From the Laboratoire de Pharmacologie Moléculaire de la Tolérance aux Opiacés, Université de Caen, Centre Hospitalier et Universitaire de Caen, 14033 Caen Cedex, France
Received for publication, January 6, 2003 , and in revised form, March 31, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most of the studies carried out on opioid receptor desensitization and internalization have been realized on transfected non-neuronal cells overexpressing opioid receptors, which is known to directly affect the rate of both sequestration and desensitization (11). The SK-N-BE cells represent a more suitable model to study these phenomena for several reasons. (i) This cell line presents the neuronal phenotype. (ii) It is a human cell line, and (iii) it expresses only endogenously -opioid receptors at physiological levels (12, 13). Upon chronic activation, hDOR undergo a rapid desensitization that was observed on the inhibition of adenylate cyclase (14, 7). Recently, we reported a differential regulation of those receptors by peptide and alkaloid agonists, which was characterized by a more robust desensitization in the presence of DPDPE and Deltorphin I than in the presence of etorphine (15) and by a differential G protein activation (16).
To address the possibility that this differential desensitization could be related to differences in opioid receptor trafficking, we performed a detailed comparison of hDOR internalization, down-regulation, recycling, desensitization, and resensitization produced by peptide (DPDPE and Deltorphin I) and alkaloid (etorphine) agonists. Visualization of receptor internalization was realized using immunofluorescence experiments within the SK-N-BE cells expressing the FLAG-tagged hDOR. Opioid receptor down-regulation and recycling were performed in SK-N-BE cells with binding studies to quantify the number of opioid binding sites. Desensitization and resensitization were determined by functional assays on the inhibition of cAMP accumulation.
In this work, we demonstrated a complex relationship between hDOR internalization/down-regulation and desensitization. We found that the profound desensitization of hDOR promoted by DPDPE and Deltorphin I was correlated with a marked down-regulation of the receptor. While etorphine promoted a strong hDOR desensitization and internalization after a 60-min period, we found no evidence for down-regulation. Exploring the postendocytic fate of hDOR demonstrated that the differential targeting, either to lysosomes for peptide agonists or to recycling endosomes for etorphine, would account for the different rate of desensitization rather than down-regulation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To visualize hDOR internalization by fluorescence and confocal microscopy, SK-N-BE cells were transfected by electroporation with pcDNA3 alone or containing the cDNA encoding for the FLAG-tagged-hDOR (pcDNA3-hDOR). Stably transfected cells were obtained after selection with 0.5 mg/ml geneticin (Sigma), and the whole pool of resistant cells was used without clonal selection.
Binding ExperimentsRadioligand binding studies were performed on attached cells as described previously (4). Briefly, SK-N-BE cells were seeded in 24-well plates at a density of 75,000100,000 cells/well. Monolayers of cell were treated for various times (30 or 60 min) in the presence of peptide or alkaloid agonists at 37 °C in DMEM/20 mM Hepes buffer. When inhibitors of either internalization (0.5 M sucrose) or lysosomal degradation (200 µM chloroquine) were used, cells were preincubated with the inhibitor for 30 or 5 min, respectively. In recycling assays, the medium was removed, and SK-N-BE cells were left in DMEM/20 mM Hepes buffer for 30 min. Before binding studies, cells were rinsed with DMEM/20 mM Hepes buffer followed by phosphate-buffered saline (PBS)/0.2% (w/v), bovine serum albumin (BSA) and incubated for 30 min at 37 °C with appropriate concentrations of [3H]diprenorphine (PerkinElmer Life Sciences) (0.055 nM) in the presence (nonspecific binding) or in the absence (total binding) of 20 µM levorphanol (Sigma), in a final volume of 300 µl of 50 mM Tris-HCl, 1% (w/v) BSA, pH 7.4. The efficiency of washing procedure was checked using control and agonist-pretreated cells. Under these conditions it is worthy to note that no modification of [3H]diprenorphine binding was detected, indicating that the agonist was completely removed (data not shown). Cells were harvested in 200 µl of 1 M NaOH and placed into vials in the presence of 3 ml of a scintillation mixture (PicoFluor-40, Packard). Vials were counted for radioactivity in a scintillation counter (Packard). Each determination was carried out in triplicate. All experiments were repeated at least three times with similar results. Scatchard analysis was performed using Ligand software (17) to calculate Kd and Bmax values.
Determination of cAMP AccumulationInhibition of adenylate cyclase was determined by measuring [3H]cAMP accumulation. SK-N-BE cells were seeded in 24-well plates at a density of 100,000150,000 cells/well in the presence of 0.6 µCi/well [3H]adenine (PerkinElmer Life Sciences) for 1215 h. Then, cells were challenged with or without different agonists, in the presence of 1 mM isobutylmethylxanthine (IBMX) and 40 µM forskolin for 5 min at 37 °C. When using inhibitors, SK-N-BE cells were pretreated for 5 min (200 µM chloroquine) or 30 min (0.5 M sucrose and 50 µM monensin). The reaction was stopped by removing the medium and by addition of 5% (w/v) trichloroacetic acid. [3H]cAMP was separated by chromatography on acid alumina columns, mixed with 8 ml of scintillation mixture (PicoFluor-40, Packard), before assaying in a scintillation counter (Packard). Percentage of inhibition was calculated according to the following formula: (1(cpm agonistcpm basal)/(cpm forskolincpm basal)) x 100, where cpm basal was determined in a medium containing only IBMX and cpm forskolin in the presence of forskolin + IBMX. All experiments were carried out in triplicate.
Western Blot AnalysisSK-N-BE cells were harvested in lysis buffer (50 mM Tris-HCl, 0.1% (v/v) Nonidet P-40, pH 7.4) and sonicated after a preincubation period of 30 min in DMEM with or without opioid agonists (10 nM Deltorphin I, 100 nM etorphine, and DPDPE). Equal amounts of protein were resolved on 10% (w/v) acrylamide gels by SDS-PAGE and transferred onto nitrocellulose membranes over 45 min at a constant voltage of 15 V. Membranes were incubated for 2 h in blocking solution (PBS, 5% (w/v) nonfat dry milk), and rabbit polyclonal antibodies directed against amino acids 317 of mDOR (Oncogene) were added for 15 h at 4 °C in a final dilution of 1:1,000 in PBS, 2% (w/v) nonfat dry milk. After washing in PBS, 0.1% (v/v) Tween-20, nitrocellulose sheets were incubated with peroxidase-conjugated anti-rabbit IgG antibodies (DAKO) (1:1,000 in PBS, 1% (w/v) nonfat dry milk) for 34 h. hDOR was revealed using the enhanced chemiluminescence system (PerkinElmer Life Sciences).
Immunolocalization of -Opioid Receptors by Fluorescence MicroscopySK-N-BE cells stably expressing FLAG-tagged hDOR were grown on alcohol-cleaned glass coverslips in 24-well plates in culture medium containing 0.5 mg/ml geneticin until 4060% confluence. Then, cells were either exposed to vehicle (control) or different agonists as described above in DMEM/20 mM Hepes. When inhibitors of internalization (sucrose), recycling (monensin), or lysosomal degradation (chloroquine) were used, cells were pretreated under similar conditions as described above. The medium was rapidly removed, and cells were fixed using a fresh solution of 4% (w/v) paraformaldehyde for 15 min and subsequently permeabilized with 0.1% (w/v) saponin. Cells were rinsed thoroughly with PBS. After blocking with PBS, 1% (w/v) BSA for 1 h at room temperature, cells were incubated with the monoclonal M2 anti-FLAG antibody (Sigma) (5 µg/ml) in the blocking buffer for1hat37 °C. After washing with PBS, cells were incubated with a 1:200 dilution of fluoresceine isothiocyanate-conjugated goat anti-mouse IgG (Sigma) for 2 h at room temperature in the blocking buffer. Coverslips were washed with PBS and mounted.
Immunostaining was visualized either with a Zeiss confocal laser microscope (LSM-310) or with a Zeiss Axiovert (Lens X63). Photographs shown in this article were obtained with a CCD camera.
Colocalization of FLAG-tagged hDOR with Lysobisphosphatidic acid (LBPA)SK-N-BE cells stably expressing FLAG-tagged hDOR were grown and exposed to different agonists as described above. The medium was rapidly removed, and cells were fixed using a fresh solution of 4% (w/v) paraformaldehyde for 15 min and subsequently permeabilized with 0.1% (w/v) saponin. Cells were rinsed thoroughly with PBS. After blocking with PBS, 1% (w/v) BSA for 30 min at room temperature, cells were incubated with the rabbit polyclonal anti-FLAG antibody (5 µg/ml) in the blocking buffer for1hat37 °C. After washing with PBS, cells were incubated with a 1:200 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) for 2 h at room temperature in the blocking buffer. Cells were rinsed with PBS and incubated with the mouse anti-LBPA antibody with a 1:150 dilution in PBS, 1% (w/v) BSA, 0.05% (w/v) saponin for 1 h at room temperature. After rinsing with PBS, cells were incubated with a 1:400 dilution of tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse IgG1 antibody (Southern Biotechnology Associates) for 2 h at room temperature in the blocking buffer. Coverslips were washed with PBS and mounted.
Images were collected with a Nikon confocal microscope (Eclipse TE-2000, lens x60) and processed with Adobe Photoshop software. After overlaying green and red labeling, we extracted only the overlapping signal that was converted into grayscale. The merged pictures represent colocalization of FLAG-tagged hDOR with LBPA.
Statistical AnalysisAll results are expressed as the mean ± S.E. of n experiments. ANOVA followed by the Bonferroni-Dunn test or Student's t test as appropriate was used to determine the statistical significance (Statview, Abacus).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Western blot analysis was performed to correlate the reduction of [3H]diprenorphine binding sites with the hDOR immunoreactivity in naive and agonist-treated SK-N-BE cells (Fig. 1B). The polyclonal mDOR antibody detected a major band of 50 kDa in the whole SK-N-BE cell extracts whereas in human fibroblast, no labeling could be observed (data not shown) demonstrating the specificity of the mDOR antibody. A decrease of hDOR immunoreactivity was evident in cells treated with peptides (Dp, Del) for 30 min compared with naive cells (cont) while no significant modification was detected after etorphine pretreatment (Fig. 1B).
Internalization of -Opioid Receptors Induced by Peptide and Alkaloid AgonistsLocalization of hDOR in SK-N-BE cells was determined by immunocytochemical experiments in naive and agonist-treated cells using the polyclonal mDOR antibody. Under these conditions, a weak but specific immunolabeling was observed as a sharp staining at the plasma membrane of naive cells. However, upon agonist treatment we could only detect a loss of the staining without clearly visualizing opioid receptor internalization. To overcome this problem, immunofluorescence experiments on FLAG-tagged hDOR cells were conducted. Binding experiments revealed about 1520-fold more opioid receptors in FLAG-tagged hDOR cells compared with SK-N-BE cells (Table I) and pcDNA3-transfected cells (data not shown). Overexpression of FLAG-tagged hDOR does modify neither the affinity of
-opioid receptors for [3H]diprenorphine nor the ability of the different agonists to promote opioid receptor desensitization (Table I).
|
While in non-transfected (data not shown) or pcDNA3-transfected SK-N-BE cells, no staining was detected (Fig. 2, pcDNA3), a strong immunofluorescent labeling could be visualized in FLAG-tagged hDOR-transfected cells (Fig. 2, Cont). Using confocal microscopy and sequential sections of cells, we confirmed that the immunostaining was confined to the plasma membrane.
|
After 30 min of DPDPE and Deltorphin I exposure, an obvious loss of hDOR immunoreactivity from plasma membrane associated with a concomitant appearance of cytoplasmic punctiform staining was observed (Fig. 2, Dp 30 and Del 30). As shown in Fig. 2 (Eto 60), when FLAG-tagged hDOR-transfected cells were incubated in the presence of the alkaloid agonist for 30 (data not shown) or 60 min, a strong vesicular labeling was observed with a marked decrease of the plasma membrane staining. Analysis using confocal microscopy confirmed the cytosolic localization of the internalized opioid receptor.
Recycling and Resensitization of hDORTo investigate the recycling process of hDOR in an active state, SK-N-BE and FLAG-tagged hDOR transfected cells were either treated for 30 min with DPDPE and Deltorphin I or 60 min with etorphine to obtain a strong internalization and almost a similar desensitization (14, 15). Then, cells were placed in an agonist-free medium for 30 min to allow the recycling of opioid receptors. Under these conditions and concerning the alkaloid agonist, immunofluorescence assay revealed a reappearance of the staining at the plasma membrane consistent with binding experiments showing an identical level of opioid receptors as in naive cells (Figs. 1A and 2, Eto 60 + 30). Conversely, for peptide agonists, a substantial loss of [3H]diprenorphine binding sites was still noticed (30 ± 7.1% for DPDPE and 28.7 ± 10% for Deltorphin I) (Fig. 1A). As depicted in Fig. 2 and in these conditions, we still noticed few scattered intracellular vesicles but associated with a progressive reappearance of the immunostaining localized at the plasma membrane demonstrating a partial recycling process (Fig. 2, Dp 30 + 30 and Del 30 + 30).
Functional experiments were further conducted on the inhibition of adenylate cyclase to ensure that the recovery of opioid binding sites was associated with a hDOR resensitization. When activated by the alkaloid and peptide agonists, hDOR inhibited cAMP accumulation by 50 and 40%, respectively. To ensure comparison between agonists, the inhibition produced by each molecule was referred as 100% in naive cells. SK-N-BE cells were first incubated for 30 or 60 min in the presence of peptides or etorphine, respectively, to promote a similar desensitization of about 8090% (Fig. 3). hDOR were allowed to resensitize in an agonist-free medium for various times (15 and 30 min), and then the inhibition produced by each agonist was measured. Fig. 3 shows that etorphine-pretreated cells displayed a faster resensitization than peptide-treated cells. However, this difference of resensitization between etorphine and peptides reached a statistical significance only after a 30-min period in agonist-free medium.
|
Lysosomal Degradation after Peptide-induced hDOR SequestrationAfter internalization, GPCR are either recycled to plasma membrane, trapped in endosomal compartments or targeted to lysosomes. The latter contains proteases that can be inhibited by chemicals such as chloroquine by elevating the luminal pH. Data presented in the Figs. 1, 2, 3 strongly suggested that peptide-induced hDOR internalization would be followed by their degradation since, unlike etorphine, no complete recovery of opioid receptors was observed when cells were left in agonist-free medium for 30 min. To assess this hypothesis, binding, immunofluorescence, and functional studies were carried out using 200 µM chloroquine, a concentration known to inhibit mDOR degradation in lysosomes (10). SK-N-BE cells were pretreated with chloroquine for 5 min and, to induce down-regulation and desensitization, agonists were then added in the same conditions as described above. As shown in Fig. 4A, chloroquine pretreatment did not affect the loss of opioid receptors induced by etorphine. In contrast, we could observe a significant decrease in the ability of peptide agonists (from 50 to 95%) to induce down-regulation of hDOR but with a more pronounced effect for Deltorphin I than DPDPE. Immunoblot experiments were conducted in the presence or in the absence of the lysosomal proteases inhibitor and showed no difference between naive cells indicating that chloroquine was devoid of any effect on basal level of hDOR. Data presented in Fig. 4B indicated that peptide-induced decrease of hDOR immunoreactivity, observed in Fig. 1B, was totally blocked by chloroquine pretreatment.
|
Effects of chloroquine were further explored in internalization experiments. SK-N-BE cells stably expressing FLAG-tagged hDOR were treated or not for 30 min with DPDPE or Deltorphin I in the presence or in the absence of chloroquine. All these results are depicted in Fig. 4C and suggest both an enhancement of the intracellular vesicle staining and the number of those vesicles compared with chloroquine-untreated transfected cells (Fig. 4C, see arrows).
To corroborate all the data obtained with chloroquine that strongly argued for an opioid receptor degradation in lysosomes after peptide pretreatment, we investigated a putative localization of the receptor with lysobisphosphatidic acid. This phospholipid is found in late endosomes and thus considered as a marker of the degradative pathway (18). After ensuring that secondary antibodies did not cross-react with the inappropriate primary antibody, we were unable to observe any colocalization of the FLAG-tagged hDOR with LBPA in naive cells (Fig. 5, Cont). In peptide-treated cells, we clearly observed a colocalization of internalized opioid receptor with the late endosomes marker (Fig. 5, Dp 30 and Del 30, arrows). Conversely, despite a massive opioid receptor internalization promoted by etorphine, colocalization of hDOR with LBPA is lacking (Fig. 5, Eto 60).
|
In order to explore the functional consequences of chloroquine effects, desensitization, and resensitization experiments were conducted. In naive cells, no marked modification of the inhibitory action of agonists was observed (data not shown). After normalization of data, no substantial effect of chloroquine was detected either on desensitization or resensitization, whatever the agonist used (Fig. 6, AC).
|
Evidence for hDOR Recycling upon Etorphine Exposure Data presented in Figs. 1 and 2 strongly suggest that upon etorphine activation, hDOR would be recycled after its endocytosis. To test this hypothesis, we examined opioid receptor desensitization, internalization and recycling in the presence or in the absence of a recycling blocker, monensin, after etorphine treatment. To avoid a maximum rate of desensitization, SK-N-BE cells were exposed for 30 min with 100 nM etorphine. In the absence of monensin, etorphine desensitized hDOR by 30% whereas a 30-min pretreatment with 50 µM monensin dramatically increased receptor desensitization by 60% (Fig. 7A). Monensin experiments were also conducted with peptide agonists. However, this recycling inhibitor dramatically reduced the inhibitory effects of peptides on adenylate cyclase by more than 50% (data not shown). Despite this unexpected effect and after normalization of values with monensin-pretreated cells, we were unable to detect any action of monensin on peptide-induced desensitization (data not shown).
|
In immunofluorescence studies, monensin pretreatment appeared to enhance both the size and the staining of cytoplasmic vesicles after etorphine exposure (Fig. 7B). When etorphine was removed and cells were left in agonist-free medium supplemented with monensin for 30 min, we noticed that FLAG-tagged hDOR failed to recycle back to the plasma membrane (Fig. 7B).
Identification of hDOR Internalization PathwaysGPCR sequestration could be mediated either by the clathrin-coated pits pathway or by an alternative endocytic pathway involving caveolin (19). The differential sorting of opioid receptor could result from distinct endocytic pathways. To answer to this question, sucrose hypertonic solution was used to block the clathrin-dependent internalization. SK-N-BE cells were pretreated for 30 min with or without 0.5 M sucrose, and the agonist-induced opioid receptor decrease was measured. After normalization of Bmax values, we did not observe any significant effect of sucrose pretreatment on the basal hDOR level in naive cells (100% for control versus 90 ± 14.6% for sucrose-pretreated cells). While all opioid agonists were able to promote loss of opioid receptors to a similar extent as shown in Fig. 1A, no substantial reduction of [3H]diprenorphine binding sites was observed when SK-N-BE cells were pretreated with hypertonic sucrose solution (Fig. 8A).
|
To ensure that 0.5 M sucrose efficiently blocks hDOR internalization, immunofluorescence experiments using FLAG-tagged-hDOR transfected cells were conducted. As depicted in Fig. 8B, sucrose pretreatment totally prevents FLAG-tagged receptor internalization induced by peptide and alkaloid agonists.
Relationship between hDOR Internalization and DesensitizationAs demonstrated for some GPCR, internalization is a prerequisite for their desensitization by reducing active receptors at the plasma membrane (8). Functional studies were conducted to establish the relationships between sequestration and desensitization in our cellular model. SK-N-BE cells were pretreated with or without 0.5 M sucrose for 30 min, then opioid receptor desensitization was induced by etorphine (100 nM, 60 min), DPDPE (100 nM, 30 min), or Deltorphin I (10 nM, 30 min). In sucrose-pretreated cells, we observed a significant reduction of hDOR desensitization by 40% only in the presence of etorphine (ANOVA, Bonferroni-Dunn test, p < 0.05) (Fig. 9). Concerning peptide agonists, we were unable to see any significant effect of hypertonic sucrose solution on opioid receptor desensitization after 30 min (Fig. 9).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To answer to this question, immunocytochemical studies were conducted to visualize hDOR internalization, and the quantification of down-regulation was achieved by binding experiments. Using SK-N-BE cells transfected with FLAG-tagged hDOR, to increase the opioid receptor fluorescence staining, we observed a massive and similar internalization after peptide agonists but also after etorphine pretreatment suggesting that the differential desensitization of opioid receptor is not directly linked to its endocytosis and that other mechanisms could occur after receptor sequestration. However, after 30-min exposure, we showed a significant difference in the loss of opioid binding sites between DPDPE and Deltorphin I, on one hand, and etorphine, on the other hand. These observations are correlated with the marked reduction of hDOR immunoreactivity detected on immunoblot experiments in peptide-pretreated cells only demonstrating a degradation of receptors. Comparison between down-regulation data and desensitization experiments suggests that the important decrease of active opioid receptors at the plasma membrane would participate in the strong desensitization produced by DPDPE and Deltorphin I. A similar observation was reported by Prather et al. (24), who observed that hDOR desensitization was associated with their down-regulation following DPDPE exposure in SH-SY5Y cells.
So, we explored the relationship between the disappearance of opioid receptors from the cell surface and the desensitization process using hypertonic sucrose solution to prevent hDOR internalization via the clathrin-coated pits pathway. If the inhibitory response of opioid agonists was directly linked to the level of opioid receptors at the cell surface, we expected to observe a decrease of desensitization in sucrose-pretreated cells. Despite of an effective blockade of hDOR internalization and down-regulation by sucrose, we were unable to observe any effect of this treatment on peptide-induced desensitization. However, a partial reduction by 50% of etorphine-induced desensitization was measured when opioid receptors endocytosis was impaired. These functional data using sucrose demonstrate that internalization is partially responsible for etorphine-induced desensitization by reducing the number of active receptors at the cell surface as demonstrated for the µ-opioid receptor activated by [D-ala2, N-MePhe4, Gly-ol5]enkephalin (25). The sucrose-insensitive desensitization would be probably due to opioid receptor uncoupling from G proteins as demonstrated for the majority of GPCRs (26). On the other hand, peptide-induced desensitization of hDOR does not require receptor endocytosis as shown by sucrose experiments. So, we can assume that desensitization would be produced either by receptor uncoupling or down-regulation at the plasma membrane as demonstrated for the 2-adrenergic receptor (27). In the case of a cell surface degradation of opioid receptors, sucrose pretreatment would be unable to affect down-regulation. However, as we observed an effective blockade of down-regulation by sucrose, we can reasonably suppose that receptor uncoupling is the main process involved in peptide-induced desensitization. These data revealed an underestimated complexity between receptor endocytosis, down-regulation, and signaling upon peptide and alkaloid agonist treatment. So we can assume that the difference in hDOR down-regulation is not per se responsible for the differential desensitization produced by chemically different agonists but probably involves other molecular mechanisms and consequently suggests an unexpected behavior of hDOR trafficking depending on the agonist.
Next, we explored the mechanisms underlying the quantitative difference in down-regulation observed between etorphine and peptides. First, we hypothesized that this differential hDOR down-regulation could result from receptor internalization by different endocytic pathways. GPCR internalization, including opioid receptors, is mainly achieved via the clathrindependent pathway (4, 6, 28) but the role of caveolae has been also demonstrated for B2 bradykinin, angiotensin II type 1, and endothelin type A receptors (2931). When examining the endocytic pathway using hypertonic sucrose solution as a classical inhibitor of clathrin-dependent internalization, we showed that whatever the agonist used, hDOR were internalized into clathrin-coated pits. These data can rule out our first hypothesis. Second, we can assume that peptides and etorphine would promote a differential sorting of hDOR after its internalization. In the presence of DPDPE and Deltorphin I, opioid receptors would be preferentially targeted to lysosomes to be degraded and consequently down-regulated while with the alkaloid agonist, these receptors would be trapped into endosomes and then recycled to the cell surface. This hypothesis is based on the studies of von Zastrow's group (10) who showed that two different GPCRs, the 2-adrenergic receptor and the mDOR, can undergo differential sorting responsible for a down-regulation in the case of opioid receptor. In Western blot experiments, we were unable to observe a decrease of hDOR immunoreactivity upon etorphine treatment suggesting that the opioid receptors were not degraded. Binding and immunocytochemical experiments showed that hDOR could recycle to plasma membrane and adenylyl cyclase assays revealed that these receptors were in an active state. In contrast, when SK-N-BE cells were challenged with peptide agonists, we showed a marked hDOR degradation on immunoblot, a poor recycling process correlated with a weak resensitization. All these data strongly argue for hDOR targeting either to lysosomes following peptide exposure or to recycling endosomes after etorphine treatment.
This differential targeting was evidenced using a recycling inhibitor, monensin and a lysosomal proteases blocker, chloroquine. Indeed, monensin both potentiated desensitization produced by etorphine and almost prevented hDOR recycling demonstrating that opioid receptors were mainly sequestrated into recycling endosomes. In this case, hDOR internalization would allow dephosphorylation of inactive receptors into endosomes and their redistribution to the plasma membrane as we previously demonstrated (4). When regarding peptide agonists, we showed that chloroquine effectively blocked hDOR degradation as observed on Western blot and in binding experiments. These data strongly suggest that DPDPE and Deltorphin I promote opioid receptor degradation in lysosomal compartments following their internalization. However, it is noteworthy that chloroquine blocked only partially DPDPE-induced down-regulation, suggesting an alternative mechanism that could involve the ubiquitin-proteasome pathway as demonstrated for the murine µ- and -opioid receptors expressed in HEK293 cells (32). So, the concept of a different hDOR regulation by peptide and alkaloid agonists seems to be attractive but regulatory mechanisms of these receptors are probably more subtle. The colocalization of internalized receptor with LBPA, described by Kobayashi et al. (18) as a late endosome marker, and thus a marker of the degradative pathway, supported the lysosomal targeting of opioid receptor upon peptide exposure. All those data clearly demonstrate that DPDPE and Deltorphin I promote hDOR degradation in lysosomes. A similar lysosomal degradation process was described for the mouse
-opioid receptor expressed in Neuro2A cells after [D-ala2, D-Leu5] enkephalin (DADLE) treatment but with longer time exposure (24 h) (5). Unexpectedly, we observed the lack of any effect by chloroquine on both desensitization and resensitization despite an effective blockade of hDOR degradation. These data suggest that once trapped into lysosomes, the receptor is unable to recycle in an active state assuming that the targeting of opioid receptor into lysosomal compartments is quite irreversible.
The time of agonist pretreatment is another essential parameter for the opioid receptor fate following internalization. Indeed, our data showed that hDOR is differentially regulated by peptides and etorphine after short time exposure (3060 min) but when the time treatment is prolonged until 4 h, the opioid receptor behavior upon etorphine exposure is similar to those observed after DPDPE and Deltorphin I. Under these conditions, resensitization and recycling processes are no longer noted indicating that hDOR are down-regulated (4). These data suggest that despite early distinct molecular processes, largely depending on chemically different agonists, the opioid receptors would converge toward down-regulation independent of the chemical nature of the agonist after long term agonist exposure.
Molecular mechanisms underlying the differential sorting of internalized GPCR either to recycling endosomes or lysosomes are still poorly understood but recent data pointed out the role of accessory proteins such as arrestins and the recently cloned GASP (GPCR-associated sorting protein) (33). By switching the C-terminal tail of 2-adrenergic and vasopressin V2 receptors, Oakley et al. (34) showed that the presence of serine/threonine residue clusters of the vasopressin V2 receptor allowed strong interactions with
-arrestins, and consequently, the co-internalization of receptors with arrestins would avoid a rapid recycling by trapping those complexes in intracellular compartments. In our cellular model, we observed that different kinases are involved in peptide and alkaloid agonist-induced hDOR desensitization.2 Accordingly, the different phosphorylation state of hDOR promoted by peptides and etorphine would modulate its affinity for
-arrestins. Upon peptide activation, the phosphorylated hDOR would form high affinity complexes with
-arrestins, which could be retained into endosomes and turned toward lysosomes. Conversely for the alkaloid agonist, the lack of co-internalization of hDOR with
-arrestins would mainly direct the receptor to the recycling pathway. Interestingly, Whistler et al. (33) discovered a new factor that acts as a post-endocytic sorting protein. Indeed, when this protein interacts with the C-terminal region of mDOR, it follows its degradation in lysosomes while in the case of the µ-opioid receptor, which weakly binds GASP, a major recycling process is observed after agonist treatment. The role of this protein should be explored in our cellular model.
In conclusion, we demonstrated that hDOR internalization and its sorting either to endosomes or lysosomes are important processes that participate in the regulation of cell responsiveness to opioid agonists. For the alkaloid agonist, hDOR internalization has a dual role: by reducing active receptors, internalization initiates desensitization, and by allowing its dephosphorylation and recycling, it reduces the rate of desensitization. While hDOR internalization is not the primary event responsible for peptide-induced desensitization, it potentiates this process by promoting its degradation into lysosomes. Our data demonstrate for the first time that the same receptor could be differently targeted to endosomes or lysosomes by chemically distinct opioid agonists. The poor propensity of a given agonist to induce tolerance would be linked to its ability to promote opioid receptor internalization and recycling as it was proposed by Finn and Whistler (35). Indeed, they showed that the "cellular tolerance" to morphine was higher in cells expressing a chimeric µ receptor targeted to lysosomes (D MOR) compared with those expressing R MOR, a mutant that recycled following its activation. In this study, we came to the same conclusions as Finn and Whistler (35), showing that opioid receptor desensitization was linked to receptor degradation.
![]() |
FOOTNOTES |
---|
Recipient of a fellowship from the Ministère de l'éducation nationale, de la recherche et de la technologie and from the Fondation pour la recherche médicale.
To whom correspondence should be addressed: Laboratoire de Biochimie A, avenue Câte de Nacre, CHU de Caen, 14033 Caen cedex, France. Tel.: 33-231064560; Fax: 33-231064985; E-mail: allouche-s{at}chu-caen.fr.
1 The abbreviations used are: MOR, µ-opioid receptors; mDOR, mouse -opioid receptors; DPDPE, [D-Pen2,5]enkephalin; Deltorphin I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2; GPCR, G protein-coupled receptor; hDOR, human
-opioid receptors; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ANOVA, analysis of variance; LBPA, lysobisphosphatidic acid.
2 S. Allouche, N. Marie, A. Hasbi, and P. Jauzac, manuscript in preparation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|