µM, Merck-Eurolab), calphostin C (1 µM; Merck-Eurolab), and wortmannin (100 nM; Merck-Eurolab) lasted for 15 min, respectively, before morphine (1 µM) was added for 5 to 60 min. HEK293 wild-type cells were challenged with opiates indicated and human EGF (50 ng/ml; Bachem, Bibendorf, Switzerland) for 5 min at 37°C.
Cell Viability Test. Glutamate (5 mM), paclitaxel (1 µM), and PD98059 (10 and 50 µM) were tested for their effects on cell viability by using the MTT test. Pretreated cells were exposed to MTT (Tetrazolium bromide, 0.5 mg/ml; Sigma Chemie) for 1 h, 37°C. After removing the medium, metabolized MTT-formazan was solubilized in dimethyl sulfoxide/ethanol (1:1) and generation of formazan was determined by photometry (
= 570 nm; Hitachi U-3200 spectrophotometer). Cell viability is expressed as percentage (absorbance treated cells/absorbance nontreated cells) x 100%. Because 50 µM PD98059 alone strongly reduced the viability of HEK293/ cells, further studies were accomplished using 10 µM of the MAP kinase blocker.
Receptor Internalization. Internalization of DORs was determined by radioligand binding to intact cells according to Eisinger et al. (2002). Cells were exposed to individual compounds solved in serum-reduced DMEM (0.1% serum). Subsequent to treatment, cells were washed three times with ice-cold phosphate-buffered saline (PBS) to remove opioids from DORs and were suspendend in binding buffer (50 mM Tris, pH 7.4). Total DOR capacity was determined by lipophilic, membrane-permeable antagonist [3H]diprenorphine (53 Ci/mmol; PerkinElmer Life Sciences, Rodgau-Jügesheim, Germany). Nonspecific receptor binding was identified in the presence of lipophilic naloxone (10 µM; Merck). Surface receptors were determined by the nonlipophilic peptide agonist [D-Ala2,D-Leu5]-enkephalin (DADLE, 1 µM; Bachem). Binding assay was performed at 4°C for 120 min and stopped by rapid filtration through Whatman GF/B filters. Filters were washed three times with ice-cold binding buffer and transferred to liquid scintillation. Radioactivity was determined using Beckmann LS1801 scintillation counter. Surface DORs were expressed in percent of total opioid receptor expression.
MAP Kinase Immunoblot. For ERK/MAP kinase stimulation assay, HEK293 wild-type and HEK293/ cells were grown in six-well plates (Techno Plastic Products AG, Trasadingen, Switzerland). Two hours before the experiments, growth medium was substituted by serum-reduced DMEM to avoid activation of MAP kinase by growth factors. For long-term pretreatment, HEK293/ cells were grown for 36 h in complete medium. Thereafter, medium was removed and cells were incubated with glutamate (5 mM) and paclitaxel (1 µM) diluted in serum-reduced DMEM for 6 to 24 h. ERK/MAP kinase stimulation was stopped by removing the medium containing indicated compounds. Cells were lysed by the addition of Lämmli buffer and samples were submitted to 10% SDS-polyacrylamide gel electrophoresis. Phosphorylated ERK1/2 was determined by Western blot analysis using phospho-specific anti-ERK1/2 antibody (New England Biolabs, Frankfurt, Germany). Total ERK1/2 was detected by anti-ERK1/2 antibody, identified by peroxidase-coupled second antibodies and enhanced chemiluminescence.
Confocal Study of DOR Internalization. For visualization of DOR internalization, HEK293 cells expressing DOR-EGFP were exposed to PD98059 (10 µM, 15 min) or glutamate (5 mM, 12 h) followed by opiate challenge for 60 min. After removing the medium, cells were washed with cold PBS buffer and fixed with paraformaldehyde (4% in PBS buffer) for 30 min at room temperatur. Subsequently, cover slips were mounted on object slides with Fluorescent Mounting Medium (DAKO Diagnostika, Hamburg, Germany) and confocal images of cell groups were studied using Zeiss LSM 510 microscope (Schulz et al., 2002).
Arrestin 2 translocation. Translocation of arrestin to plasma membranes upon DOR stimulation was determined as previously described (Eisinger et al., 2002). Briefly, HEK293/ cells were exposed to substances as indicated for 10 min, 37°C. In some experiments, cells were pretreated with PD98059 for 15 min before opiate exposure for 10 min. Subsequently, cells were washed with PBS (4°C) and homogenized. Cell homogenate was centrifuged at 1000g for 10 min at 4°C to remove nuclei and unbroken cells. Subsequently, the supernatant was centrifuged at 20,000g for 30 min, 4°C. The resulting pellet was resuspended in PBS buffer containing protease inhibitor mixture (Complete; Boehringer, Mannheim, Germany). Subsequently, membrane proteins were solubilized in sample buffer, separated by 10% SDS-PAGE (10 µg protein/lane) and arrestin was determined by Western blot analysis using anti-mouse arrestin 2 antibody (Transduction Laboratories, Lexington, KY). To control equal membrane protein loading, blots were reprobed with anti-G antibody (Ammer and Schulz, 2000) recognizing G subunits being associated with plasma membranes in intact cells.
[35S]GTP
S binding. For [35S]GTPS binding, HEK293/ cells exposed to opiates with or without PD98059 pretreatment (10 µM, 15 min) were washed and homogenized using a Polytron homogenizator. Cell membranes were isolated by centrifugation (1000g, 10 min; 20,000g, 30 min) and suspended in GTPS-buffer (50 mM Tris, 5 mM MgCl2, 0.2 mM EGTA, 120 mM NaCl, 50 µM GDP; pH 7.4). For binding reaction, membrane proteins (20 µg) were incubated with [35S]GTPS (0.1 nM; specific activity 12.5 mCi/ml; PerkinElmer Life Sciences) and etorphine (100 nM) at 25°C for 30 min. Nonspecific binding was determined in presence of cold GTPS (10 µM). After rapid filtration through GF/B filters, the filters were washed with ice-cold 50 mM Tris/5 mM MgCl2 (pH 7.4) buffer, and bound radioactivity was determined by Beckmann LS1801 scintillation counter.
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cells exposed to etorphine (100 nM) or morphine (1 µM) for different periods (Fig. 1), determining the fraction of [3H]diprenorphine binding displaced by [D-Ala2,D-Leu5]-enkephalin. The binding technique used enables differentiation between receptor internalization (loss of surface receptors) and total loss of binding capacity by receptor down-regulation. Agonist exposure (etorphine and morphine) for 5 min revealed no effect on the fraction of surface DORs (Fig. 1) from total receptor expression (average 1.1 pmol/106 cells). Extension of etorphine exposure progressively provoked a loss of surface binding without changing total [3H]diprenorphine binding. Thus, etorphine treatment induces DOR internalization, whereas morphine (1 µM) failed to affect the capacity of surface DORs at any time of exposure. Even increasing morphine concentration up to 10 µM and extending the time of exposure up to 2 h had no effect on both surface (control, 52.8 ± 2.1%; 10 µM morphine, 56.3 ± 3.1%; 2-h morphine (1 µM), 53.3 ± 0.3%) and total DOR capacity (average 1.3 pmol/106 cells).
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Phosphorylation of ERK/MAP Kinases in HEK293/ Using HEK293/ cells, we examined the effect of DOR stimulation on ERK/MAP kinase activity. Activation of those kinases occurs through their phosphorylation, which was determined by phospho-specific anti-ERK1/2 antibody in Western blot analysis. In the absence of any opioid, serum-starved HEK293/ cells exhibit no phosphorylation of ERK/MAP kinases (Fig. 2A, top and middle; 0 min). DOR stimulation with etorphine (100 nM) and morphine (1 µM), respectively, brought about highest phosphorylation levels of ERK1/2. However, ERK1/2 phosphorylation declined in presence of etorphine within 60 min (Fig. 2A, top) to almost predrug level. In contrast, morphine exposure caused a prominent phosphorylation of ERK1/2 for the duration of drug exposure (Fig. 2A, middle). In HEK293 cells not expressing DORs, incubation with opiates failed to stimulate ERK phosphorylation (Fig. 2B, top), although these cells exhibit functional ERK/MAP kinases being activated by EGF (50 ng/ml; 5 min). Thus, ERK phosphorylation in HEK293/ cells is mediated by DORs. Blots probed with ERK1/2 antibodies revealed that total ERK1/2 expression in HEK293/ and wild-type HEK293 cells was not affected by opiate treatment (Fig. 2, A and B, bottom).
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DOR Internalization and ERK1/2 Activation. Internalization of GPCRs impairs responsiveness of cells to their respective agonist (Ferguson and Caron, 1998). Thus, loss of DOR-mediated MAP kinase activation by etorphine might be due to a decline of functional receptors (internalization) caused by this ligand. To identify the link of DOR internalization and decrease of MAP kinase activation, ERK1/2 phosphorylation was examined in the presence of con A and sucrose (suc). Both compounds inhibit internalization of GPCRs (Mundell and Kelly, 1998) and were tested for their effects on DOR sequestration under the experimental conditions used here (Fig. 3A). Pretreatment with both con A (250 µg/ml; 30 min) and suc (0.4 M; 2 h) had no effect on total receptor expression, but the rate of surface DORs slightly increased. In both cases, subsequent exposure to etorphine (100 nM; 60 min) did not bring about DOR internalization contrasting the opiate effect in controls.
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The effect of con A and sucrose on ERK1/2 phosphorylation after short-(5 min) and long-term (60 min) exposure to etorphine was explored next (Fig. 3B). Compared with significant increase of MAP kinase phosphorylation upon etorphine challenge, preexposure to both con A (250 µg/ml; 30 min) and sucrose (0.4 M; 2 h) did not increase ERK1/2 phosphorylation (Fig. 3B, top). Furthermore, both sequestration inhibitors did not interfere with the phosphorylation of ERK/MAP kinases by short-term etorphine exposure (100 nM; 5 min). Phosphorylation of ERK/MAP kinases also declined during prolonged cell exposure to etorphine in presence of con A or suc (Fig. 3B; 60 min). Thus, inhibition of etorphine-promoted DOR internalization by either con A or sucrose did not prevent decrease of MAP kinase activation (60 min; Fig. 3B). Total ERK/MAP kinase expression was left unaffected by any treatment tested (Fig. 3B, bottom).
Morphine-Induced DOR Internalization in the Presence of PD98059. The significance of the sustained phosphorylation of MAP kinase by morphine exposure on DOR internalization was tested by the MAP kinase blocker PD98059. The compound inhibits the ERK/MAP kinase activating enzyme MAP kinase kinase (MEK), and in consequence the stimulation of ERKs. Because pretreatment of HEK293/ cells with PD98059 (10 µM; 15 min) exerted no effects on cell viability as judged by the MTT test (control, 100%; PD treated, 100.2 ± 0.69%), it totally blocked morphine-stimulated ERK/MAP kinase phosphorylation (Fig. 4A).
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We next examined the effect of ERK blockade on DOR regulation (Fig. 4B). Apparently, PD98059 (10 µM; up to 60 min) failed to affect the quantity of surface DORs (Fig. 4B, open columns). However, cell exposure to PD98059 (10 µM; 15 min) followed by morphine challenge resulted in a significant decrease of surface DORs within 60 min (1 µM; Fig. 4B, closed columns), whereas total receptor expression was unchanged. Thus, blockade of ERK/MAP kinase enabled morphine to strongly induce DOR internalization by more than 85%.
Permanent Activation of MAP Kinases Inhibits Etorphine-Induced -Receptor Internalization. Permanent phosphorylation of ERK/MAP kinase, brought about by morphine, was observed to prevent opioid receptor internalization. We therefore hypothesized that persistent MAP kinase activation should block DOR internalization. Glutamate has been reported to stimulate ERKs in HT22 cells (Stanciu et al., 2000), causing us to expose HEK293/ cells with the amino acid. As shown in Fig. 5A, incubation with glutamate (5 mM) for 6 h increased phosphorylation of ERK/MAP kinase, and this effect was even more enhanced extending glutamate exposure to 12 h. Glutamate stimulates ERK phosphorylation via the MEK/ERK pathway, which is inhibited by the MEK blocker PD98059 (10 µM). Paclitaxel, a further compound enhancing ERK/MAP kinase activity (Okano and Rustgi, 2001), was tested in addition. Paclitaxel rapidly stabilizes microtubules (Schiff et al., 1979), but ERK phosphorylation occurred with a latency of 7 h (1 µM), reaching a maximum after 24 h of exposure. Activation of ERKs by paclitaxel was also dependent on MEK stimulation and is inhibited by PD98059 (Fig. 5A).
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The effect of glutamate and paclitaxel on DOR internalization by etorphine was analyzed next. Glutamate itself (12 h) did not affect the number of surface DORs (Fig. 5B), but etorphine (100 nM; 60 min) completely failed to internalize DORs in glutamate-pretreated cells. Similar findings were obtained for etorphine in cells exposed to paclitaxel for 24 h. However, when cells were preteated with paxlitaxel for 2 h only, etorphine still brought about sequestration of surface DORs by about 80%, compared with controls (absence of paclitaxel minus 83%). In cells incubated with these ERK activators (glutamate and paclitaxel) and PD98059 (Fig. 5B), etorphine regained its ability to efficiently internalize surface opioid receptors (-90 or -88%, respectively).
Depending on the cell line, sustained ERK activity may either initiate apoptosis or may prevent from cell death (Stanciu et al., 2000; Okano and Rustgi, 2001). To validate that failure of etorphine to induce internalization is not due to a cytotoxic effect of glutamate or paclitaxel, HEK293/ cells were tested for viability in the presence of these drugs. As revealed by the MTT test, neither glutamate nor paclitaxel exposure revealed any alteration in cell viability (control, 100%; glutamate, 103.2 ± 4.1%; paclitaxel, 98.2 ± 2.6%).
Receptor Trafficking upon Glutamate and PD98059 Pretreament. Using HEK293 cells stably expressing DOREGFP (Fig. 6), effects of glutamate and PD98059 on receptor trafficking can be visualized by confocal microscopy. In absence of any drug, the green fluorescent opioid receptor is localized in the plasma membrane and thus defines clearly the cell shape (Fig. 6). The inability of morphine to sequester opioid receptors is confirmed by this technique because exposure to this agonist (1 µM; 60 min) did not change the membrane-associated localization of DOR-EGFP. In contrast, challenge of the cells with etorphine (100 nM; 60 min) resulted in vesicle-like accumulation of EGFP-tagged DORs in the cytosol, representing receptor internalization (Schulz et al., 2002). Sustained phosphorylation of ERK/MAP kinases by glutamate showed no effect on receptor distribution between plasma membranes and cytosol, and neither etorphine nor morphine exposure altered DOR-EGFP localization in presence of this amino acid. Also cell challenge with PD98059 (10 µM; 60 min) did not affect membrane-fixed localization of DOR-EGFP. However, when cells were pretreated with PD98059 for 15 min and then exposed to etorphine (100 nM; 60 min), many more vesicles appeared in the cytosol than by etorphine treatment without this MAP kinase blocker (Fig. 6). Obviously, PD98059 enhanced DOR-EGFP internalization by this opiate. Exposure to morphine (1 µM; 60 min) in presence of PD98059 resulted in vesicle-like accumulation of DORs in the cytosol, confirming receptor endocytosis as determined by radioligand binding.
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Calphostin C and Tyrphostin Facilitate DOR Internalization by Morphine. GPCRs use multiple pathways to activate ERK/MAP kinases. With respect to µ-opioid receptors, stimulation of ERK/MAP kinases depends on activation of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K; Ai et al., 1999) as well as transactivation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR; Belcheva et al., 2001). To examine the mechanism of activating ERK/MAP kinases and controlling DOR internalization in HEK293/ cells, phosphorylation of MAP kinases by morphine was analyzed in presence of specific inhibitors for PKC, PI3K, and EGFR transactivation (Fig. 7A). Significant phosphorylation of ERKs by morphine exposure (1 µM; 5 min) was impaired when cells were pretreated with both calphostin C (1 µM; 15 min), an inhibitor of PKC activity, and by tyrphostin (5 µM; 15 min), an inhibitor of EGFR transactivation. Similarly, challenge with wortmannin (100 nM; 15 min), a selective and cell-permeable inhibitor of PI3K, abolished morphine-mediated ERK phosphorylation.
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Because morphine uses PKC, PI3K, and EGFR transactivation for MAP activation in HEK293/ cells, inhibition of these kinases may also enable DOR internalization by this opiate. Therefore, cells were pretreated with the kinase inhibitors for 15 min before morphine (1 µM) was added for 60 min. Subsequent receptor binding experiments revealed that calphostin C (1 µM) facilitated DOR internalization by morphine, resembling findings with PD98059 (Fig. 7B). Furthermore, prechallenge with tyrphostin (5 µM) also brought about a loss of surface DORs in presence of morphine. In contrast, morphine (1 µM; 60 min) completely failed to promote DOR internalization after pretreatment of cells with PI3K inhibitor wortmannin (100 nM). Exposure of cells to individual inhibitors alone at the concentrations mentioned did not affect the amount of surface DORs.
For control experiments, all kinase inhibitors used were tested for their effects on DOR internalization by etorphine (100 nM; 60 min). Calphostin C and tyrphostin did not affect the magnitude of DORs vanished from the cell surface (not shown), but etorphine totally lost its ability to sequester the receptor after wortmannin pretreatment (untreated, 57.9 ± 7.9%; wortmannin + etorphine, 56.7 ± 1.1%)
Morphine-Stimulated Arrestin Translocation. Arrestins are cytosolic adaptor proteins that are translocated to the cell membrane upon opioid receptor activation (Zhang et al., 1998). When arrestins bind to GPCRs, they direct receptors to clathrin-coated pits for their internalization. Therefore, the effect of MAP kinases blocker on arrestin translocation was examined (Fig. 8). HEK293/ cells were exposed to etorphine (100 nM) and morphine (1 µM) for 10 min, respectively, and plasma membranes were tested for associated arrestin 2 by Western blot analysis. Clearly, activation of DOR by etorphine increased the amount of arrestin at the plasma membrane, and this effect is prevented by naloxone (1 µM). Using morphine (1 µM) and PD98059 (10 µM), respectively, we failed to detect membrane-associated arrestin 2 (Fig. 8). However, marked arrestin accumulation at plasma membranes was observed when cells were challenged with morphine (1 µM; 10 min) in the presence of PD98059 (10 µM). Thus, inhibition of ERK/MAP kinase accounts for arrestin 2 accumulation at the cell membrane after activation of DORs by morphine. Etorphine (100 nM) alone already provoked maximal accumulation of arrestin 2 at plasma membranes, which is not further enhanced by PD98059 pre-treatment.
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Morphine-Induced DOR Desensitization in Presence of PD98059. Arrestin binding to GPCRs prevents receptor coupling to G proteins, which are no longer activated. Thus, morphine-induced arrestin binding in presence of PD98059 should induce receptor uncoupling as can be determined by [35S]GTPS binding (Fig. 9). Exposure to etorphine (100 nM), which activates DOR-associated G proteins, resulted in a significant increase of GTPS binding to isolated cell membranes over basal levels (Fig. 9). Pretreatment of HEK293/ cells with PD98059 (10 µM, 60 min) slightly reduced G protein activation by etorphine. When HEK293/ cells were pre-challenged with etorphine (100 nM; 60 min), subsequent DOR stimulation by this opiate failed to activate G proteins, indicating uncoupling. Receptor uncoupling also occurred after etorphine treatment in presence of PD98059 (10 µM). Because morphine is unable to translocate arrestin, pretreament with this opiate (1 µM; 60 min) only slightly affects receptor-G protein interaction. In contrast, after cell treatment with morphine and PD98059, GTPS binding was not enhanced upon DOR stimulation by etorphine anymore. Thus, morphine not only induces internalization but also DOR uncoupling in presence of the MAP kinase inhibitor PD98059.
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-opioid receptors in HEK293 cells. Apparently, the duration of ERK/MAP kinase activation (phosphorylation) may be critical for the decision to either induce receptor internalization or to leave the receptor at the cell surface. Etorphine, a ligand with very high affinity to the opioid receptors, only transiently activates ERK/MAP kinases but strongly internalizes -opioid receptors. In contrast, morphine permanently activates these kinases and fails to bring about receptor internalization. When ERK/MAP kinase activation was inhibited by the MAP kinase blocker PD98059, sequestration by etorphine is improved, and, probably more importantly, morphine gains the ability to strongly induce internalization of DORs. This line of evidence is strengthened by the ERK/MAP kinase activator glutamate and paclitaxel, which almost completely prevent etorphine to internalize DORs. Furthermore, we showed that ERK/MAP kinases control DOR internalization by affecting arrestin translocation to activated receptors.
Acute stimulation of DORs by both etorphine and morphine immediately phosphorylates ERK1/2-MAP kinases. However, prolonged exposure to one of these opiates reveals important differences in the state of phosphorylation of these kinases. Etorphine only transiently phosphorylates the kinases, and these cells exhibit an efficient sequestration of DORs. Thus, loss of ERK/MAP kinase activity may be linked to DOR internalization. Prolonged morphine exposure contrasts with these findings because this opiate brings about a continuous ERK/MAP kinase phosphorylation, but fails to internalize -receptor. However, when etorphine-induced receptor internalization was blocked by the sequestration inhibitors con A and sucrose (Mundell and Kelly, 1998), the opiate-induced MAP kinase activation still declined exhibiting a similar time course as observed in the absence of the sequestration inhibitors. Notably, con A, which inhibits receptor mobility in plasma membranes (Henis and Elson, 1981), and hypertonic sucrose, which prevents the formation of clathrin-coated pits (Heuser and Anderson, 1989), do not interfere with MAP kinase activity by themselves (Budd et al., 1999; Shah et al., 2002). Thus, ERK/MAP kinase activity seems not to be regulated by mechanisms responsible for the process of opioid receptor internalization, and similar results were reported for m3-cholinergic receptors (Budd et al., 1999). Because the activity of ERK/MAP kinases is balanced by phosphorylation and dephosphorylation, the divergent kinetics of ERK activation observed by exposure to opioids is believed to relate to individual responses of ERK regulators such as ras and c-raf (Johnson and Lapadat, 2002). In this regard, morphine might induce a different receptor conformation compared with any other opioids, e.g., etorphine, resulting in activation of distinct G proteins and signaling pathways. However, sustained MAP kinase activation by morphine may also be due to impaired dephosphorylation of ERKs. This notion is strengthen by findings that morphine reduces the action of certain phosphatases (Sood and Mohanakumar, 1985) by an increased generation of a phosphatase inhibitor (Liu et al., 2002).
ERK/MAP kinases are activated through phosphorylation by MEK, a mechanism blocked by PD98059. This MEK blocker was also shown to abolish internalization of MOR and DOR (Kramer et al., 2000; Schmidt et al., 2000) by using concentrations of the compound that are considered to bring about cytotoxic effects (Sano and Kitajima, 1998). We report here that PD98059 (10 µM) fails to affect cell viability and enables morphine to strongly induce DOR internalization. It was thus hypothesized that permanent phosphorylation of MAP kinases during morphine exposure prevents receptor internalization. This notion was strengthen by results obtained with cells exposed to the ERK activators glutamate (Stanciu et al., 2000) and paclitaxel (Okano and Rustgi, 2001). Indeed, glutamate provably brings about sustained phosphorylation of ERK/MAP kinases in HEK293/ cells. High concentrations of extracellular glutamate inhibit cystine uptake through ubiquitary expressed glutamate/cystine transporter, and activate ERK/MAP kinases by generating oxidative stress as already discussed for HT22 cells (Stanciu et al., 2000). Under these conditions even etorphine almost completely lost its strong potency to sequester DORs. A similar experimental outcome was observed with paclitaxel, which is known to both activate ERKs (Okano and Rustgi, 2001) and to stabilize microtubules (Schiff et al., 1979). However, both mechanisms require distinct kinetics because polymerization of tubulin is observed within 1 h (Rao et al., 1999), but ERK phosphorylation is delayed with about 24 h of exposure. Internalization of DORs was affected only after long-term challenge (24 h) with the compound. Inhibition of both glutamate and paclitaxel promoted ERK activation by PD98059 restored receptor sequestration by etorphine, strengthening the notion that sustained MAP activity prevents DOR internalization. Thus, DOR displays similar characteristics as the excitatory -adrenergic receptors, which also fails to internalize upon prolonged activation of ERK/MAP kinases (Lin et al., 1999).
Depending on the cell line, GPCR uses mutliple pathways to stimulate ERK/MAP kinase (Pierce et al., 2001). As demonstrated herein, activation of ERKs through DORs by morphine depends on PKC, PI3K, and EGFR activity. Examination of these individual pathways revealed differences with respect to the link of ERK activation and inhibition of DOR internalization. Elimination of PKC activity by calphostin C and blockade of EGFR transactivation by tyrphostin were found to enable morphine to internalize DORs. Opioid receptors mediate EGFR transactivation by activation of PKC (Belcheva et al., 2001), and active EGFRs in turn stimulate PKC (Yarden and Sliwkowski, 2001). Because blockade of these PKC-involving pathways as well as inhibition of MEK-mediated ERK/MAP phosphorylation facilitate morphine-induced DOR internalization, we believe that PKC-mediated ERK/MAP kinase activation prevents DOR internalization. A PKC-dependent control of receptor internalization was already suggested by Ueda et al. (2001), who demonstrated a morphine-induced endocytosis of MORs after PKC inhibition. Notably, PI3K stimulation also accounts for ERK/MAP kinase activation by DORs, but inhibition of the kinase by wortmannin failed to facilitate receptor internalization by morphine. Opioid receptors may activate PI3K by both the release of G subunit of activated G proteins associated with DORs, and by transactivation of EGFRs, which promotes PI3K stimulation (Yarden and Sliwkowski, 2001). These two distinct pathways of PI3K activation might bring about distinct kinase effects with respect to signaling and modulation of cellular responses. Stimulation of PI3K by EGFR leads to MAP kinase activation (Daub et al., 1997), and wortmannin might block ERK phosphorylation interfering with morphine promoted EGFR signaling. Moreover, PI3K accounts for membrane trafficking by regulating phosphoinositides (Gaidarov and Keen, 1999). These complex interactions are likely to involve also arrestin, whose function depends on phosphoinositide binding (Gaidarov et al., 1999) and thus PI3K activity (Naga Prasad et al., 2002). This adaptor protein promotes recruitment of receptors to clathrin-coated pits and sequestration (Bünemann and Hosey, 1999). With regard to this critical function of PI3K, inhibition of the kinase thus blocked receptor internalization, including the -receptor.
Opioid receptor internalization draws upon receptor binding to arrestins. In contrast to etorphine, we observed only a weak translocation of arrestin 2 in the presence of morphine. Inhibition of morphine-mediated ERK/MAP kinase activity, however, strongly facilitated arrestin 2 accumulation at the DOR carrying plasma membrane. Thus, impaired arrestin translocation may be linked to an activation of ERKs. This assumption is confirmed by studies possibly offering an explanation for our observation. For instance, phosphorylated and thus active ERK/MAP kinases form cytosolic complexes with arrestins (DeFea et al., 2000). These complexes may be retained in the cytoplasm (Tohgo et al., 2002), preventing a sufficient concentration of arrestin at the receptors located in the plasma membrane. By decrease or loss of ERK activity (e.g., by PD98059), ERK-arrestin binding might be abrogated, allowing translocation of sufficient amounts of arrestin to the cell membrane where it promotes internalization of GPCRs. This notion is strengthened by morphine induced DOR uncoupling in presence of PD98059. Besides induction of internalization, arrestin binding prevents G protein coupling to receptors. Morphine does not desensitize opioid receptors because of its failure to translocate arrestin (Whistler and von Zastrow, 1998). In the presence of PD98059, sufficiently arrestin 2 is translocated for receptor desensitization by morphine, which is convincingly observed in cells overexpressing arrestin (Whistler and von Zastrow, 1998). In summary, our results show that prolonged ERK/MAP kinase activation prevents uncoupling and internalization of opioid receptors by morphine. Because the failure of morphine to desensitize and internalize opioid receptors implicates a persistent opioidergic signal transduction and thus may add to the high addiction liability of morphine, further analysis has to clarify the role of ERK/MAP kinases for the mechanism underlying opiate addiction.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; DOR, -opioid receptor; MOR, µ-opioid receptor; ERK1/2, extracellular signal-regulated protein kinase 1/2; MAP, mitogen-activated protein; HEK, human embryonic kidney; EGFP, enhanced green fluorescent protein; PD98059, 2'-amino-3'-methoxyflavone; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; con A, concanavalin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS, phosphate-buffered saline; EGFR, epidermal growth factor receptor; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; GTPS, guanosine 5'-O-(3-thio)triphosphate; suc, sucrose; MEK, mitogen-activated protein kinase kinase.
Address correspondence to: Dr. Daniela A. Eisinger, Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Koeniginstrasse 16, 80539 Munich, Germany. E-mail: eisinger{at}pharmtox.vetmed.uni-muenchen.de
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