From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Received for publication, October 19, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Prolonged activation of opioid receptors leads to
their phosphorylation, desensitization, internalization, and
down-regulation. To elucidate the relationship between µ-opioid
receptor (MOR) phosphorylation and the regulation of receptor activity,
a series of receptor mutants was constructed in which the 12 Ser/Thr
residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells
and exhibited similar expression levels and ligand binding properties.
Among those 12 Ser/Thr residues, Ser363,
Thr370, and Ser375 have been identified as
phosphorylation sites. In the absence of the agonist, a basal
phosphorylation of Ser363 and Thr370 was
observed, whereas
[D-Ala2,Me-Phe4,Gly5-ol]enkephalin
(DAMGO)-induced receptor phosphorylation occurs at
Thr370 and Ser375 residues. Furthermore, the
role of these phosphorylation sites in regulating the internalization
of MOR was investigated. The mutation of Ser375 to Ala
reduced the rate and extent of receptor internalization, whereas
mutation of Ser363 and Thr370 to Ala
accelerated MOR internalization kinetics. The present data show that
the basal phosphorylation of MOR could play a role in modulating
agonist-induced receptor internalization kinetics. Furthermore, even
though µ-receptors and Opioid alkaloids, as well as endogenous opioid peptides, exert
their multiple biological effects on target tissues by interacting with
specific cell surface receptors including the Using chimeric, truncated, or mutated opioid receptors, several studies
reported the crucial role of the C-tail of opioid receptors in
regulating their activities and trafficking (11, 16-20). Whereas
several potential phosphorylation sites were suggested to be involved
in regulating the activity and trafficking of opioid receptors, the
actual phosphorylation of these residues was not demonstrated. Only
recently, Maestri-El Kouhen et al. (21) have reported the
identification of phosphorylation sites of the From our own studies and other reports, agonist-induced opioid receptor
phosphorylation has been shown to be time- and
dose-dependent and could be blocked by antagonist naloxone
(23-25). We have previously reported that rapid phosphorylation of MOR
does not directly correlate with the relatively slow rate of receptor
desensitization as measured by the loss of inhibition of adenylyl
cyclase activity (23). We recently reconciled that such a difference
could be attributed to the expression level of the receptor as well as
the rapid recycling and resensitization of MOR after etorphine
treatment (26). By limiting the recycling of the receptor and receptor
density on the cell surface, rapid desensitization of MOR was observed
(26). Whether MOR phosphorylation plays a role in these cellular events needs to be established unequivocally. Therefore, it is imperative to
understand the phosphorylation mechanisms and identify specific phosphorylation sites and their involvement in the cellular regulation of MOR.
In the present work, we examine the role of phosphorylation of
individual Ser/Thr residues in agonist-induced MOR internalization. Our
mutational analysis indicates that Ser363,
Thr370, and Ser375 residues are phosphorylation
sites, and we clearly show that these sites are differentially
phosphorylated in basal and agonist-induced conditions. Subsequently,
we provided evidence that phosphorylation of these specific Ser/Thr
residues differentially regulates agonist-induced MOR internalization.
This study reveals that agonist-induced internalization of MOR is
modulated by site-specific phosphorylation.
Materials--
Oligonucleotides were synthesized by an automated
DNA synthesizer (Millipore model 8905). Taq polymerase and
restriction enzymes were obtained from Roche Molecular
Biochemicals. Expression vector pcDNA3 and
cloning vector pCRII were from Invitrogen (San Diego, CA). Cell culture
reagents, Dulbecco's modified Eagle's medium, minimal
essential medium, phosphate-free Dulbecco's modified Eagle's medium,
fetal calf serum, and Geneticin (G-418) were purchased from Life
Technologies, Inc. [3H]Diprenorphine (39.0 Ci/mmol) was
supplied by Amersham Pharmacia Biotech.
[32P]Orthophosphate (400-800 mCi/ml) was supplied by ICN
(Costa Mesa, CA). DAMGO and other opioid ligands were supplied by the
National Institute on Drug Abuse. All other chemicals were purchased
from Sigma.
Construction of Mutants of the µ-Opioid Receptor--
The
human influenza virus hemagglutinin epitope-tagged rat µ-opioid
receptor (MOR1TAG) (27) subcloned into the expression vector
pcDNA3 (Invitrogen) was used to generate most point
mutations. Additional point mutations, alone or in combination, were
constructed in multiple steps using the QuickChangeTM
site-directed mutagenesis method as outlined by Stratagene (La Jolla,
CA). Forward and reverse oligonucleotides with the desired mutation and
a designed endonuclease site were synthesized. The nucleotide sequences
of all mutants were confirmed by dideoxynucleotide termination
reactions using Sequenase II.
Stable Transfection of HEK293 Cells with Wild Type and Mutant
µ-Opioid Receptors--
HEK293 cells were cultured in minimal
essential medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin under humidified
atmosphere at 5% CO2. Cells were stably transfected by the
Ser/Thr mutants or the wild type rat µ-opioid receptor-1 cDNAs
using the calcium phosphate precipitation method (28). To avoid any
position effect because of random integration of cDNAs into the
chromosomes, colonies of transfected HEK293 cells surviving 1 mg/ml
Geneticin (G-418) selection were pooled. Confirmation of µ-opioid
receptor expression was determined by whole cell binding assay using
[3H]diprenorphine in 25 mM HEPES buffer, 5 mM MgCl2, pH 7.6. Specific binding is defined
as the difference between the radioactivity bound to the cells in the
presence and absence of naloxone (100 µM).
Receptor Phosphorylation--
Phosphorylation of HEK293 cells
stably expressing the Ser/Thr mutant or wild type µ-opioid receptors
was determined as described previously (23). Cultured cells in 6-well
plates were washed with phosphate-free Dulbecco's modified Eagle's
medium and incubated with 100 µCi/ml
[32P]orthophosphate for 2 h at 37 °C in 10%
CO2. 5 µM DAMGO was added as indicated. The
reactions were terminated on ice at which point the cells were washed
with ice-cold phosphate-buffered saline and subsequently lysed in 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 with 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and 0.1 mM sodium
vanadate as phosphatase inhibitors). After solubilization, insoluble
debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C. Supernatants were eluted 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
nonbound radioactive proteins, and the MOR-containing fraction was
eluted with 3 ml of buffer A containing 0.5 M
N-acetylglucosamine and the protease/phosphatase inhibitors
as indicated above. These purified samples were incubated in the
presence of 3 µg of rat hemagglutinin-monoclonal antibody 3F10 (Roche
Molecular Biochemicals) and prewashed immunopure protein G-agarose
beads (Pierce) overnight at 4 °C. The final samples were eluted from
the agarose beads and separated on a 10% SDS-PAGE (23). After
electrophoresis, the gels were dried, and phosphorylated bands were
visualized and quantified using the PhosphorImager Storm 840 system
(Molecular Dynamics, Sunnyvale, CA). Phosphorylation levels obtained
for mutant and wild type receptors were normalized according to protein
amount as well as receptor expression level within the starting cell materials.
Opioid Receptor Binding--
Characterization of the µ-opioid
receptor binding sites in HEK293 cells expressing wild type and mutated
receptors was carried out with membranes (100,000 × g × 60-min membrane preparations minus nuclei) in 25 mM HEPES buffer, pH 7.6, containing 5 mM
MgCl2 at 24 °C for 90 min as described previously (29).
The Kd and Bmax values for
[3H]diprenorphine were determined for each mutant by
saturation binding. IC50 and Ki values
for DAMGO were determined by competition binding studies using
increasing concentrations of DAMGO to compete for the binding of 1 nM [3H]diprenorphine. Data from saturation
and competition binding were fitted by nonlinear curve fitting using
the data analysis program GraphPad Prism.
Cyanogen Bromide Cleavage of µ-Opioid
Receptor--
Radiolabeled µ-opioid receptor bands were excised from
the gel, and receptor protein was eluted in Tris-glycine buffer (25 mM Tris, 192 mM glycine, and 0.1% w/v SDS) at
4 °C. Receptor fractions were first dialyzed and lyophilized and
then reduced and alkylated before treatment in the presence of CNBr
(160 mg/ml in 70% formic acid) (30). CNBr cleavage products were
separated on a Tricine-SDS-PAGE (16.5% acrylamide, 6% bis-acrylamide)
(31), and phosphopeptide bands were visualized by PhosphorImager.
Quantitative Analysis of µ-Opioid Receptor Internalization by
Fluorescence Flow Cytometry--
A pool of stably transfected HEK293
cells expressing wild type or mutant hemagglutinin epitope-tagged
receptors was incubated in the presence of 5 µM DAMGO for
the indicated time intervals. Cells were then chilled on ice to stop
membrane trafficking, and all subsequent incubations were performed at
4 °C. Residual cell surface receptors were monitored by incubating
cells in the presence of monoclonal anti-hemagglutinin antibody (clone
16B12) (Babco, Richmond, CA) for 2 h followed by an incubation
with the Alexa 488-conjugated anti-IgG antibodies (Molecular Probes,
Inc., Eugene, OR) for 1 h. Surface receptor staining intensity of
antibody-labeled cells was analyzed using fluorescence flow cytometry
(FACScan, Becton Dickinson, Palo Alto, CA). Each data point represents
the average of mean fluorescence values ± S.E. of receptor
internalization determined in an analysis of 10,000 cells performed in triplicate.
Previously, we reported that DAMGO-induced MOR
phosphorylation occurs rapidly with maximum phosphorylation obtained
after 20 min of agonist treatment (23). To localize the intracellular domains containing the phosphorylation sites, phosphorylated MOR was
purified and chemically cleaved in the presence of cyanogen bromide as
described under "Experimental Procedures." The theoretical CNBr
cleavage after Met residues generates several fragments with different
sizes including the C-tail domain of the receptor (Fig. 1A). To perform total protein
digestion, phosphorylated receptors were incubated with CNBr for up to
24 h. After CNBr cleavage, samples containing generated peptides
were resolved on 16.5% Tricine-SDS-PAGE, and phosphorylated bands were
visualized using a PhosphorImager (Fig. 1B). Only one
phosphorylated band migrating at ~13 kDa, a size corresponding to the
C-tail domain of MOR, could be detected (Fig. 1B). This
result strongly indicated that DAMGO-induced phosphorylation occurs
only within the cytoplasmic tail of MOR. A direct sequencing of the
purified C-tail of MOR to determine the exact phosphorylation sites was
not feasible because of the insufficient amount of purified peptide.
Therefore, we used a site-directed mutagenesis approach to identify
phosphorylated Ser/Thr residues within the C-tail of MOR.
-opioid receptors have the same motif
encompassing agonist-induced phosphorylation sites, the different
agonist-induced internalization properties controlled by these sites
suggest differential cellular regulation of these two receptor subtypes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-, µ-, and
-opioid receptors (1). These opioid receptors belong to the superfamily of G protein-coupled receptors
(GPCRs).1 The µ-opioid
receptor (MOR) serves as the principle physiological target for most
clinically important opioid analgesics, such as morphine and fentanyl
(2, 3). Although many opioid alkaloids exert their pharmacological
effects via MOR, their binding affinity for the receptor and potency to
activate the receptor do not always correspond to their abilities to
induce tolerance (4-8). This suggests that other cellular processes
that modulate MOR responsiveness, such as receptor desensitization and
internalization, may contribute to opioid tolerance and dependence.
Like many other GPCRs, opioid receptors are regulated by
agonist-dependent processes and undergo receptor
phosphorylation, desensitization, internalization, and down-regulation
(1). Interestingly, in addition to the subtype-specific regulation of
opioid receptors (9-12), individual opioid receptors are
differentially regulated by distinct opioid agonists (5-8, 13, 14). In
the case of MOR, opioid agonists demonstrating equivalent ability to
activate receptor signaling exhibit remarkable differences in their
abilities to functionally desensitize (5, 6) and induce internalization
of the receptor in both transfected cells and neurons (7, 8, 13-15).
However, the detailed molecular events underlying this differential
regulation of MOR by distinct agonists remain unclear.
-opioid receptor
(DOR) in human embryonic kidney (HEK293) cells. These results show the
implication of precise phosphorylation sites (Thr358 and
Ser363) in regulating the internalization and
desensitization of DOR (21). Additionally, agonist-induced
internalization of opioid receptors seems to be cell
type-dependent (22), indicating a recruitment of specific
cellular factors in regulating opioid receptor trafficking.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CNBr cleavage of phosphorylated MOR.
A, theoretical CNBr digestion profile of µ-opioid
receptor. The size of generated peptides (>500 dalton) as well as the
corresponding position were determined using the Swiss Protein Database
server. The asterisk indicates a 13-kDa fragment
corresponding to the COOH-terminal part of MOR. B,
phosphoreceptor protein was chemically cleaved in the presence of CNBr
as described under "Experimental Procedures." The three
lanes correspond to increasing amounts of cleavage products of
32P-labeled MOR loaded on a gel. A phosphopeptide band
corresponding to the COOH-terminal domain of MOR is indicated by an
arrow. These data are representative of three
independent experiments.
There are 12 Ser/Thr residues within the C-tail of rat MOR that are potential phosphorylation sites. To identify which Ser/Thr residues are phosphorylated, we constructed a number of mutated receptors in which these residues were substituted to Ala, either individually or in combination. Each of these mutated receptors as well as wild type MOR were stably expressed in HEK293 cells. To avoid any phenotype effect as a result of random integration of the plasmid into the chromosomes, a mixture of cell clones stably expressing either receptor was used in the present study as described under "Experimental Procedures." Receptor expression levels (Bmax) obtained were high for all mutated and wild type receptors ranging from 0.9 to 6.9 pmol/mg protein (Table I). Kd values of [3H]diprenorphine were similar among all these receptors, whereas small variations in the affinities of certain mutated receptors for DAMGO were observed (Table I).
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To define the region within the C-tail domain of MOR containing the
phosphorylation sites, we first used a group of mutated receptors in
which we progressively substituted clusters of Ser/Thr residues to Ala
(Fig. 2C). After 20 min of
treatment in the presence of a saturating concentration of DAMGO (5 µM), phosphorylated receptors were purified and resolved
on 10% SDS-PAGE as described under "Experimental Procedures" (Fig.
2A). Phosphorylated bands were quantified and normalized
according to total protein amount as well as receptor expression levels
measured in the presence of [3H]diprenorphine. As shown
in Fig. 2, A and B, the substitution of the first
four Ser/Thr residues (Thr354-Thr357) to Ala
(mutant 354-357) did not affect agonist-induced MOR phosphorylation when compared with that of wild type receptor. Substitution of the six
residues Thr354-Thr357, Ser363,
and Thr364 to Ala (mutant 354-364) reduced the level of
phosphorylation to approximately 60% of wild type receptor.
Phosphorylation of mutant 354-376, in which the nine residues
Thr354-Thr357, Ser363,
Thr364, Thr370, Ser375, and
Thr376 were replaced by Ala, was completely abolished. As
expected, no phosphorylation signal could be detected with mutant
354-394 in which all 12 Ser/Thr residues of the C-tail were changed to Ala (Fig. 2). These results clearly support our finding from CNBr experiments and show that phosphorylation occurs only at the C-tail of
MOR. In addition, these results indicate that the phosphorylation sites
are localized within the region
Ser363-Thr376.
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With the next group of mutated receptors, we focused on the region
between Ser363 and Thr376 by substituting the
five potential phosphorylation sites (Ser363,
Thr364, Thr370, Ser375, and
Thr376) to Ala, either individually or in combination (Fig.
3C). The phosphorylation level
of mutant 363-376, in which all of the five potential sites were
replaced by Ala, was nearly abolished (Fig. 3, A and
B). However, when compared with mutant 354-376 that
completely abolishes receptor phosphorylation (Fig. 2), mutant 363-376
still presents a weak phosphorylation signal (<5% of maximal
phosphorylation). This slight difference in phosphorylation
signals between these two mutants could be because of a minor
phosphorylation site present outside of the
Ser363-Thr376 region. Nevertheless, this
result indicates that major phosphorylation sites are located within
the Ser363-Thr376 region.
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Mutation of the doublet Ser363-Thr364 to Ala (mutant 363/364) significantly reduced phosphorylation level of the receptor to approximately 60%. The single mutation S363A (mutant 363) also reduced the level of phosphorylation, but the T364A mutation (mutant 364) did not, suggesting that Ser363 is the only phosphorylation site within this doublet (Fig. 3). Similarly, the substitution of Ser375-Thr376 (mutant 375/376) reduced the phosphorylation level to approximately 60%. Only the S375A (mutant 375) but not the T376A (mutant 376) mutation reduced the phosphorylation level of the receptor (Fig. 3). This indicates that Ser375 is also a phosphorylation site. Finally, the substitution of Thr370 into Ala (mutant 370) reduced the phosphorylation level to approximately 70%, suggesting that this residue is a phosphorylation site as well (Fig. 3). Taken together, these results indicate that among the 12 potential phosphorylation sites present in the C-tail of MOR, three residues, Ser363, Thr370, and Ser375, are actually involved in MOR phosphorylation.
The possibility that the observed reduction in phosphorylation level
with these mutated receptors could be a result of conformational changes of the C-tail domain or a modification of a kinase
recognition motif cannot be ruled out. Therefore, to visualize
the phosphorylation of each of these three residues
(Ser363, Thr370, and Ser375), we
constructed a new set of mutated receptors in which we replaced two of
these three Ser/Thr residues with Ala, leaving the third residue wild
type (Fig. 4B). Thus, we were
able to analyze both qualitatively and quantitatively the
phosphorylation of those three sites in the absence (basal) and in the
presence of DAMGO.
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Substantial basal phosphorylation of MOR in the absence of agonist activation has been reported by several groups including ours (13, 23, 24). As shown in Fig. 4, substantial basal phosphorylation of wild type receptor in the absence of agonist was observed. This basal phosphorylation was increased to approximately 3-fold in the presence of DAMGO (Fig. 4). Phosphorylation of mutant 363/370/375, in which the three phosphorylation sites were mutated to Ala, is completely abolished in the basal condition, whereas a weak phosphorylation signal can still be observed in the presence of DAMGO (Fig. 4, lanes 4 and 9). This remaining signal is not significant and is comparable with that observed with mutant 363-376 (Fig. 3), indicating the presence of a minor phosphorylation site outside of the Ser363-Thr376 region. However, the majority of DAMGO-induced phosphorylation signal (>95%) is abolished with this mutant (363/370/375). Interestingly, when Ser363 is left wild type (mutant 370/375), this mutant shows a significant basal phosphorylation, and this observed pattern of phosphorylation remains identical to that obtained in the presence of DAMGO. Thr370 (mutant 363/375 leaving Thr370 wild type) was weakly phosphorylated in basal conditions, and this phosphorylation pattern was increased significantly to approximately 2-fold in the presence of agonist. Finally, in the case of Ser375 (mutant 363/370 leaving Ser375 wild type), no basal phosphorylation could be detected, although this site is strongly phosphorylated in the presence of DAMGO (Fig. 4). The levels of DAMGO-induced phosphorylation observed with all these mutants were dramatically reduced in the presence of the antagonist naloxone (10 µM) to comparable levels with these obtained in basal conditions, whereas incubation with naloxone alone had no effect on basal phosphorylation itself (data not shown). Taken together, these results confirm that Ser363, Thr370, and Ser375 are phosphorylated, and they indicate that these residues are phosphorylated in an independent manner. Additionally, our data clearly point out that those three sites are phosphorylated differentially: Ser363 is phosphorylated only in the basal condition, Thr370 is phosphorylated both in the absence and in the presence of DAMGO, and Ser375 is phosphorylated only in the presence of DAMGO (Fig. 4). Therefore, it is tempting to propose that these three sites may participate to different extents in regulating MOR activity.
Previously, we reported that the rapid receptor phosphorylation (min) does not directly correlate with the relatively slow rate of desensitization (h) of MOR (23). Subsequently, we demonstrated that the relatively slow rate of desensitization was because of rapid recycling and resensitization of MOR (26). The rapid desensitization of MOR was observed only when the receptor expression level was low and receptor recycling was blocked. This study suggests that MOR internalization participates in receptor desensitization. Several studies have suggested a role of agonist-induced phosphorylation in opioid receptor internalization (8, 14, 32, 33). Therefore, it is important to clearly delineate the role of phosphorylation in regulating the loss of cell surface receptors. In this regard, it is important to examine the potential role of each individual phosphorylated Ser/Thr in regulating MOR internalization.
DAMGO-induced loss of cell surface wild type or mutated receptors was
monitored using flow cytometry as described under "Experimental Procedures." Kinetic analysis showed that MOR internalizes rapidly in
response to DAMGO activation (t1/2 ~16 min) and
that approximately 50% of the total cell surface receptors were
internalized after 1 h of treatment (Fig.
5). The S375A mutation (mutant 375)
significantly reduced the rate and extent of DAMGO-induced
internalization of receptor compared with wild type MOR. In this case,
only approximately 20% of the receptors were internalized after 1 h of DAMGO activation, p < 0.001 (Fig. 5B).
However, internalization kinetics of the T370A mutant
(t1/2 ~18 min, extent internalization ~50%)
were similar to those observed for the wild type receptor (Fig.
5A). Interestingly, the S363A mutation accelerated the rate
(t1/2 ~10 min, p < 0.01) but did
not increase the extent of DAMGO-induced receptor internalization (Fig.
5A). As a mixture of HEK293 clones stably expressing wild
type or mutated receptors was used for this study, the observed
differences in receptor internalization among those mutated receptors
could not be attributed to a phenotype effect. Thus, these results
clearly show that phosphorylation plays an important role in
controlling MOR internalization. Additionally, it appears clearly that
phosphorylation of these three sites contributes differentially in
regulating MOR internalization.
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We next examined the effects of mutating either two or three of those phosphorylation sites to Ala (Fig. 4B) on receptor internalization. Mutation of both Ser363 and Thr370 (mutant 363/370) further increases the rate and extent of internalization compared with that of single mutant S363A. This result suggests that the phosphorylation of both residues (Ser363 and Thr370) plays a role in attenuating receptor internalization (Fig. 5A). However, all mutants containing S375A mutation (mutants 363/375, 370/375, and 363/370/375) showed a reduced rate and extent of MOR internalization when compared with wild type receptor (Fig. 5B). These results show clearly that the phosphorylation of Ser375 plays a critical role in promoting the internalization of activated MOR. It should be noted that internalization kinetics observed in the case of mutants 363/375, 370/375, and 363/370/375 were slightly faster than those of the S375A single mutant (Fig. 5B). This finding confirms that phosphorylation of Ser363 and Thr370 has an opposite effect to that of the Ser375 mutation in regulating MOR internalization. Additionally, 363/370/375 mutations did not completely block the internalization of MOR (Fig. 5B), indicating that other signals or motifs could participate in these cellular events.
To avoid any differential rate of recycling of mutant receptors, which
may interfere with measured internalization kinetics, a receptor
recycling blocking agent monensin was included in this study to monitor
only internalized receptors. A 1-h pretreatment with monensin (50 µM) prior to the addition of DAMGO had no significant effect on the internalization kinetics of mutant or wild type µ-opioid receptors in HEK293 cells (data not shown). Thus, in our
present study, measurement of DAMGO-induced loss of cell surface receptors is mainly because of the internalization phenomena. Our results clearly show differential involvement of phosphorylation sites in regulating µ-opioid receptor internalization.
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DISCUSSION |
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The present study represents a significant step toward understanding the molecular events associated with the regulation of MOR. We first demonstrated that of the 12 Ser/Thr residues present within the C-tail of MOR, 3 residues (Ser363, Thr370, and Ser375) are phosphorylated. The role of these specific phosphorylated sites in receptor internalization was delineated. Systematic studies of the corresponding Ser/Thr mutants revealed differential regulation of MOR internalization by phosphorylation.
Several studies have reported agonist-induced phosphorylation of opioid
receptors, which has been shown to be time-dependent and
agonist concentration-dependent in different cell systems (23-25, 33). Whereas all these studies suggest that phosphorylation plays a role in opioid receptor regulation, i.e.
desensitization, internalization, and down-regulation, molecular events
related to receptor phosphorylation/regulation are still poorly
understood. In addition, differential phosphorylation/regulation of
opioid receptors by opioid agonists has been observed (6, 11, 14, 33).
These reports suggest that different agonist-activated receptor
conformations may exist and that specific phosphorylation site(s) may
participate in regulating receptor activity and trafficking. Thus,
several Ser/Thr residues within the COOH terminus of opioid receptors
have been suggested to be involved in the regulation of these
receptors. Thr394, Ser353, and
Ser369 have been shown to be important for the regulation
of µ-opioid receptors (20, 34, 35), -opioid receptors (18, 19), and
-opioid receptors (36), respectively, although phosphorylation of these residues has not been determined. Recently, a T394A mutation was shown to significantly block DAMGO-induced MOR phosphorylation to
approximately 10% of the wild type receptor and impair receptor desensitization in Chinese hamster ovary cells (37), indicating that
this residue is a phosphorylation site. However, in HEK293 cells, the
mutation of Thr394 to Ala reduced the DAMGO-induced
phosphorylation level to approximately 80% of maximal phosphorylation
obtained with wild type receptor (data not shown). This finding clearly
shows that major phosphorylation sites of MOR expressed in HEK293 cells
are located in residues other than Thr394. DAMGO-induced
phosphorylation of mutant 354-376 (leaving Thr394 wild
type) is completely abolished (Fig. 2). These results clearly indicate
that DAMGO does not induce phosphorylation of Thr394 in
HEK293 cells, but this site might participate in the agonist-induced phosphorylation of MOR as a recognition binding motif for a kinase or a
regulatory protein. Taken together, these results suggest that cell
type-specific differences may exist in the agonist-induced receptor
phosphorylation. Our current mutational analysis indicated that at
least in HEK293 cells Thr370 and Ser375 are the
residues phosphorylated in the presence of agonist, whereas Thr394 is the residue that is phosphorylated in Chinese
hamster ovary cells (37). Considering that the T394A mutation did not
completely block agonist-induced MOR phosphorylation in Chinese hamster
ovary cells (37), other sites involved in receptor phosphorylation in
this cell line need to be identified.
Several kinases have been postulated to be involved in opioid receptor phosphorylation including protein kinase A (38, 39), protein kinase C (33, 40), GRKs (25, 34), mitogen-activated protein kinase (41, 42), calmodulin kinase II (43, 44), and tyrosine kinase (32). Recently, our group has reported a [D-Pen2-D-Pen5]enkephalin-induced hierarchical phosphorylation mechanism of DOR in HEK293 cells (21). Ser363 has been shown to be the primary agonist-induced phosphorylation site allowing subsequent phosphorylation of Thr358. Thr361 was shown to be involved in regulating phosphorylation of DOR without being phosphorylated. In addition, mutation of Thr361 to Ala impairs phosphorylation of Thr358, suggesting that Thr361 could participate in a recognition binding motif for a kinase or regulatory protein (21). Although MOR and DOR have differences in their C-tail primary sequence, it is interesting to note that a common motif encompassing agonist-induced phosphorylated sites exists for both receptor subtypes. The organization of motif (357VTACTPSD364) containing Thr358 and Ser363 of DOR is analogous to the motif (369NTREHPST376) containing Thr370 and Ser375 of MOR. The presence of acidic or charged amino acids surrounding those agonist-induced phosphorylated sites suggests that they are potential GRK-phosphorylation sites. Furthermore, a conserved Pro residue directly present upstream of Ser363 and Ser375 in DOR and MOR, respectively, may confer a specific spatial conformation of the C-tail, and these phosphorylation sites may be accessible to specific kinase(s). Whether the same kinase(s) is involved in the phosphorylation of both receptor subtypes remains to be demonstrated. Clearly, the two sites being phosphorylated in the presence of the agonist do not represent a mitogen-activated protein kinase site. Several groups including our own have reported basal phosphorylation of opioid receptors in the absence of agonist (13, 23, 24, 33), suggesting a correlation between this basal phosphorylation and the observed basal activity of these receptors. Our present results show that Ser363 is specifically phosphorylated in the absence of DAMGO treatment. Thr370 appears to be phosphorylated in both basal and agonist-induced conditions. However, Ser375 is phosphorylated only in the presence of DAMGO. The present identification of phosphorylation sites in MOR will greatly facilitate determination of the kinase responsible for phosphorylation of each specific site as well as associated protein(s) involved in regulating receptor activities.
Previous reports show no effects of mutated or truncated COOH termini of opioid receptors on desensitization and/or internalization of these receptors (22, 36, 45, 46). These studies raise the fundamental question of whether or not phosphorylation is actually required for agonist-induced desensitization and internalization of opioid receptors. The present identification of MOR phosphorylation sites enabled us not only to dissect the complex phosphorylation mechanisms but also to investigate the role of each individual phosphorylation site in receptor internalization. For the first time, we show that the rate and extent of MOR internalization are differentially modulated by site-specific phosphorylation. Phosphorylation of Ser375 plays an important role in promoting the internalization of activated receptor, whereas phosphorylation of Ser363 and Thr370 may synergistically attenuate this receptor internalization. Segredo et al. (14) have shown that truncation of µ-opioid receptor at positions 363 and 354 differentially regulates DAMGO-induced receptor internalization in HEK293 cells. This study suggests that phosphorylation sites located in the C-tail domain play distinct roles both in promoting agonist-induced endocytosis of activated receptors and preventing receptor endocytosis in the absence of the agonist (14). The same study shows that a truncated mutant at position 354 internalized constitutively and that receptor internalization is enhanced in presence of DAMGO (14). Similarly, Trapaidze et al. (46) have shown that a truncated MOR lacking Thr394 continued to internalize in response to DAMGO but not the mutant lacking the last COOH-terminal 24 amino acids. Consistent with our findings, these results indicate the presence of positive and negative endocytotic signals within the C-tail of MOR. In addition, the current study provides evidence that a single phosphorylated Ser/Thr mutation could differentially alter MOR internalization. Although the vast majority of endocytotic signals described previously are positive signals, the presence of endocytotic negative signals within the C-tail of the receptors has been reported for several GPCRs including somatostatin (47), parathyroid hormone/parathyroid hormone-related receptors (48), and luteinizing hormone/human choriogonadotropin (49) receptors. Thus, these results and our findings indicate that receptor-mediated endocytosis is a highly complex and regulated process with multiple receptor domains playing facilitatory as well as inhibitory roles.
As for many GPCRs, opioid receptors are endocytosed in a dynamin-dependent manner via clathrin-coated pits (9, 33, 36, 40). Our findings suggest that at least two mechanisms of phosphorylation may exist and contribute differentially to MOR endocytosis in HEK293 cells. The first mechanism of phosphorylation is agonist-induced GRK-mediated phosphorylation of Ser375, and maybe Thr370, which involves the binding of arrestin and subsequent internalization of MOR. This is consistent with a recent report that blocking DAMGO-induced phosphorylation but not basal phosphorylation of MOR in HEK293 cells prevents the receptor from being internalized (42). The second mechanism is the phosphorylation of Ser363, and maybe Thr370, in the absence of agonist that may involve kinase(s) other than GRKs and could play a regulatory role in modulating basal or constitutive MOR internalization. As shown by Segredo et al. (14), both constitutive and agonist-induced internalization of MOR could be mediated by the clathrin-coated pit pathway. Receptor recycling to the cell surface after DAMGO treatment was negligible in the current study (data not shown) in contrast to the substantial receptor recycling after etorphine treatment as reported previously (26). This finding reflects the differential regulation of MOR by opioid agonists including desensitization, internalization, and down-regulation of the receptors (5, 6, 8, 50). Structurally, distinct domains of the rat MOR were shown to be involved in the binding of receptor selective alkaloids and peptides (51, 52). In addition, the level of overall MOR phosphorylation was reported to be agonist-dependent (6, 33). Taken together, these findings strongly indicate that different agonist-activated receptor conformations exist and that receptor phosphorylation plays an important role in the structure-function of this receptor. Thus, identification of phosphorylation sites induced by other agonists will help considerably to delineate molecular mechanisms controlling the structure-function of MOR.
In conclusion, the present studies have brought new insights into the
complex function of MOR phosphorylation. We have identified three
phosphorylated sites within the C-tail of MOR and delineated their role
in regulating receptor endocytosis. Interestingly, we have found that
phosphorylation of Ser375 is critical for DAMGO-induced MOR
internalization. However, phosphorylation of Ser363 and
Thr370 represents a negative regulatory signal in this
internalization process. Whether these regulation mechanisms are
conserved among other members of the GPCR family remains to be
demonstrated. Nevertheless, the present work makes a significant step
toward the elucidation of molecular events related to opioid receptor
regulation and thus to opioid tolerance and dependence.
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FOOTNOTES |
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* This research is supported in part by National Institutes of Health Grants DA00564, DA07339, DA01583, and DA11806.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church
St., S.E., Minneapolis, MN 55455. Tel.: 612-624-6691; Fax:
612-625-8408; E-mail: elkou001@tc.umn.edu.
§ Present address: Boehringer Ingelheim Pharmaceuticals, Inc., P.O. Box 368, 900 Ridgebury Rd., Ridgefield, CT 06877.
¶ Recipient of K05DA70554 award from the National Institute on Drug Abuse and the F. and A. Stark Fund of the Minnesota Medical Foundation.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009571200
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G
protein-coupled receptor;
MOR, µ-opioid receptor;
DOR, -opioid
receptor;
HEK, human embryonic kidney;
DAMGO, [D-Ala2,Me-Phe4,Gly5-ol]enkephalin;
PAGE, polyacrylamide gel electrophoresis;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
GRK, G
protein-coupled receptor kinase;
C-tail, carboxyl tail.
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