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J. Biol. Chem., Vol. 278, Issue 38, 36848-36858, September 19, 2003
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From the Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455-0217
Received for publication, February 13, 2003 , and in revised form, June 3, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-opioid receptors (MOR and DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. By exchanging the carboxyl tail domains of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A cells, it could be demonstrated that the carboxyl tail domain is not the sole determinant in directing the intracellular trafficking in these Neuro2A cells. Deletion of the dileucine motif (Leu245-Leu246) within the third intracellular loop of DOR or the mutation of Leu245 to Met slowed the lysosomal targeting of these -opioid receptors. Meanwhile the mutation of Met264 to Leu increased the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the -opioid receptor carboxyl tail chimera of the µ-opioid receptor. These studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-adrenergic and A2-adenosine receptor required the receptor internalization and trafficking to the endosomes (5, 6). Activation of the mitogen-activated protein (MAP) kinases by 2-adrenergic receptor could be blocked by inhibitors of receptor endocytosis, such as dominant negative arrestin (7). Activation of the MAP kinases by 
2-adrenergic receptor was reported also to be dependent on the endocytosis of the receptor but appears to be cell line-specific (8-10). The fate of the activated MAP kinases was shown to be dependent on the internalized GPCR. This was demonstrated by the nonendocytosed mutant of PAR2 receptor-activated MAP kinases to translocate to nucleus, whereas the endocytosed PAR2 receptor-activated MAP kinases remained in the cytosol (11). Thus, the GPCR signaling and the consequence of the signals are influenced by the receptor trafficking.
Opioid receptor belongs to the rhodopsin subfamily of the superfamily of GPCRs. From the early studies reported by Chang et al. (12) and Law et al. (13), it is clear that opioid agonist would induce -opioid receptor (DOR) internalization and subsequent down-regulation in neuroblastoma cells. Similar down-regulation of µ-opioid receptor (MOR) was observed in the neuroblastoma SHSY5Y cells (14). Further, the internalized receptors were trafficked to the endosomes and subsequently to lysosomes as demonstrated with the intracellular accumulation of the ligand-receptor complexes in the presence of chloroquine (15). Such itinerary of the opioid receptor was substantiated after the cloning of the opioid receptors. Using epitope-tagged opioid receptor, the agonist-induced endocytosis of the receptors was demonstrated to involve the arrestin- and dynamin-dependent clathrin-coated pits pathway (16-20), with the internalized DOR trafficked rapidly to the lysosomal compartments (21), whereas MOR and its spliced variants have distinct properties to recycle and resensitize (22-25).
Similar to other GPCRs, the importance of the carboxyl tails in the trafficking of the opioid receptors has been established. This was best exemplified by the ability of agonist to down-regulate the µ/-opioid receptor chimeras more rapidly (26), and morphine could internalize the µ/ receptor chimeras but not the wild type receptor (27). Truncation of the carboxyl tail sequences also resulted in the blockade of agonist-induced MOR internalization (28) or the release of a brake mechanism in the DOR internalization (29). The importance of the carboxyl tail is demonstrated recently as the recognition motif for cellular proteins such as GASP that participate in lysosomal trafficking of DOR (30).
The exact motif within the carboxyl tail sequence in directing the opioid receptor lysosomal targeting remains unknown. Previous studies indicated that the mutation of Ser356 and Ser363 to Ala resulted in the blockade of MOR down-regulation (28). However, other receptor sequences could participate also in the intracellular trafficking of the receptor. Mutation of Tyr336 of MOR within the highly conserved NPX2-3Y motif of GPCR resulted in the attenuation of receptor down-regulation (31). This NPXY motif, where X is any amino acid, has been identified as consensus binding sequence for adenosine diphosphateribosylation factor (32), which has a regulatory role in the endocytosis and recycling of the transferrin receptor (33) and targets the recycling vesicles to the plasma membrane (34). Further investigation of the NPX2-3Y motif revealed that it was required for receptor interaction with agonist, G proteins, and GPCR kinases (35, 36), the three components that are involved in receptor internalization. Thus, other motifs in addition to carboxyl tail could participate in the intracellular trafficking of the opioid receptors.
Hence, in the present studies, the cellular motifs in directing the lysosomal targeting of opioid receptors are examined in the neuroblastoma N2A cell model. This cell model was used to mimic the presynaptic and dendritic location of the endogenous opioid receptors. By expressing the wild type receptor, µ/ receptor chimeras, and the mutated receptors in the N2A cells, we determined that the carboxyl tail of the receptor alone could not direct the lysosomal targeting of the receptors. A di-leucine motif within the third intracellular loop is identified in conjunction with the carboxyl tail sequence to regulate the intracellular trafficking of the opioid receptors.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction—The human influenza virus HA, YPYDVPDYA, epitope-tagged opioid receptors HA-MOR and HA-DOR, and the receptor chimera constructs with the carboxyl tail exchanged (HAMOR/DT=HA-MOR with the DOR tail (DT) and HA-DOR/MT=HADOR with the MOR tail (MT)) were constructed as reported (26) and utilized in the construction of further mutant receptors. Point mutations were accomplished using the QuikChangeTM site-directed mutagenesis method as outlined by Stratagene (La Jolla, CA). The methioline amino acid at position 264 of HA-MOR and HA-MOR/DT opioid receptors and leucine amino acid at position 245 of HA-DOR and HADOR/MT opioid receptors were mutated to leucine as the mutant M264L (5'-CTCAAGAGCGTTCGATTCCTATCGGGCTCCAAAG-3') and methioline as mutant L245M (5'-GCCTGCGCAGCGTACGAATGCTGTCCGGTTCC-3'). Forward and reverse primers with the desired mutation and a designed endonuclease site were synthesized (bold letters represent mutated bases). After PCR, 10 units of DpnI endonuclease of each reaction were used to remove the original templates. Finally, 5 µl of the resulting incubation mixtures were transformed into competent XL-Blue cells. The mutants were confirmed by sequencing.
Cell Culture and Stable Transfection of N2A Cell with Wild Type and Mutant Opioid Receptors Plasmids—Mouse neuroblastoma N2A cells were cultured at 37 °C in DMEM supplemented with 15 mM glucose, 0.43 M NaHCO3, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum in a 10% CO2 incubator. Approximately 1 x 105 N2A cells were seeded in a 6-well clustered plate <24 h prior to transfection. 0.4 µg of plasmid DNA were transfected into these N2A cells with the Effectene transfection kit (Qiagen) according to the manufacturer's recommendation. 24-48 h after the transfection, the cells were passed at a 1:5 to 1:10 dilution into selective DMEM containing 1 mg/ml G418 (Geneticin; Invitrogen). When visible clones were observed, single clones were then isolated, and these clones were screened for the level of receptor expressed determined by radioactive ligand binding assay as described previously (26).
Fluorescence Flow Cytometry—The HA-tagged receptors on the plasma membrane was quantified by determining the immunofluoresence on the cell surface using FACS analysis. Briefly, Neuro2A cells stably expressing the indicated HA-tagged receptors were preincubated with 25 µl of 95% ethanol or with 25 µl of monensin (50 µM) in 95% ethanol for 1 h at 37 °C. Then cells were treated with 1 µM etorphine for different time period to induce receptor internalization. After rinsing twice rapidly with serum-free DMEM at 4 °C, the cells were incubated at 4 °C for 60 min in serum-free DMEM with the anti-HA antibody (1:500 dilution). Afterward, the cells were washed twice with serum-free DMEM at 4 °C and then incubated with Alexa 488-labeled goat anti-mouse IgG secondary antibody (1:400 dilution) at 4 °C for additional 1 h. After washing the cells to remove the excess secondary antibodies, the cells were fixed with 3.7% formaldehyde prior to FACS analysis. Receptor immunofluorescence was quantitated by FACScan (Becton Dickinson, Palo Alto, CA). Fluorescence intensity of 10,000 cells was collected for each sample. Cell Quest software (Becton Dickinson) was used to calculate the mean fluorescence intensity of the cells population. All experiments were conducted at least three times with triplicate samples in each time point of etorphine treatment.
Confocal Microscopy—N2A cells expressing the wild type, receptor chimeras, or mutant receptors were grown on glass coverslips that were acid-cleaned and pretreated with 0.1 mg/ml poly-lysine (Sigma) until 50% confluent. The cells were incubated with primary 16B12 anti-HA antibody at 4 °C for 1 h so as to label the receptors. After washing to remove excess antibodies, 1 µM etorphine and 0.25 µM LysoTracker were added. After incubating at 37 °C for the designated time, the medium was removed, and the cells were fixed with 3.7% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. The cell were then incubated with blocking buffer PBS diluent (0.3% Triton X-100, 1% normal donkey serum, 1% bovine serum albumin, and 0.01% sodium azide, pH 7.2) for 1 h and then incubated with Alexa 488-conjugated secondary antibody for 1 h at room temperature. The coverslips were dipped in distilled water and mounted by NO-FADE (PBS:glycerol 9:1, 0.1% p-phenylenediamine, pH 8.0). Confocal fluorescence microscopy was carried out using a Bio-Rad MRC 1024 and a 60x objective. As for biotin staining, cells were biotin-labeled, etorphine-treated, and stripped as described below. Afterward the cells were fixed and permeabilized with PBS diluent and then incubated with Alexa 594-conjugated streptavidin for 1 h. After washing, the cells were incubated with primary mouse anti-HA and secondary goat anti-mouse Alexa 488 antibodies sequentially.
Surface Biotinylation—To distinguish the recycling opioid receptors from the newly synthesized receptors, a cell surface biotinylation labeling system was applied. Stably transfected N2A cells expressing HA-tagged receptors were grown in 10-cm dishes to 90% confluence. After washing twice with PBS at 4 °C, the cells were biotinylated by incubating with 300 µg/ml sulfo-NHS-SS-biotin in PBS for 30 min at 4 °C. Excess biotin was quenched and removed by washing twice with TSB (10 mM Tris, pH 7.4, 154 mM NaCl). Then DMEM at 37 °C was added to the biotinylated cells, and the cells were treated with 1 µM etorphine for designated time. After washing away the excess drug, the remaining cell surface biotin molecules were stripped in a solution containing 50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, and 1% fetal bovine serum at 4 °C for 30 min. The cells were then extracted with cell lysis buffer (0.2% (v/v) Triton X-100, 10 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, protease inhibitors mixture tablet Complete (Roche Applied Science; 1 tablet/50 ml)). Cellular debris was removed by centrifuging at 12,000 x g for 10 min at 4 °C. The clarified cell extracts were precipitated with streptavidin immobilized on agarose at 4 °C overnight. After washing five times with the cell lysis buffer, the beads were extracted with SDS sample buffer, and the eluted proteins were resolved by SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred to polyvinylidene difluoride membrane (Amersham Biosciences) in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol) and blocked for 60 min in a blocking solution (10% dry milk, 0.1% Tween 20 in TBS). The receptors were detected by sequential 60-min incubation with mouse anti-HA (1:2000) and goat anti-mouse antibody conjugated to AP (1:5000). Receptor band on the membrane were detected by the ECF substrate supplied by Amersham Biosciences and scanning the membrane with Storm 840 (Molecular Dynamics, Sunnyvale, CA). Band intensities were quantitated and analyzed using the ImageQuant software supplied by Molecular Dynamics.
For measuring the degradation kinetics, N2A cells were biotin-labeled as before. The cells were preincubated with 50 µM monensin with or without 100 µM leupeptin for 1 h and then treated with 1 µM etorphine for another 1 h to trap the receptor at the endosome. After washing twice with PBS, the cells were stripped to removed surface biotin and incubated in serum-free medium for the desired time at 37 °C in the presence or absence of leupeptin. Then cells were lysed, and the biotinylated receptors were immunoprecipitated by streptavidin immobilized on agarose.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Opioid Receptors Internalized at Different Rates in N2A Cells—To examine the molecular basis for the neuronal regulation of opioid receptor trafficking, the wild type MOR and DOR were expressed in a neuronal cell model, neuroblastoma N2A cells. These N2A cells do not express a detectable level of opioid receptor, and when these cells were transfected with the HA epitope-tagged receptors, the expressed receptors exhibited similar ligand selectivity and function as reported with other cell models (37). After examining various transfecting techniques and reagents, because of the relative low efficiency in transfecting the plasmid DNAs into these N2A cells, stable cell lines were established, and clones expressing similar levels of wild type and mutant receptors were selected to carry out the current studies. The rate and the magnitude of receptor internalization were examined in two or three clonal cell lines for each plasmid construct, with no significant difference observed among cell lines expressing the same construct.
When the N2A cells were treated with the opioid receptor nonselective agonist etorphine, similar to previous reports (26), MOR and DOR internalized with different profiles. When the cell surface receptor level was determined using FACS analyses, 1 µM etorphine induced MOR to internalize in a biphasic manner (Fig. 1A). The amount of receptor internalization (45 ± 6.0%) peaked after 20 min of etorphine treatment. Subsequently, this was followed by a decline to a steady state level (
41%) (Table I). The level of MOR being internalized increased to 49% after 4 h of etorphine exposure (data not shown). In contrast, DOR endocytosed in a monophasic time course that asymptotically increased toward a maximum level of 76% receptor internalized after 1 h of etorphine treatment (Fig. 1A and Table I). Although the maximal levels of receptors being internalized were different between the MOR and DOR, the initial rates of etorphine-induced receptor endocytosis were rapid for both receptors, with t
< 5 min (Table I).
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The differences in the maximal level of MOR and DOR being internalized could be caused by the rapid recycling of the MOR as reported previously (22). If this is the case, then monensin, a Na+ ionophore that blocks glycoproteins secretion and traps the receptors at endosomes, should increase the etorphine-induced MOR internalization in N2A cells. As shown in Fig. 1A, preincubating the N2A cells with 50 µM monensin altered the etorphine-induced MOR internalization rate to monophasic and increased the magnitude of receptor being internalized to 56% (Table I). However, even with comparable rates, the magnitude of MOR being internalized in the presence of monensin was significantly lower than that observed with DOR when measured in the absence of monensin. Such observation suggested that the endocytosis of the opioid receptor in N2A cells could be affected by the trafficking of the receptors to other cellular compartments, such as targeting to lysosomes. This hypothesis is partially supported by the decrease in the rate of DOR endocytosis to t = 11 ± 1.2 min in the presence of 50 µM monension (Fig. 1A and Table I). Etorphine binding to the DOR was not altered by monensin.
The FACS analyses determined the steady state levels of the cell surface receptors during etorphine treatment. Such analysis was unable to distinguish the newly synthesized receptor population from that internalized and subsequently recycled. Thus, prior to the addition of etorphine, the cell surface receptor pools were labeled with the sulfo-NHS-SS-biotin as described under "Experimental Procedures." After etorphine treatment, all of the cell surface biotin labels were stripped. Hence, only the internalized receptors would contain the biotin labels and could be immunoprecipitated by streptavidin. As shown in Fig. 1B, both MOR and DOR were biotinylated similarly in the absence of etorphine. The stripping conditions removed >90% of the biotin labels from MOR (Table II), suggesting that without agonist treatment, a minimal amount of the cell surface MOR was internalized. However, during the 30 min of incubation at 37 °C after biotinylation, >50% of the biotin on DOR could not be stripped (Table II). The presence of biotin on DOR was not due to incomplete stripping of the biotin-labeled cell surface receptor but rather caused by the rapid constitutive endocytosis of DOR. The amount of intracellularly located biotinylated DOR increased with prolonged incubation at 37 °C (data not shown). When the cellular location of the biotin-labeled proteins was examined with confocal microscopy, as expected, immediately after biotin labeling, the labels colocalized with the DOR and MOR at the cell surface (Fig. 2). Treating the N2A cells with the stripping solution resulted in the redistribution of some biotin-labeled proteins intracellularly, with no apparent colocalization of the biotin labels and the opioid receptors. Treating the cells with etorphine resulted in the endocytosis of the biotin labels that could not be removed by the stripping solution, and these labels colocalized with the opioid receptors (Fig. 2). Hence, the residual biotin-labeled DOR after stripping reflected the pools of DOR located intracellularly. As shown in Fig. 1B and Table II, 85% of the biotinylated DOR and 42% of MOR could not be stripped after 30 min of etorphine treatment, thus suggesting the agonist induced endocytosis of these receptors in N2A cells. Interestingly, the addition of monensin increased the amount of internalized biotin-labeled MOR, whereas monensin decreased the amount of intracellular located biotinylated DOR (Table II). These results were in agreement with the FACS analyses and supported the notion that MOR recycled after agonist-induced internalization in the N2A cells.
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To further characterize the intracellular sorting of MOR and DOR in N2A cells, confocal microscopy was used to visualize directly the HA-tagged receptor. The N2A cells were prelabeled with anti-HA antibody at 4 °C and then incubated with 1 µM etorphine for 30 min. As shown in Fig. 1C, both MOR and DOR located primarily on the plasma membrane in the absence of agonist. After incubating the N2A cells with agonist for 30 min, antibody-labeled receptors redistributed from the plasma membrane into cytoplasmic vesicles. Because lysosomes have been shown to be the primary targets for opioid receptor degradation (15), these compartments were labeled with red fluorescent LysoTracker during etorphine treatment. Colocalized staining (yellow) of DOR HA epitope (green) and lysosome probe LysoTracker (red) was observed after 30 min of etorphine treatment (Fig. 1C). Complete colocalization of DOR staining and the LysoTracker was observed after 2 h of etorphine treatment. In contrast, the internalized MOR did not colocalize with lysosome after 30 min of etorphine treatment and even after 60 min of etorphine exposure. After 2 h of etorphine treatment, some degree of lysosomal sorting of MOR was observed in minor fraction of the N2A cells. Thus, these confocal microscopy data support the conclusions from the FACS analyses and the biotinylation experiments that DOR is sorted to the lysosomal compartment rapidly, whereas MOR is recycled in N2A cells as reported previously with other cell model systems (21, 22).
Carboxyl Tail Domain of the Opioid Receptor Is Not the Sole Determinant in Regulating the µ- and -Opioid Receptors Endocytosis—The carboxyl tail domains have the greatest sequence diversity among the opioid receptors and have been reported to contribute to the cellular sorting and down-regulation of the receptors (26). As summarized in Fig. 3, swapping of the carboxyl tails between MOR and DOR could alter the etorphine-induced receptor internalization kinetics in the N2A cells. When MOR carboxyl tail was replaced with that of DOR resulting in the receptor chimera HA-MOR/DT, etorphine-induced receptor internalization kinetic became monophasic (Fig. 3A). The maximal amount of the receptor chimera being internalized was determined to be 68 ± 1.4%, which was significantly greater than those observed with the wild type MOR but significantly less than that of the wild type DOR (Table I). Further, the amount of the HA-MOR/DT chimera being internalized increased in the presence of monensin (Fig. 3A), suggesting that this chimera recycled in the presence of agonist. In contrast, when the carboxyl tail of DOR was swapped with that of MOR, generating the receptor chimera HA-DOR/MT, the ability of etorphine to induce receptor internalization in N2A cells was greatly impeded (Fig. 3B). This was not a result in the change in the functional integrity of the chimera receptor. Etorphine binds with similar affinity and inhibits with similar potency the forskolin-stimulated intracellular cAMP production in N2A cells expressing the HA-DOR/MT chimera as cells expressing the wild type DOR (26). However, the internalization kinetics of the HA-DOR/MT chimera did not resemble that of the wild type MOR or DOR. The kinetic of the etorphine-induced HA-DOR/MT internalization was much slower than that observed with the wild type MOR, and the maximal amount of this receptor chimera being internalized could be increased by the monensin treatment (Table I). Again, these results suggested that the HA-DOR/MT could recycle after the agonist-induced receptor internalization.
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Both HA-MOR/DT and HA-DOR/MT recycled after agonist-induced endocytosis were demonstrated with the biotin labeling experiments. As shown in Fig. 3C, in contrast to DOR, both the HA-DOR/MT and the HA-MOR/DT exhibited lower levels of constitutive endocytosis. Nevertheless, 20-30% of these receptor chimeras were internalized in the absence of ligands after 30 min of incubation at 37 °C (Table II). Further, in the presence of monensin, a 2-fold increase in the intracellularly located HA-DOR/MT was observed, whereas the amount of intracellularly located HA-MOR/DT remained unchanged during the 37 °C incubation in the absence of agonist. These data suggested that HA-DOR/MT not only internalized but also recycled constitutively, whereas HA-MOR/DT did not recycle once it endocytosed constitutively. On the other hand, 30 min of etorphine treatment increased the endocytosis of cell surface and located HA-DOR/MT to a level similar to that of MOR (Fig. 3C and Table II), as suggested by relatively slow rate of internalization determined by FACS analyses (Table I). The agonist-induced internalized HA-DOR/MT receptors also did not appear to recycle, because monensin pretreatment did not increase the amount of the intracellularly located receptor after agonist treatment (Table II). Such results were in direct contrast to that obtained with DOR or MOR in which monensin altered the amount of biotinylated receptor located intracellularly (Fig. 1B and Table II). Thus, these data indicated that both HA-MOR/DT and HA-DOR/MT acquired some but not all MOR and DOR phenotypes.
The confocal microscopy studies also indicate that the carboxyl tail domains of MOR and DOR are not sufficient in directing the intracellular trafficking of these proteins in N2A cells. If the carboxyl tails were the only determinants in lysosomal targeting, then the swapping of the carboxyl tails would direct the chimera receptors from the recycling to the degradation pathway or vice versa. The exchange of MOR carboxyl tail with that of DOR resulted in the rapid internalization and lysosomal targeting of the HA-MOR/DT receptor chimera, as indicated by the colocalization of the anti-HA with LysoTracker dye (Fig. 4). However, the percentage of receptor colocalizing with the LysoTracker stained lysosomes was much less than that observed with DOR. Further, although the HA-DOR/MT receptor chimera has the MOR carboxyl tail sequence, treatment with etorphine for 30 min resulted in the routing of these internalized chimeras to the LysoTracker positive lysosomes (Fig. 4). Together with the data obtained with FACS analyses and biotin labeling experiments, these confocal microscopy data suggest that the intracellular trafficking of the opioid receptors in N2A cells requires sorting signals in addition to the carboxyl tail domains.
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Sorting of Opioid Receptors Requires the Interplay between a Di-leucine Motif and the Carboxyl Tail—The different internalization rates and intracellular locations of HA-DOR, HA-MOR/DT, HA-MOR, and HA-DOR/MT suggested that additional signals must exist for the intracellular lysosomal targeting of these opioid receptors. Di-leucine motif is an important sorting signal present in the endosome/lysosome-targeted proteins (38). Di-leucine-based signals consist of an invariant leucine in the first position and a hydrophobic residue, Leu, Ile, Val, or Met, in the more tolerant second position (39). Substituting the first Leu residue in the motif with Met would lose its function (40). When examining the amino acid sequences of MOR and DOR, we notice that a consensus lysosomal targeting sequence of (D/E)XXXLL exists within the third intracellular loop of DOR with Arg substituting the Asp/Glu residue as a variant. MOR has the Met264-Leu265 sequence, whereas DOR has the corresponding Leu245-Leu246 sequence. Instead of Arg, MOR has Lys four residues upstream of the Met264. The variation within the putative di-leucine lysosomal targeting sequences between these two opioid receptors could contribute to the differences in the intracellular trafficking of MOR and DOR.
To test this hypothesis, a mutant DOR with a deletion of five amino acids sequence: Val243-Ser247 (HA-DOR
VRLLS) was constructed. As shown in Fig. 5, there was a significant reduction in the amount of HA-DORVRLLS being internalized as determined by FACS analyses. 1 µM of etorphine induced internalization of 76 ± 0.8% of HA-DOR receptor after 1 h (Table I). On the other hand, only 43 ± 1.9% of HA-DORVRLLS were endocytosed after 1 h of etorphine exposure (Fig. 5). The t of HA-DORVRLLS was 41 ± 5.0 min, which is much slower than that observed with DOR (5.0 ± 0.3 min). Such decreases in both the magnitude and the rate of receptor internalization could not be attributed to the rapid recycling of the mutant receptor. For monensin did not increase the rate or the amount of the deletion mutant receptor being internalized (Fig. 5), as in the case of MOR, which has been shown to recycle (Fig. 1 and Tables I and II). These results implicated that the deletion of the di-leucine motif within the third intracellular loop did not affect the recycling of the receptor and may contribute to lysosomal targeting.
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The importance of this di-leucine motif within the third intracellular loop of DOR in directing the lysosomal targeting was examined by single amino acid mutational analyses. We first examined if the Leu-Leu sorting signal was sufficient for directing the opioid receptor trafficking to the lysosomes. The Leu245 in DOR was mutated to Met (HA-DORL245M), whereas the corresponding Met264 in MOR was mutated to Leu (HAMORM264L). As summarized in Fig. 6A and Table I, etorphine induced the rapid internalization of the HA-MORM264L with a monophasic kinetic. The maximal receptor being internalized after 1 h of etorphine was 54 ± 2.3%, a value that was similar to that obtained with MOR in the presence of monensin (Table I). Monensin treatment did not increase the etorphine-induced internalization of the HA-MORM264L further (Table I), suggesting that this di-leucine motif has minimal effect on recycling of MOR. The FACS analyses data were substantiated by the biotin labeling experiments, in which monensin did not increase the percentage of the HA-MORM264L localized intracellularly (Fig. 6B and Table II).
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Mutation of the Leu245 in DOR to Met did not alter the magnitude of the etorphine-induced endocytosis of the mutant receptor when compared with that of wild type (Fig. 6A and Table I). Instead, the rate of the etorphine-induced internalization was significantly reduced. The t of the HA-DORL245M was determined to be 15 ± 1.2 min. Monensin pretreatment further decreased the agonist-induced internalization rate of this receptor mutant (Table I). Such reduction in the rate of endocytosis was in agreement with that observed with HADORVRLLS (Fig. 5) and implicated the participation of the Leu-Leu sequence in agonist-induced internalization of DOR. Similar to DOR, HA-DORL245M exhibited constitutive endocytosis in the absence of agonist as shown by the biotin labeling experiments (Fig. 6B and Table II). However, distinctly different from the wild type, monensin increased the amount of intracellularly located HA-DORL245M, both in the absence and in the presence of etorphine (Fig. 6B and Table II). Thus, the disruption of the putative di-leucine motif within the third intracellular loop of DOR enabled the mutant receptor to recycle.
The interplay between this di-leucine motif and the carboxyl tail domain in regulating the opioid receptor trafficking could be demonstrated best with the mutation of the Met264 in HAMOR/DT. As shown in Figs. 3 and 4, the receptor chimera HA-MOR/DT did not have the identical endocytosis kinetics or intracellular location as DOR. If the interplay between the di-leucine motif and the carboxyl tail exists, then the mutation of the Met264 residue in this chimera to Leu should generate a receptor mutant that has endocytosis kinetics comparable with that of DOR. Indeed, when the N2A cells expressing the HA-MOR/DTM264L mutant were treated with 1 µM etorphine, the maximal level of receptor being internalized significantly increased from that observed with the HA-MOR/DT (Fig. 6A and Table I). Further, the rate and the magnitude of receptor being internalized compared favorably with those values obtained with DOR. Monensin pretreatment did not alter the rate or magnitude of the agonist-induced mutant receptor internalization, suggesting that similar to DOR, the HA-MOR/DTM264L did not recycle. This conclusion was confirmed with the biotin labeling experiments. In contrast to the minimal constitutively internalization activity of HA-MOR/DT, HA-MOR/DTM264L constitutively endocytosed in a magnitude similar to that of DOR (Fig. 6B). In the presence or absence of monensin, etorphine induced 100% of the initially cell surface-located HAMOR/DTM264L to internalize. Hence, MOR with the carboxyl tail of DOR and a di-leucine motif in the third intracellular loop is more efficiently internalized by etorphine. Further, the internalized HA-MOR/DTM264L mutant receptors were targeted to the lysosomes. As shown in Fig. 7, after 30 min of etorphine treatment, a high degree of colocalization between the receptor positive intracellular vesicles and the LysoTracker positive lysosomes was observed. This was in contrast with the relatively low percentage of colocalization between the HAMOR/DT staining and the LysoTracker dye positive lysosomes after treating the cells with etorphine for the same period of time (Fig. 4). However, a similar percentage of colocalization between HA-MOR/DT and LysoTracker positive lysosomes was observed when the cells were treated with etorphine longer, 1-2 h (data not shown), suggested that the di-leucine motif increased the lysosomal targeting kinetic of the HA-MOR/DT.
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If the di-leucine motif indeed is involved in directing the receptor traffic toward the lysosomal degradation pathway, then this motif should enhance the trafficking between endosomes and lysosomes. Hence, N2A cells were prelabeled with biotin and then treated with etorphine for 1 h in the presence of 50 µM monensin. Afterward, both agonists and monensin were removed, the cell surface biotin labels were stripped, and the fate of the internalized receptor trapped at the endosomes was examined. As shown in Fig. 8, there was a time-dependent decrease in the intracellular located biotin-labeled DOR. 1 and 2 h after the removal of monensin, 62 and 28% of the biotin-labeled DOR remained, respectively. The decrease was attenuated by leupeptin, a lysosomal proteases inhibitor (Fig. 8). The internalized HA-MOR/DT also exhibited a time-dependent decrease, albeit in a slower rate. After 1 h, 83% of the biotin-labeled receptor remained, and 2 h after the removal of monensin, 71% of the intracellular located labels remained. Interestingly, the rate of decrease in biotin-labeled receptor was increased by the M264L mutation, with HA-MOR/DTM264L having similar kinetics as the DOR (Fig. 8). After 1 h of monensin removal, 61% of the intracellularly located mutant chimera receptor remained. Approximately 28% of the mutant chimera receptor remained 2 h after washing. Again, such a decrease could be attenuated by leupeptin (Fig. 8), indicating that the mutant chimera was trafficked to the lysosomes for degradation. Thus, the di-leucine motif in the third intracellular loop of HA-MOR/DT greatly enhanced the targeting of this receptor to the lysosomes.
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In parallel with the mutation of Met264 in HA-MOR/DT to Leu, Leu245 in HA-DOR/MT was mutated to Met. As summarized in Figs. 3 and 4, although the receptor chimera with the carboxyl tail of MOR exhibited a decrease in the rate and magnitude of agonist-induced internalization, the HADOR/MT chimera still was trafficked to lysosomes within 30 min of etorphine treatment. Thus, if the di-leucine motif within the third intracellular loop is the lysosome targeting signal, then mutation of the Leu245 in HA-DOR/MT to Met should modify the receptor trafficking. As summarized in Fig. 6A and Table I, the magnitude of the etorphine-induced internalization of the HA-DOR/MTL245M mutant receptor paralleled that of MOR. The endocytosis rate of the mutant receptor was drastically slower than that observed with DOR (Table I). Furthermore, monensin pretreatment reduced the maximal level of the agonist-induced HA-DOR/MTL245M endocytosis. These data suggested that in contrast to MOR, the rapid recycling of the mutant chimera receptor did not cause the reduction in the etorphine-induced receptor endocytosis. Rather the di-leucine motif has the dramatic effect on the HA-DOR/MT internalization, as suggested by the data obtained with HA-DORL245M and HA-DORVRLLS. Such a conclusion is supported by the biotin labeling experiments in which the percentage of intracellularly located HA-DOR/MTL245M was similar in the presence or absence of agonist, although this mutant chimera receptor exhibited constitutive endocytosis activity (Fig. 6B and Table II). Confocal microscopy studies also indicated the plasma membrane location of the receptor after 30 min of etorphine treatment, and the majority of the internalized receptor did not colocalize with the LysoTracker positive lysosomes (Fig. 7). Thus, the disruption of the di-leucine motif within the third intracellular loop of the HA-DOR/MT reduced the etorphine-induced internalization and targeting of the receptor to lysosomes.
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An interesting difference between the current N2A cell model and the ones reported previously is that DOR could internalize in the absence of agonist. This was revealed by the presence of biotin labels on DOR after 30 min of incubation at 37 °C followed by stripping of the cell surface biotin (Fig. 1B). The inability to observe biotin staining on the cell surface by confocal microscopy studies (Fig. 2) and the drastic reduction in the biotin-labeled receptor in plasma membrane preparation (data not shown) after stripping suggested that the current paradigm was able to strip the biotin from cell surface DOR. Such a conclusion was supported by an increase in the amount of intracellular located DOR with prolonged incubation at 37 °C. Thus, the presence of biotinylated DOR after stripping suggested intracellularly located receptors and suggested that DOR could be constitutively internalized. On the other hand, MOR did not constitutively internalized in N2A cells. The failure to observe intracellularly located MOR could be attributed to the rapid recycling of this receptor. However, trapping of the internalized receptor at the endosomes with monensin could increase slightly the intracellular located MOR. This is surprising because previous reports have suggested that both MOR and DOR exhibit constitutive activities, as reflected by the GTP
S binding assays (41, 42). Our current biotin labeling experiments clearly indicate that at least with the ability to internalize, only DOR exhibits robust constitutive activity in the N2A cells.
The role of the carboxyl tail domains in directing the opioid receptor intracellular trafficking has been established in other cell models (26, 43, 44). In the present studies, we also demonstrated that the carboxyl tails of MOR and DOR participate in regulating the etorphine-induced receptor endocytosis kinetics. The splicing of the DOR carboxyl tail to MOR could increase the rate and magnitude of the receptor chimera internalization. The splicing of the MOR carboxyl tail to DOR slowed the endocytosis of the receptor chimera. Biotin labeling experiments reveal that these carboxyl tail domains contain the motifs that manifest some phenotypes of these receptors. The most obvious one is that the carboxyl tail of the MOR contains the recycling motif. This notion is best supported by the observations that monensin increased the intracellular localization of all the chimera receptors containing carboxyl tail with the exception of the HA-DOR/MTL245M chimera (Table II). However, dissimilar to previous reports (45), in N2A cells, domains in addition to the carboxyl tail domains of these two opioid receptors control the intracellular trafficking of these receptors. If the carboxyl tail domain is the sole determinant in directing the receptor traffic, then the HA-DOR/MT and HAMOR/DT should have phenotypes identical to those of MOR and DOR, respectively. This is not the case. Not only were the rates and magnitudes of etorphine-induced receptor internalization of these receptor chimeras not identical to that of the corresponding wild type receptors, but also the HA-DOR/MT receptor chimera could internalize constitutively, whereas MOR could not. Moreover, even with the ability to recycle, the HA-DOR/MT was shown to target to lysosome during the 1 h of etorphine treatment, whereas MOR did not. These data and others suggest that multiple motifs within the opioid receptor regulate the targeting of these receptors to lysosomes.
One of these motifs is the di-leucine motif in the third intracellular loop that we demonstrated to be involved in lysosome targeting of DOR in the present studies. The di-leucine motif ((D/E)XXXLL) has been demonstrated to be one of the signals in sorting membrane proteins to lysosomes (46). The motif has been reported to be important for the internalization and/or lysosomal targeting of the epidermal growth factor receptor (47), vasopressin V1a receptor (48), - and -chain of T cell receptor complex, interferon -receptor, tyrosinase, and membrane trafficking of syntaxin (49-51). The di-leucine motif has also been implicated in the endocytosis-promoting activity of ubiquitin when linked to integral membrane proteins (52). Interestingly, although the di-leucine motif by itself could affect DOR endocytosis kinetics, it has minimal effects on the trafficking of MOR. The mutation of Met264 to Leu in MOR did not result in the rapid targeting of the mutant receptor to lysosomes, as in the case of DOR. The HA-MORM264L was observed to recycle rapidly by the biotin labeling experiments. The presence of a strong recycling signal in the carboxyl tail domain of MOR and the absence of acidic residues four amino acids upstream of the di-leucine residues could contribute to intracellular trafficking characteristics of HA-MORM264L. Lys260 is in the place of Asp/Glu in the (D/E)XXXLL motif generated by the M264L mutation of MOR. In other membrane proteins trafficking, as in the case of GLUT4 and IRAP (insulin-regulated aminopeptidase), two proteins localized to insulin-regulated storage compartment, Arg has been reported to substitute for the acidic residues Asp/Glu (53, 54). Only when both the di-leucine motif and the DOR carboxyl tail domain were present together in the receptor chimera HA-MOR/DTM264L, were the phenotypes, constitutive and rapid agonist-induced endocytosis, and rapid lysosomes targeting observed. Thus, in the N2A cells, interplay between the carboxyl tail domain and the di-leucine motif is needed in directing the opioid receptor trafficking. The interplay between a di-leucine motif and other signals in regulating protein trafficking has been shown with the studies of tyrosinase and IRAP. Tyrosinase targeted to the lysosome via a di-leucine and a tyrosine-based signal (50). In contrast, rapid internalization of IRAP required two or three distinct motifs: Met15-Met16, Asp64-Glu65-Asp66, and Leu76-Leu77. The di-leucine sequence and DED are part of the motifs that regulate recycling of these proteins (54).
The di-leucine motif probably also participates in the initial endocytosis of the opioid receptor. Adaptor protein AP-2 can bind directly to the di-leucine signals at the plasma membrane leading the protein into endocytic pathway (55, 56). This might be the reason for the decrease in the rate of etorphine-induced internalization of the HA-DORL245M and the HADORVRLLS mutant receptors. However, mutation of the Met264 to Leu in MOR could not increase the rate of endocytosis. Again, these data support the conclusion that interplay between the carboxyl tail domain and the di-leucine motif is the key. It is interesting to note that the di-leucine motif in question is located within the third intracellular loop of the receptor, whereas the di-leucine motif reported to regulate trafficking is located at the carboxyl domains of other GPCRs (48, 57-60). The third intracellular loop of the opioid receptor has been implicated in the coupling of G proteins (61-63). The interaction with G proteins appeared to regulate the agonist-induced receptor internalization and down-regulation (64). Although the deletion of the 263RMLSG267 sequence within the third intracellular loop of MOR resulted in a decrease of coupling efficiency of the mutant, the agonist-induced down-regulation of the mutant receptor was not altered (65). Thus, any possible alteration in G protein and receptor interaction as a result of di-leucine mutation would have minimal effect in the agonist-induced opioid receptor endocytosis.
Probably other functions of the third intracellular loop, such as calmodulin binding and putative phosphorylation by the calmodulin-dependent kinases, and arrestin binding could contribute to the di-leucine motif regulating the receptor trafficking (66). Pull-down experiments using glutathione S-transferase fusion proteins and BIACORE studies revealed the -arrestin interaction with the third intracellular loop (67, 68). The third intracellular loops of the opioid receptors contain the consensus sequence (69) that is responsible for arrestin interaction, i.e. Arg254 and Arg257 in DOR and the corresponding Arg residues in MOR. Arrestin controls not only the desensitization and internalization of GPCRs, it also controls the rate of receptor resensitization, the latter correlating with the ability of arrestin to dissociate from the receptor (70). Pull-down assays also demonstrated the involvement of the opioid receptor carboxyl tail domains in arrestin binding (68). Thus, a model can be proposed in which the di-leucine motif in the third intracellular loop, and the carboxyl tail of the receptor could both participate in the arrestin interaction. The interaction of the arrestin with the clathrin has been firmly established (71, 72). With the di-leucine motif forming the AP2 binding site, an arrestin/clathrin complex can be formed involving the third intracellular loop and carboxyl tail domain of the receptor. Such a complex could regulate the initial endocytosis of the opioid receptor, thus providing an explanation for the decrease in the endocytosis rate of the HA-DORL245M and HADORVRLLS mutant receptors. However, whether the same complex participates in the lysosomes targeting remains to be demonstrated. Recent studies suggested that other sorting proteins such as GASP participate in the sorting of DOR from the endosomes to multi-vesicular bodies (30). The GASP interacts preferably with the carboxyl tail domain of DOR. Whether the di-leucine motif in the third intracellular loop also is a probable site for GASP5 interaction is unknown.
In conclusion, in N2A cells, MOR and DOR are being regulated differently. The internalized MOR recycles rapidly, whereas the internalized DOR is being targeted to lysosomes for degradation. There are multiple signals residing within the primary sequence of the receptors that regulate the trafficking of the receptors. We have identified that in the third intracellular loop of DOR, a consensus (D/E)XXXLL motif with R in the place of acidic residue participates in the lysosomal targeting of the receptor proteins. This does not imply that these are the only two motifs that participate in the lysosome targeting. Because of the many proteins that might involve in the sorting of the proteins from endosomes to multi-vesicular bodies, multiple domains within the opioid receptor must participate in concert for the eventual delivery of the receptor to the lysosomes for degradation.
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FOOTNOTES |
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Recipient of National Institutes of Health Grant K05 DA70544.
Recipient of National Institutes of Health Grant K05 DA00513. To whom correspondence should be addressed: Dept. of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455-0217. Tel.: 612-626-6539; Fax: 612-625-8408; E-mail: lawxx001{at}umn.edu.
1 The abbreviations used are: GPCR, G protein-couple receptor; N2A, murine neuroblastoma Neuro2A cells; HA, the hemagglutinin epitope, YPTDVPDYA; DOR, -opioid receptor; MOR, µ-opioid receptor; FACS, fluorescence-activated cell sorter; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GTPS, guanosine 5'-3-O-(thio)triphosphate.
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and 
) or with (• and 
) 50 µM monensin of 1 h and then incubated with 1 µM etorphine for times shown. After drug treatment, the cells were chilled to 4 °C to arrest further trafficking and stained with an anti-HA monoclonal antibody (16B12) and an AlexaTM 488-conjugated anti-mouse antibody. The cells were fixed and analyzed on a FACScan flow cytometry. The values are the means ± S.D. of at least three independent experiments. B, intracellular location of opioid receptor as detected by biotinylation. Stably transfected N2A cells labeled with sulfo-NHS-SS-biotin were pretreated with or without 50 µM monensin 1 h. After etorphine treatment 30 min, the remaining cell surface biotinylated receptors were stripped to remove the biotin. The proteins were then precipitated with immobilized streptavidin. The receptors were detected by sequential incubation with mouse anti-HA and goat anti-mouse antibody. The protein bands were detected by ECF substrate (
