The Absence of a Direct Correlation between the Loss of [D-Ala2,MePhe4,Gly5-ol]Enkephalin Inhibition of Adenylyl Cyclase Activity and Agonist-induced µ-Opioid Receptor Phosphorylation*

Rachid El KouhenDagger , Odile Maestri-El Kouhen, Ping-Yee Law, and Horace H. Loh

From the Department of Pharmacology, University of Minnesota, Medical School, Minneapolis, Minnesota 55455

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Chronic activation of the µ-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when µ- and delta -opioid (DOR1TAG) receptors were expressed in the same system, overexpression of beta -adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of beta -adrenergic receptor kinase 2 and beta -arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.

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Phosphorylation of membrane receptors, such as tyrosine kinase receptors or G-protein coupled receptors (GPCRs),1 leads to an alteration in the receptor activities (1). While autophosphorylation of the tyrosine kinase receptors by the associated kinases results in the initiation of the signal cascades, agonist-induced phosphorylation of the GPCRs by protein kinases, recruited to the vicinity of the receptors, blunts the signal cascades. Rapid phosphorylation of these GPCRs, by specific protein kinases, i.e. G-protein-coupled receptor kinases, GRKs, and the subsequent binding of arrestins, has been considered to be a mechanism for the agonist-induced homologous desensitization of these receptors (2, 3).

From the cloning of the receptors, it is clear that µ-, delta -, and kappa -opioid receptors belong to the superfamily of GPCRs (4, 5). As with other GPCRs, prolonged agonist treatment results in an attenuation of effector responses and down-regulation of the receptor, both in clonal cell lines that express the opioid receptor endogenously (6, 7) or in cell lines that stably express cloned opioid receptors (8, 9). Chronic exposure to opioid agonist results in a reduced receptor affinity for the agonist, as well as diminished ability of the agonist to either inhibit cAMP accumulation (10-12), or to open inwardly rectifying potassium channels (GIRK-1) (13, 14). This reduction in the opioid agonist activity has been suggested to involve receptor phosphorylation. Pei and colleagues (15) demonstrated phosphorylation of the delta -opioid receptor that appeared to parallel the loss in agonist inhibition of adenylyl cyclase activity in HEK293 cells. Transient transfection of the dominant negative mutant of beta ARK1 into these cells reduced the degree of receptor phosphorylation which suggested the involvement of GRKs in DOR1TAG phosphorylation. Phosphorylation of the µ-opioid receptor in the presence of agonist has been demonstrated both in HEK293 (16) and in Chinese hamster ovary cells (13, 17). Furthermore, a parallel time course was reported between µ-opioid receptor phosphorylation in Chinese hamster ovary cells and the loss in µ-opioid receptor regulation of GIRK-1 in Xenopus oocytes (13). Co-injection of the cDNAs of DOR1TAG, beta -arrestin 2, beta ARK2, and GIRKs has been shown to result in an increase in the rate of agonist-induced homologous desensitization of the delta -opioid receptor (14). These data would tend to support the hypothesis that phosphorylation of the opioid receptor is the probable mechanism for the observed receptor desensitization.

There are some inconsistencies, however, within the reported data on opioid receptor desensitization and phosphorylation. Although the phosphorylation of the opioid receptor has been reported to be very rapid, the agonist-induced loss of receptor activity as measured by either adenylyl cyclase activity or GIRK-1 channel activity, especially in the case of µ-opioid receptor, occurs rather slowly (6, 14). Comparing the rates of desensitization of the two splice variants of the µ-opioid receptor, MOR-1 and the shorter isoform MOR-1B receptors, Zimprich and colleagues (18) showed that MOR-1B desensitized more slowly than MOR-1. The authors attributed this difference to the absence of Thr394 in the splice variant MOR-1B. Subsequently, Pak et al. (19), reported that the mutation of Thr394 in the wild-type MOR-1 to Ala resulted in the complete blockade of the agonist-induced receptor desensitization. These data are in contrast with those reported in which the agonist could induce receptor desensitization in a µ-opioid receptor mutant in which all the Ser/Thr residues of the third intracellular loop and the carboxyl tail are mutated to alanine (20). It should be noted, however, that in neither of these studies was receptor phosphorylation determined and therefore, it is uncertain whether the reported mutations have affected the overall phosphorylation of the receptor. Furthermore, the observed µ-opioid receptor phosphorylation in Chinese hamster ovary cells was not blocked by protein kinase C (PKC) inhibitors, thus suggesting the involvement of GRKs (21). However, opioid receptor regulation of phospholipase C in Xenopus oocytes appeared to involve PKC (13), and µ-opioid receptor desensitization in Xenopus oocytes, as measured with GIRK-1 regulation, was potentiated by PKC activation (13, 21). Consequently, the manner by which these data contribute to the role of receptor phosphorylation in the loss of opioid receptor activity remains to be demonstrated.

In light of these discrepancies, we chose to investigate both receptor phosphorylation and desensitization in the same system. Exposure of HEK293 or neuro2A cells stably expressing the µ-opioid receptor to the opioid agonist DAMGO resulted in a rapid phosphorylation of the receptor protein, which is time- and agonist concentration-dependent. However, the phosphorylation of the receptor does not correlate directly to the loss of the µ-opioid receptor mediated inhibition of adenylyl cyclase activity. Furthermore, alteration in the phosphorylation state of the delta - and µ-opioid receptors, expressed in the same system, induced differential regulation of these two receptor subtypes. These data suggest a pathway in which µ-opioid receptor phosphorylation may not be the major determinant in the eventual loss of agonist response.

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Materials-- Expression vector pCDNA3 was from Invitrogen (San Diego, CA). DMEM, Met/Cys-free DMEM, phosphate-free DMEM, and Geniticin (G-418) were purchased from Life Technologies, Inc. (Grand Island, NY). [3H]Diprenorphine (39.0 Ci/mmol), [3H]adenine (15-25 Ci/mmol), and [35S]Met/Cys (5.0 Ci/mmol) were supplied by Amersham. [32P]Orthophosphate (400-800 mCi/ml) was supplied by ICN (Costa Mesa, CA). 125I-Acetylated cAMP was purchased from Linco Research (St. Charles, MO). Polyclonal antibodies for the cAMP radioimmunoassay were a generous gift from Dr. T. Gettys (Medical University of South Carolina, SC). The polyclonal antibodies to beta -arrestin were a generous gift from Dr. R. J. Lefkowitz (Howard Hughes Medical Institute, Durham, NC). cDNAs for the bovine beta ARK2 (GRK3) and beta ARK1K220R (GRK2K220R) were generous gifts from Dr. J. Benovic (Thomas Jefferson University Medical School, Philadephia, PA) and were subcloned into the expression vector pCDNA3. cDNA encoding the beta -arrestin 2 was the generous gift of Dr. S. G. Ferguson (Dept. of Cell Biology, Duke University Medical Center, Durham, NC). Forskolin was purchased from Calbiochem (La Jolla, CA). DAMGO and other opioid ligands were supplied by NIDA, National Institutes of Health. All other chemicals were purchased from Sigma.

Cell Culture and Transfections-- Human embryonic kidney HEK293 cells and neuroblastoma neuro2A cells were stably transfected with the rat µ-opioid receptor cDNA, MOR-1, subcloned into the EcoRI/XbaI sites of the expression vector pCDNA3 by the calcium phosphate precipitation method (22). In order to facilitate the identification of the µ-opioid receptor with both polyclonal and monoclonal antibodies, a hemagglutinin (HA) epitope tag (YPYDVPDYA), recognized by the 12CA5 or 3F10 monoclonal antibodies (Boehringer Mannheim), was spliced into the NH2-terminal immediately after the initial methionine codon as described earlier (23). Cell colonies stably expressing the µ-opioid receptor were isolated by selection in the presence of 1 mg/ml Geniticin (G418) for 10 to 14 days, and confirmation of µ-opioid receptor expression was determined by whole cell binding using [3H]diprenorphine (specific activity 39.0 Ci/mmol) in 25 mM HEPES buffer, pH 7.6. Specific binding is defined as the difference between the radioactivity bound to the cells in the presence and absence of 100 µM naloxone. The selected clones, identified as MOR1TAGID2 (for neuroblastoma neuro2A cells) or HEKMT (for HEK293 cells), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 250 µg/ml G418 under humidified atmosphere with 10% CO2. For transient transfection, the cDNA was introduced into the cells also by the calcium phosphate method and the assays were performed 48 h after transfection.

Opioid Inhibition of Intracellular cAMP Level-- Two separate methods were used in the determination of the opioid agonist effect on the intracellular cAMP. The experiments in which neuro2A MOR1TAGID2 cells were used, the effect of DAMGO on cAMP levels was determined by measuring the conversion of the [3H]adenine-labeled ATP pools to [3H]cAMP as described (6). In the experiments when HEKMT were used, the intracellular cAMP level was determined by radioimmunoassay using 125I-acetylated cAMP and rabbit polyclonal antibodies which recognize the acetylated cAMP. In either method, cells were seeded in 24-well plates (2 × 105 cells/well) 48 h prior to experiments. In the [3H]adenine assay, the cells were prelabeled for 2 h in 0.5 ml of DMEM supplemented with 29.3 mM NaHCO3, 15.3 mM glucose, 15.4 mM NaCl, 2.5 µCi of [3H]adenine, and 0.25 mM 3-isobutyl-1-methylxanthine. Immediately before challenging the cells, in both methods, the plates were placed on ice, the radioactive or growth media was removed and replaced by 0.5 ml of ice-cold Krebs-Ringer-HEPES buffer (KRHB: 110 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 25 mM glucose, 55 mM sucrose, 10 mM HEPES, pH 7.4) containing 0.25 mM 3-isobutyl-1-methylxanthine, 5 µM forskolin (or no forskolin in case of basal determination) with the indicated concentrations of DAMGO. The plates were subsequently incubated at 37 °C for 15 min and the reaction terminated by addition of 50 µl of 3.3 N perchloric acid. In the case of the [3H]adenine assay, [32P]cAMP was added as an internal standard and the amount of [3H]cAMP synthesized was then separated from the other 3H-labeled nucleotides by double column chromatography as described by White and Karr (24). In the case of radioimmunoassay, the perchloric acid in each well was neutralized with 120 µl containing M KOH, 1 M Tris, and 60 mM EGTA. The amount of cAMP in each well was determined by comparing the ability of the diluted samples to compete for 125I-acetylated cAMP binding to the antibodies with that of standard concentrations of acetylated cAMP.

In the experiments in which the rate and extent of desensitization were determined, cells were pretreated with the indicated concentrations of DAMGO for various time intervals (30 min to 4 h). The same concentration of DAMGO was included during the 2-h labeling of the ATP pools with [3H]adenine. After the incubation period, the media was removed and replaced with 0.5 ml of KRHB containing forskolin, 3-isobutyl-1-methylxanthine, and DAMGO. The level of inhibition using 1 µM or 20 nM DAMGO in these chronic agonist-treated cells was determined by averaging the results from at least 3 passages of cells.

Receptor Phosphorylation-- Neuro2A (MOR1TAGID2) or HEK293 (HEKMT) cells were seeded at 70-80% confluence in 100-mm plates 24 h prior to receptor phosphorylation assay. On the day of the assay (90-100% confluence), cells were washed twice with phosphate-free DMEM and incubated in 4 ml of the same medium for 1 h at 37 °C and 10% CO2. Then, the cells were incubated with 100 µCi/ml [32P]orthophosphate for 2 h at 37 °C and 10% CO2. Agonists and other compounds were added as indicated. The reaction was stopped by putting the plates on ice, at which point the labeling medium was rapidly removed, the cells were washed twice with ice-cold phosphate-buffered saline and harvested with 1 ml of lysis buffer (25 mM HEPES, pH 7.4, 1%, v/v, Triton X-100, 5 mM EDTA, with 100 µg/ml bacitracin, 10 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin A, and 20 µg/ml benzamidine as protease inhibitors and 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and 0.1 mM sodium vanadate as phosphatase inhibitors) in microcentrifuge tubes. The receptor was solubilized by rotating the samples for 1 h at 4 °C and the insoluble cellular debris was then removed by centrifuging the samples at 14,000 × g for 15 min at 4 °C. Supernatants were diluted to twice the volume with lysis buffer (without Triton X-100) before loading onto 1-ml wheat germ lectin affinity columns pre-equilibrated with Buffer A (25 mM HEPES, pH 7.4, 100 mM NaCl, and 0.1% Triton X-100). The columns were then washed with 10 ml of Buffer A to remove non-bound radioactive proteins and the µ-opioid receptor-containing fraction was eluted with 3 ml of Buffer A containing 0.5 M N-acetylglucosamine, and the protease/phosphatase inhibitors as indicated above. The samples were incubated in the presence of either 2.5 µg of HA-monoclonal antibody or 5 µl of polyclonal antisera (552G) developed against the COOH-terminal of the receptor (23), and 60 µl of slurry (50%) of prewashed immunopure protein A-agarose (or protein G-agarose) beads (Pierce) overnight at 4 °C. The beads were subsequently washed twice with Buffer A and three times with Buffer A without NaCl. Afterward, receptor protein was dissociated from the beads by adding 50 µl of SDS-PAGE sample buffer (62.5 mM Tris buffer, pH 6.8, 2% SDS, 3 M urea, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue). The samples were heated at 42 °C for 1 h and then separated on a 10% SDS-PAGE. After electrophoresis, gels were dried and the phosphorylated proteins were visualized and quantified by using the PhosphorImager Storm 840 system (Molecular Dynamics, Sunnyvale, CA).

Data Analysis-- Saturation binding data were analyzed using the computer program Ligand, providing estimates of receptor density (Bmax) and agonist affinity (Kd). Mean values from individual treatment groups were statistically analyzed by a one-way analysis of variance (ANOVA) with subsequent comparisons among treatment groups and from their control by the Student's t test.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Time-dependent Loss of the Agonist Inhibition of Forskolin-stimulated Intracellular [3H]cAMP Production upon DAMGO Pretreatment-- In cell lines stably expressing the µ-opioid receptors, prolonged treatment with agonists resulted in a gradual loss in the receptor activities (7). This loss in agonist activity has been attributed to the desensitization and down-regulation of the µ-opioid receptor. In order to investigate the role of receptor phosphorylation in such cellular adaptation processes, a hemagglutinin (HA) epitope was introduced at the NH2 terminus of µ-opioid receptor so as to facilitate the immunoprecipitation of the receptor with antibodies other than the polyclonal antibodies developed against the last 15 amino acids of the carboxyl tail sequence (23, 25). For HEK 293 clone (HEKMT), the Kd and Bmax values for [3H]diprenorphine were determined to be 1.3 ± 0.16 nM (n = 2) and 13.1 ± 0.54 pmol/mg of protein (n = 2), respectively.2 In previous studies, the Kd and Bmax values with the neuro2A clone (MOR1TAGID2) were determined to be 0.33 ± 0.02 nM (n = 3) and 1.9 pmol/mg of protein (n = 3), respectively (26). There was no observable difference between epitope-tagged (MOR1TAG) or wild-type (MOR1 WT) receptor in the DAMGO affinity for the receptor and the potency of DAMGO to inhibit forskolin-stimulated cAMP production in the neuro2A cells (25). The EC50 to inhibit adenylyl cyclase activity and Kd of DAMGO in HEKMT cells have a value of 7.5 ± 1.6 nM (n = 3) and 4.2 ± 0.51 nM (n = 3), respectively,2 and compare favorably with those obtained with the MOR1TAGID2 cells.

In the HEK 293 cells expressing the wild-type µ-opioid receptor, prolonged exposure to DAMGO resulted in the loss of the agonist ability to inhibit forskolin-stimulated cAMP production. The kinetics of the loss in agonist activity were monophasic and the rates were relatively slow. The receptor was completely desensitized after 24 h of DAMGO pretreatment (Fig. 1A). Similar loss in the µ-opioid receptor activity in the neuro2A MOR1TAGID2 cells was observed (25). For our studies, we tested the ability of 20 nM and 1 µM DAMGO to inhibit adenylyl cyclase activity. These concentrations correspond to the sigmoidal and the asymptotic region of the log dose-response curve, respectively (25). As shown in Fig. 1B, there was a time-dependent decrease in the ability of DAMGO to inhibit the intracellular cAMP production after 5 µM DAMGO pretreatment in both the MOR1TAGID2 and the HEKMT cells. The desensitization rate was very slow in both cell lines. It is not surprising that the loss of 1 µM DAMGO response was significantly slower than that in the loss of 20 nM DAMGO response, after 4 h of DAMGO pretreatment. However, it is surprising, that even the decrease in the activity of 20 nM DAMGO took a relatively long period of time (Fig. 1B).


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Fig. 1.   Time-dependent loss in the agonist inhibition of forskolin-stimulated intracellular cAMP production upon DAMGO pretreatment. Neuro2A or HEK293 cells stably expressing MOR1TAG were pretreated with 5 µM DAMGO for the indicated time intervals. A and B, HEKMT were challenged with 1 µM DAMGO () and internal cAMP was measured by radioimmunoassay method as described. B, Neuro2A MOR1TAGID2 cells were challenged with 1 µM (triangle ) or 20 nM (black-triangle) DAMGO and accumulation of cAMP was measured by column chromatography assay as described under "Experimental Procedures." *, p < 0.05. Data shown represent mean ± S.E. of at least three independent experiments each performed in triplicate.

Phosphorylation of HA Epitope-tagged µ-Opioid Receptor in the Presence of DAMGO-- In order to investigate the phosphorylation of the µ-opioid receptor in either the MOR1TAGID2 or HEKMT cells, immunoprecipitation of the receptor proteins with either the polyclonal antisera (552G), which recognize the TAPLP epitope of the carboxyl terminus of the µ-opioid receptor (23), or the monoclonal antibody (12CA5 or 3F10), which recognizes the HA epitope, was carried out. The problem of nonspecific phosphorylated proteins being immunoprecipitated by these antibodies was overcome by partial purification of the µ-opioid receptor. This partial purification was carried out by the adsorption of the receptor and other glycoproteins onto a wheat germ agglutinin column, followed by subsequent elution of the receptor from the columns with N-acetylglucosamine. Thus, in all our receptor phosphorylation studies, the Triton X-100 extracts of the total cellular proteins were partially purified with the wheat germ agglutinin columns as described under "Experimental Procedures" prior to immunoprecipitation.

When the MOR1TAGID2 cells were radiolabeled with [32P]orthophosphate and treated with 5 µM DAMGO, purified receptor was resolved on SDS-PAGE and revealed a diffused phosphoprotein band migrating at approximately 65-75 kDa (Fig. 2, second lane). The migration of this phosphoprotein band in the SDS-PAGE corresponded to that of the HA epitope-tagged µ-opioid receptor when Western blot analysis was carried out with either polyclonal antiserum 552G or monoclonal antibody 12CA5 (23). The incorporation of [32Pi] into the immunoprecipitated epitope-tagged µ-opioid receptor in the presence of DAMGO was dramatically increased compared with the basal control level. This increase in the phosphorylation can be demonstrated to be associated with the µ-opioid receptor from the following observations: (a) the antagonist naloxone can completely block the DAMGO-induced increase in phosphorylation (Fig. 2, third lane), and (b) in the untransfected neuro2A control cells, no phosphoprotein with these molecular weights was immunoprecipitated from cells treated with DAMGO for the same period of time (Fig. 2, fourth lane).


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Fig. 2.   DAMGO-induced phosphorylation of MOR1TAG. Untransfected neuro2A cells (N2A) or stably transfected with MOR1TAG (MOR1TAGID2) were labeled with [32Pi] and then treated with 5 µM DAMGO with or without 25 µM naloxone for 30 min at 37 °C as indicated. Phosphoprotein bands were resolved on 10% SDS-PAGE. The phosphoreceptor band corresponding to MOR1TAG is indicated by an arrow.

DAMGO-induced µ-opioid receptor phosphorylation was rapid, occurring within the first few minutes of agonist addition and reaching a maximum within 20 min of DAMGO exposure (Fig. 3, A and B). The low levels of basal receptor phosphorylation in the control (without agonist treatment) were dramatically increased when the cells were treated with 5 µM DAMGO for 20 min. A similar level of increase was observed when the HEKMT cells were treated with DAMGO for 30 min (Fig. 3C). The amount of phosphorylated receptor stayed at the maximum level during the first hour of DAMGO treatment. During this time period, the DAMGO-induced phosphorylation was concentration-dependent (Fig. 4). After 30 min of cell exposure to 100 nM DAMGO, there was a detectable increase in phosphorylation. This increase reached a plateau at 3 µM, giving an EC50 of 450 ± 120 nM (Fig. 4B). The EC50 value for DAMGO to induce 50% of maximal phosphorylated µ-opioid receptor was markedly higher than the 3.1 ± 0.9 nM required to elicit 50% of maximal adenylyl cyclase inhibition, under identical conditions.


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Fig. 3.   Time course of DAMGO-dependent MOR1TAG phosphorylation. After [32Pi] labeling, the cells were treated with 5 µM DAMGO for the indicated time intervals as described under "Experimental Procedures." The samples were resolved on 10% SDS-PAGE and the phosphoreceptor bands were quantified and analyzed by PhosphorImager Storm (Molecular Dynamics). A, extracted phosphoreceptor from MOR1TAGID2, corresponding to different DAMGO treatment period. Pictured is the result of a single experiment representative of at least three performed. B, receptor phosphorylation bands were quantitatively analyzed with PhosphorImager and band intensities expressed as a percentage of the maximum phosphorylation (20 min). Data shown are mean ± S.E. *, p < 0.05; **, p < 0.01 compared with the maximum phosphorylation. C, quantitation analysis of time course of DAMGO-induced MOR1TAG phosphorylation in HEKMT. Data shown are mean ± S.E. of three independent experiments. **, p < 0.01.


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Fig. 4.   MOR1TAG phosphorylation is agonist concentration dependent, reaching a plateau at 3 µM with an EC50 of 450 nM. A, MOR1TAGID2 cells were radiolabeled and treated for 30 min with increasing concentrations of DAMGO. Lanes 1-8 correspond to 1, 10, 100, 300, 1,000, 3,000, 10,000, and 30,000 nM, DAMGO, respectively. Pictured are the results of a single experiment representative of at least three performed. B, phosphoreceptor band was quantitatively analyzed with PhosphorImager and band densities expressed as percentage of maximal phosphorylation. Data shown are mean ± S.E. of at least three separate experiments.

After 1 h, there was a progressive decrease in the amount of phosphorylated µ-opioid receptor in the MOR1TAGID2 cells (Fig. 3, A and B). The phosphorylation level decreased to 58 ± 19% after 4 h of DAMGO treatment when compared with that after 20 min of DAMGO exposure, p < 0.01 (Fig. 3B). A similar decrease (70 ± 8.5%) was observed in the HEKMT cells (Fig. 3C). This observed phosphorylation decrease could be a result of (a) loss of receptor molecules, (b) decrease in the affinity of 552G for the TAPLP epitope due to conformational changes, or (c) dephosphorylation of the phosphorylated receptor molecules. A decrease in the affinity of antiserum 552G for the phosphorylated receptor was ruled out by using anti-HA monoclonal antibody for immunoprecipitation of the µ-opioid receptor. Using 12CA5 to immunoprecipitate the receptor, receptor phosphorylation shows a decrease to 61 ± 19% (n = 3) after 4 h of agonist treatment (data not shown) which was similar to that observed when 552G was used.

The decrease in the overall cellular receptor level during chronic agonist treatment as the mechanism for the observed decrease in µ-opioid receptor phosphorylation can be eliminated by determining the receptor level by either [3H]diprenorphine binding or the 35S-labeled Met/Cys labeling of the receptor. After 4 h of DAMGO treatment, [3H]diprenorphine binding was decreased by 19 ± 4% (n = 3) and the 35S-Met/Cys labeled receptor level was decreased by 26 ± 2.6% (n = 3) as quantitated by the radioactivity incorporated into the 65-75-kDa band in SDS-PAGE after immunoprecipitation (data not shown). Thus, the level of µ-opioid receptor being down-regulated was insufficient to explain the phosphorylation decrease during the prolonged agonist treatment (58 ± 19%), and was most likely due to the dephosphorylation of the receptor (Fig. 3).

Modulation Effect of Phosphorylation on µ-Opioid Receptor Desensitization-- Although the DAMGO-induced phosphorylation of the receptor reached its maximal level by 20 min, there was a minimal loss in the ability of either 20 nM or 1 µM DAMGO to inhibit adenylyl cyclase activity after 5 µM DAMGO treatment (Fig. 1B). If the receptor phosphorylation is a determinant in the loss of activity, then an alteration in the phosphorylation level of the receptor should change the extent of the receptor desensitization. Thus, we sought to modify the level of the µ-opioid receptor phosphorylation either by the inhibition of receptor dephosphorylation or by enhancement of phosphorylation by overexpression of the GRKs.

Since there was a time-dependent decrease in the level of receptor phosphorylation which could not be explained completely by the decrease in the receptor protein level, then it is possible that the change indicates an alteration in the equilibrium between the activities of the protein kinase(s) and the phosphatase(s). Thus, calyculin A, a potent inhibitor of phosphatase I and II was included in the phosphorylation experiments in order to increase the phosphorylated receptor level. When HEKMT cells were pretreated with 20 nM calyculin A for 30 min prior to the addition of DAMGO, the phosphorylated receptor level was dramatically increased (Fig. 5A). The concentration of DAMGO used for this experiment (0.5 µM) corresponds to the EC50 of the maximal phosphorylation level (Fig. 4B). A similar increase was observed with MOR1TAGID2 cells were pretreated with calyculin A (data not shown). Interestingly this calyculin A-induced increase in phosphorylation did not alter the degree of receptor desensitization. The ability of DAMGO to inhibit the adenylyl cyclase activity was not altered significantly after 30 min of 0.5 µM DAMGO treatment in the presence of 20 nM calyculin A in both HEKMT (Fig. 5B) and MOR1TAGID2 cells (data not shown). Higher concentrations of calyculin A could not be used because of its relative toxicity toward the cell lines used. Nevertheless, as summarized in Fig. 5, 20 nM calyculin A, which had a pronounced effect on the magnitude of receptor phosphorylation, did not significantly alter the acute or chronic effects of DAMGO.


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Fig. 5.   Effects of calyculin A on MOR1TAG phosphorylation and desensitization. HEKMT cells were treated, as indicated, with 0.5 µM DAMGO in the presence or absence (control) of 20 nM calyculin A. A, effects of calyculin A on DAMGO-induced MOR1TAG phosphorylation. Pictured are the results of a single experiment representative of three performed. B, effects of calyculin A on DAMGO-induced MOR1TAG desensitization. HEKMT cells either untreated (dark bars) or treated with 0.5 µM DAMGO for 30 min (hatched bars) were challenged with 0.5 µM DAMGO in the presence of 5 µM forskolin. 20 nM calyculin A or vehicle was added 30 min prior to pretreatment with DAMGO. The results were normalized as a percentage of maximal inhibition of forskolin-stimulated adenylyl cyclase activity (the DAMGO-mediated inhibition in untreated cells). Data shown represent mean ± S.E. of three separate experiments performed in triplicate.

From our own studies and those reported (13), it is clear that protein kinase C is not involved in the DAMGO-induced phosphorylation of the µ-opioid receptor. Similar to previous studies performed with DOR1TAG (15), depletion of PKC by pretreatment with the phorbol ester, PMA, did not affect DAMGO-induced phosphorylation but attenuated the PMA-induced MOR1TAG phosphorylation, p < 0.005 (n = 3) (Fig. 6). Furthermore, the inclusion of a PKC-specific inhibitor, chelerythrine chloride (10 µM), inhibited the PMA-induced, but not the DAMGO-induced, phosphorylation of the µ-opioid receptor, p < 0.05 (n = 3) (Fig. 6). Since activation of the opioid receptor resulted in a decrease in the intracellular cAMP level, DAMGO-induced phosphorylation must involve protein kinases other than PKC and the cAMP-dependent kinase, PKA. The presence of GRK consensus sites within the opioid receptor amino acid sequence, and the implication of GRKs in the phosphorylation of GPCRs, suggests that the observed results could be due to the GRK phosphorylation of the agonist-activated µ-opioid receptor. More specifically, the implication of beta ARK2 in phosphorylation of several GPCRs, and the ability of co-injected beta ARK2 to accelerate delta -opioid receptor desensitization in the Xenopus oocytes (14) argues for a probable involvement of this protein kinase in µ-opioid receptor phosphorylation.


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Fig. 6.   Activated PKC can phosphorylate MOR1TAG but is not involved in DAMGO-induced phosphorylation. MOR1TAGID2 cells were pretreated without (control) or with (PKC-depleted) 1 µM PMA for 24 h before labeling with [32Pi]. The cells were then stimulated for 30 min with 5 µM DAMGO (hatched bars), 1 µM PMA (dark bars), or vehicule (basal) (white bars). PKC inhibitor, chelerythrine chloride (10 µM), was added 30 min before adding phosphorylation stimuli. Receptors were then purified and resolved on SDS-PAGE as described under "Experimental Procedures." Phosphoreceptor bands were quantitatively analyzed with a PhosphorImager and expressed as a percentage of the basal phosphorylation level seen in control cells. Data shown represent mean ± S.E. of three separate experiments. *, p < 0.05; **, p < 0.005 compared with the control cell value.

As mentioned above, another approach to alter the receptor phosphorylation level would be to overexpress beta ARK2 in HEKMT cells. After 30 min treatment with 0.5 µM DAMGO, the phosphorylated receptor level was increased compared with the basal phosphorylation level (Fig. 7A). The expression of beta ARK2 significantly elevated the phosphorylation level in an agonist-dependent manner, while the co-expression of the mutant of beta ARKs, beta ARK1K220R, abolished the phosphorylation effect of beta ARK2 (Fig. 7A). The expression of this mutant alone does not affect dramatically the endogenous phosphorylation level. In the same cell line, under the same conditions, a minimal loss in DAMGO-induced inhibition of forskolin-stimulated adenylyl cyclase activity was detected after 30 min of DAMGO pretreatment. Overexpression of beta ARK2 alone, or with beta ARK1K220R mutant, did not alter DAMGO-induced MOR1TAG desensitization (Fig. 7B). The same results were obtained when increasing amounts of beta ARK2 or beta ARK1K220R cDNAs (up to 30 µg of cDNA) were used (data not shown).


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Fig. 7.   Effects of overexpressing beta ARK2 on DAMGO-induced phosphorylation and desensitization of µ-opioid receptor. HEKMT cells were transiently transfected with 2.5 µg of beta ARK2 and/or 10 µg of beta ARK1K220R mutant cDNA as indicated. A, after [32Pi] labeling, the cells were treated with 0.5 µM DAMGO, as indicated, for 30 min and phosphoreceptor bands were resolved on SDS-PAGE as described under "Experimental Procedures." B, DAMGO-mediated MOR1TAG desensitization was carried out in HEKMT cells, in the absence (control) or presence of either beta ARK2 or beta ARK2 and beta ARK1K220R mutant (beta ARK2/Mutant). Untreated () or treated () cells with 0.5 µM DAMGO for 1 h were challenged with 0.5 µM DAMGO in the presence of 5 µM forskolin. The basal level (black-square) (control in the absence of agonist and forskolin) and the forskolin stimulated level () of cAMP were measured for each transfection condition.

One of the consequences of GPCR phosphorylation is the enhancement of beta -arrestin binding to the protein, resulting in further uncoupling of the receptor from the G-proteins (2). The failure to observe any receptor desensitization after maximal receptor phosphorylation could be attributed to an insufficient endogenous beta -arrestin level in the HEKMT cells. Studies with other GPCRs (27, 28) and the delta -opioid receptor desensitization in Xenopus oocytes (14) have suggested that overexpression of beta -arrestin can potentiate receptor desensitization. Hence, beta -arrestin 2 was overexpressed in HEKMT cells in order to investigate whether an increase in the level of this protein would accelerate the loss of the µ-opioid receptor-mediated inhibition of adenylyl cyclase activity. As shown in Fig. 8A, Western blotting revealed that HEKMT cells do endogenously express beta -arrestin 2. The expression level of beta -arrestin 2 was increased when HEKMT cells were transfected with 0.01 or 0.1 µg of beta -arrestin 2 cDNA. However, even in presence of overexpressed beta -arrestin 2, the desensitization of MOR1TAG after 1 h treatment with DAMGO was not altered as compared with the vehicle-transfected HEKMT cells (Fig. 8B). As a result of the failure to observe any effect of beta ARK2 or beta -arrestin 2 on DAMGO-induced MOR1TAG desensitization, we considered the functional activity of these two proteins in our system. Thus, we chose delta -opioid receptor as an internal positive control.


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Fig. 8.   Effects of overexpressing beta -arrestin 2 on DAMGO inhibition of forskolin-stimulated adenylyl cyclase activity. A, HEKMT cells were transiently transfected with increasing amounts of beta -arrestin 2 cDNA and samples from whole cell extract were loaded on 10% SDS-PAGE. Immunoblotting using polyclonal anti-beta -arrestin 2 antibodies revealed increasing band intensities corresponding to beta -arrestin 2. B, under the same transfection conditions, HEKMT cells were subjected to adenylyl cyclase assays by measuring DAMGO inhibition of cAMP production. Untreated () or treated () cells with 0.5 µM DAMGO for 1 h were challenged with 0.5 µM DAMGO in the presence of 5 µM forskolin. The basal level (black-square) (control in the absence of agonist and forskolin) and the forskolin stimulated level () of cAMP were measured for each beta -arrestin 2 transfection condition.

To eliminate any transfection artifacts, HEKMT cells, which stably express MOR1TAG were transfected with HA-tagged delta -opioid receptor (DOR1TAG). The expression level of DOR1TAG in HEKMT was 1.42 ± 0.16 (n = 6) pmol/mg of protein. The inserted hemagglutinin epitope at the NH2 terminus of both receptor subtypes enabled us to purify and immunoprecipitate both receptors with the same monoclonal antibody. After 30 min treatment with 0.5 µM DAMGO and 1 µM DPDPE, phosphorylated receptor proteins were resolved on 12% SDS-PAGE and revealed two diffuse bands corresponding to MOR1TAG (65-75 kDa) and DOR1TAG (50-60 kDa). There was a small, although not significant, increase in basal phosphorylation of DOR1TAG in the presence of overexpressed beta ARK2. However, as expected, DPDPE-induced DOR1TAG phosphorylation was markedly increased when beta ARK2 was overexpressed to a level about 3-fold of that in the absence of agonist (p < 0.01) (Fig. 9A). These data would argue that the beta ARK2-mediated potentiation of phosphorylation is agonist dependent. Co-expression of beta ARK2 and beta ARK1K220R mutant significantly reduced the DOR1TAG phosphorylation level (p < 0.01).


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Fig. 9.   Effects of overexpressing beta ARK2 and beta -arrestin 2 on agonist-induced phosphorylation and desensitization of MOR1TAG and DOR1TAG. A, HEKMT cells stably expressing MOR1TAG were transiently co-transfected with 10 µg of DOR1TAG, 2.5 µg of beta ARK2, and 10 µg of beta ARK1K220R mutant cDNA as indicated. After [32Pi] labeling, the cells were treated with 0.5 µM DAMGO and 1 µM DPDPE for 30 min and phosphoreceptor bands corresponding to MOR1TAG and DOR1TAG were resolved on 12% SDS-PAGE as described under "Experimental Procedures." The graph shown represents mean ± S.E. for MOR1TAG (gray bars) and DOR1TAG (dark bars) phosphorylation of at least three independent experiments quantified by PhosphorImager analysis. Data were normalized to the corresponding basal phosphorylation level in the absence of agonist treatment and kinase overexpression. **, p < 0.01; *, p < 0.05 compared with the corresponding controls without kinase overexpression or to the sample co-expressing beta ARK2 and beta ARK1K220R. B, HEKMT cells transiently transfected with DOR1TAG (, black-square) or with DOR1TAG, beta ARK2, and beta -arrestin 2 (open circle , ) were treated with 0.5 µM DAMGO (, open circle ) or with 1 µM DPDPE (black-square, ) for the indicated time intervals. The same agonist and concentration were used for the challenge in the presence of 5 µM forskolin as described under "Experimental Procedures." Data shown represent mean ± S.E. of three to five separate experiments performed each in triplicate. *, p < 0.05.

Similar to the HEK 293 cells expressing MOR1TAG alone (Fig. 7), a small, but significant increase (p < 0.05), was observed when DAMGO-induced MOR1TAG phosphorylation was determined in the presence of beta ARK2 and DOR1TAG. This increase was abolished in the presence of beta ARK1K220R mutant. Overexpression of the beta ARK1K220R mutant alone did not have any significant effect on the phosphorylation level of either receptor, compared with the corresponding controls (Fig. 9A). Consistent with data reported above (Fig. 7A), this result indicates that the transient expression of this mutant might be unable to compete with the endogenous GRKs. Alternatively, these data suggest the involvement of different kinases, other than beta ARKs, in opioid agonist-induced receptor phosphorylation.

In the same system expressing both receptors, agonist-induced receptor desensitization was carried out by measuring agonist-induced inhibition of forskolin-stimulated adenylyl cyclase activity. After pretreatment with 0.5 µM DAMGO or 1 µM DPDPE, Fig. 9B shows agonist-mediated time-dependent desensitization for µ- and delta -opioid receptors. As expected, the rate and magnitude of DPDPE-induced DOR1TAG desensitization were faster than those for DAMGO-induced MOR1TAG desensitization. The co-expression of beta ARK2 and beta -arrestin 2 with DOR1TAG in the HEKMT cells significantly enhanced both the rate and magnitude of the DPDPE-induced DOR1TAG desensitization (Fig. 9B). After 2 h of 1 µM DPDPE treatment, the extent of the loss in delta -opioid receptor was significantly increased from 37 ± 4% in the control cells (HEKMT/DOR1TAG) to 68 ± 12% in HEKMT cells transfected with DOR1TAG/beta ARK2/beta -arrestin 2 (p < 0.05) (Fig. 9B). Intermediate responses in DPDPE-induced receptor desensitization were obtained when beta ARK2 (or beta -arrestin 2) alone was expressed in HEKMT cells in the presence of DOR1TAG (data not shown). The failure to observe a complete DOR1TAG desensitization within the 2-h agonist treatment may be due to the recycling/resensitization of the sequestered receptors or to newly synthesized receptors. However, there was no observable effect on DAMGO-induced MOR1TAG desensitization when beta ARK2 and beta -arrestin 2 were co-expressed (Fig. 9B). These results show that µ- and delta -opioid receptors are under differential regulation by the beta ARK2 and beta -arrestin 2. It is clear also that modulation of receptor phosphorylation does not affect DAMGO-induced MOR1TAG desensitization, as measured by agonist inhibition of adenylyl cyclase activity.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The evidence in support of the phosphorylation of GPCRs as the essential step in the agonist-mediated desensitization of these receptors is compelling. Direct phosphorylation experiments with various GPCRs, especially those with the beta 2-adrenergic receptor, clearly indicate a direct correlation between the degree of receptor phosphorylation and the extent of desensitization (29, 30). The dephosphorylation of the beta 2-adrenergic receptor with phosphatase treatment resulted in resensitization of receptor-stimulated adenylyl cyclase activity (31). These data were further supported by the experiments in which the beta 2-adrenergic receptor desensitization could be attenuated by Ser/Thr mutations (32). In addition, the identification of a family of receptor kinases, beta ARKs, which phosphorylate only the agonist-activated receptor, as well as the ability of a inactive mutant of the beta ARKs to block receptor phosphorylation and subsequently receptor desensitization, indicate unequivocally the important role of phosphorylation of GPCRs in the agonist-induced receptor desensitization, particularly for receptors such as the beta 2-adrenergic receptor (33, 34).

As opioid receptors belong to the GPCR family (1, 5), it is logical to hypothesize that the loss of opioid responses during chronic treatment follows a mechanism similar to that reported with the beta 2-adrenergic receptor and other GPCRs. Previous reports have demonstrated a direct relationship between receptor phosphorylation and agonist-induced opioid receptor desensitization (11, 13, 15, 16). Receptor phosphorylation as the mechanism for µ-opioid receptor desensitization was supported also by a recent report (19) in which mutation of Thr394 or Glu393 to Ala was shown to result in the complete block of agonist induced loss of receptor activity. Since GRKs are acidokinases (34), the results reported by Pak et al. (19) suggest that phosphorylation of the receptor by GRKs is the mechanism of µ-opioid receptor desensitization.

In the present work, we provide evidence in contrast to a direct correlation between µ-opioid receptor phosphorylation and desensitization. By measuring, under identical conditions in the same system, the loss of opioid receptor activities and receptor phosphorylation, we were able to demonstrate a difference in the time course of these processes. The rate of a loss of DAMGO inhibition of forskolin-stimulated adenylyl cyclase activity in either MOR1TAGID2 or HEKMT cells was relatively slow in comparison with the agonist-induced receptor phosphorylation in both cell lines (Figs. 1 and 3). Since both neuro2A and HEK293 cells stably expressing the µ-opioid receptor have similar rates, this relatively slow rate of µ-opioid receptor desensitization could not be attributed to the phenotypic differences between the present observations and those previously reported. Furthermore, this slow desensitization rate was also observed with the human neuroblastoma SH-SY5Y cells endogenously expressing both the µ- and delta -opioid receptors (7). The difference in rates of receptor phosphorylation and desensitization could also be demonstrated with endogenously expressed receptors. Maximal phosphorylated µ-opioid receptor in human neuroblastoma SH-SY5Y cells can be obtained by incubating these cells in the presence of 1 µM DAMGO for 30 min (data not shown).

The absence of a direct correlation between µ-opioid receptor phosphorylation and desensitization was further demonstrated by altering the level of receptor phosphorylation. The inclusion of calyculin A or the overexpression of beta ARK2 significantly increase the level of MOR1TAG phosphorylation. However, this increase did not affect the DAMGO-induced receptor desensitization (Figs. 5 and 7). The lack of an effect due to overexpression of beta ARK2 on µ-opioid receptor desensitization does not exclude the possible involvement of other known GRKs. Different effects of the overexpression of various GRKs in other GPCRs such as dopamine (35), beta -adrenergic (27), and chemokine (CCR-5) (36) receptor desensitization have been reported. With the presence of multiple putative GRK sites within the carboxyl tail of µ-opioid receptor alone, the sites in the receptor that are being phosphorylated in the presence of agonist alone could be different from those in the presence of the overexpressed beta ARK2. Overexpression of other GRKs could have a potentiating effect on the DAMGO-induced receptor desensitization. However, in an effort to identify a probable mechanism of phosphorylation and desensitization, our studies continued to evaluate the effects of beta -arrestin. This approach was used because an increase in GPCR phosphorylation by GRKs directly enhances the affinity of beta -arrestin for the receptor and thus, promoting a further uncoupling of the receptor from the G-proteins (2, 37). Agonist-induced receptor desensitization can be potentiated subsequently by overexpression of the beta -arrestin in the system (27, 28). Our present study shows that co-expression of beta ARK2 and beta -arrestin potentiated the desensitization rate of DOR1TAG but not of MOR1TAG. These data are consistent with the homologous desensitization of the G-protein-coupled inward rectifying K+ channels reported by Kovoor et al. (14, 15), and indicate differential regulation of these two opioid receptor subtypes, by beta ARK2 and beta -arrestin 2. Since both µ- and delta -opioid receptors were expressed in the same cells that were co-transfected with beta ARK2 and beta -arrestin 2, the differences between the regulation patterns of these two opioid receptors could not be attributed to phenotypic or transfection artifacts. From the carboxyl tail truncation mutant studies with the delta -opioid receptor (38) and our own studies (data not shown), it is apparent that agonist-induced phosphorylation of the opioid receptors occurs at the carboxyl tail domain. Thus, the difference in the regulation of these two opioid receptor subtypes can be attributed to the difference in intrinsic domains of the receptor protein, specifically, the varying COOH termini of these two receptors. Furthermore, for µ-opioid receptor, the magnitude of phosphorylation induced by various opioid ligands appeared to correlate with the ability of these ligands to modulate the response, by measuring adenylyl cyclase activity (17) or receptor internalization (39). As high concentrations of agonists were used in these studies, the observed difference in the overall phosphorylation is most likely due to the agonist-induced phosphorylation of specific sites on µ-opioid receptor. This observation indicates that different agonist-activated conformations of µ-opioid receptor may exist. Subsequently, the receptor can modulate the intracellular responses by this specific phosphorylation. Identification of phosphorylated sites will help to elucidate the phosphorylation mechanism(s) and to understand the role of phosphorylation in µ-opioid receptor signal transduction.

As for µ-opioid receptor and other GPCRs (40), the lack of correlation between receptor phosphorylation and desensitization does not imply that receptor phosphorylation does not have a role in the receptor desensitization. The differences between the time course of receptor desensitization and phosphorylation, and the inability of the alteration in the phosphorylation state of the receptor to influence µ-opioid agonist-induced receptor desensitization, only suggests that an alternative mechanism(s) is involved in the cellular adaptation processes during chronic DAMGO treatment. One probable receptor desensitization mechanism is receptor internalization/sequestration. MOR1B, a splice variant of MOR-1, has been reported to desensitize at a slower rate than MOR-1 (41). The disappearance of the MOR-1B receptor in the presence of agonist also occurred at a slower rate than MOR-1. However, both the rates of desensitization and internalization of MOR-1B could be reverted to that of MOR1 if monensin was included during the incubation, suggesting the rapid recycling of the receptor proteins (41). Since the regulation of the adenylyl cyclase by µ-opioid agonist has been reported to be highly dependent on the receptor density (19), the internalization/sequestration of the µ-opioid receptor could be a critical factor in the observed loss of response. Such a mechanism is suggested by the fact that the rate of overall dephosphorylation of the µ-opioid receptor parallels that of receptor desensitization (Figs. 1 and 3). Dephosphorylation of GPCRs in sequestered vesicles has been suggested, although dephosphorylation has been proposed to be the resensitization mechanism (42). The overall phosphorylation state of all cellular µ-opioid receptors should reflect the trafficking of the receptors into the sequestered vesicles. However, whether the sequestration/internalization of the µ-opioid receptor is the alternative mechanism for the observed loss in DAMGO inhibition of adenylyl cyclase activity remains to be demonstrated. Nevertheless, our current studies clearly demonstrate that phosphorylation of the µ-opioid receptor is not the obligatory event for the DAMGO-induced receptor desensitization, as measured by the regulation of adenylyl cyclase activity.

    FOOTNOTES

* This work was supported by NIDA, National Institutes of Health Research Grants DA-00546, DA-01583, DA-05695, KO5-DA-70554, and the A & F Stark Fund of the Minnesota Medical Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: Dept. of Pharmacology, University of Minnesota, Medical School, 3-249 Millard Hall, 435 Delaware St. S.E., Minneapolis, MN 55455. Tel.: 612-624-6691; Fax: 612-625-8408; E-mail: elkou001{at}tc.umn.edu.

2 A. Burd, L. Erickson, L. Dicker, C. Sheen, R. El Kouhen, E. Miller, H. Loh, and P. Law, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GPCRs, G-protein-coupled receptors; DAMGO, [D-Ala2,MePhe4,Gly5-ol]enkephalin; DPDPE, [D-Pen2-D-Pen5]enkephalin; beta ARKs, beta -adrenergic receptor kinases; GRKs, G-protein-coupled receptor kinases; MOR1TAG, µ-opioid receptor HA epitope-tagged; DOR1TAG, delta -opioid receptor HA epitope-tagged; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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