Heterologous Activation of Protein Kinase C Stimulates
Phosphorylation of
-Opioid Receptor at Serine 344, Resulting in
-Arrestin- and Clathrin-mediated Receptor Internalization*
Bin
Xiang
,
Guo-Hua
Yu§,
Jun
Guo
,
Li
Chen
,
Wei
Hu
,
Gang
Pei§, and
Lan
Ma
¶
From the
National Laboratory of Medical Neurobiology,
Fudan University Medical Center, Shanghai 200032, and
§ Shanghai Institute of Biochemistry and Cell Biology,
Shanghai Institutes of Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, People's Republic of China
Received for publication, July 13, 2000, and in revised form, October 6, 2000
 |
ABSTRACT |
The purpose of the current study is
to investigate the effect of opioid-independent, heterologous
activation of protein kinase C (PKC) on the responsiveness of opioid
receptor and the underlying molecular mechanisms. Our result showed
that removing the C terminus of
opioid receptor (DOR) containing
six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate
13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation
levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were
comparable to that of the wild-type DOR, whereas S344G substitution
blocked PMA-induced receptor phosphorylation, indicating that
PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated
Ser-344 phosphorylation was also induced by activation of
Gq-coupled
1A-adrenergic receptor or increase in intracellular Ca2+ concentration. Activation of
PKC by PMA,
1A-adrenergic receptor agonist, and
ionomycin resulted in DOR internalization that required phosphorylation
of Ser-344. Expression of dominant negative
-arrestin and hypertonic
sucrose treatment blocked PMA-induced DOR internalization, suggesting
that PKC mediates DOR internalization via a
-arrestin- and
clathrin-dependent mechanism. Further study demonstrated
that agonist-dependent G protein-coupled receptor kinase
(GRK) phosphorylation sites in DOR are not targets of PKC.
Agonist-dependent, GRK-mediated receptor phosphorylation
and agonist-independent, PKC-mediated DOR phosphorylation were
additive, but agonist-induced receptor phosphorylation could inhibit
PKC-catalyzed heterologous DOR phosphorylation and subsequent
internalization. These data demonstrate that the responsiveness of
opioid receptor is regulated by both PKC and GRK through
agonist-dependent and agonist-independent mechanisms and
PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.
 |
INTRODUCTION |
Opioid receptors are G
protein-coupled receptors (GPCR)1 and include
,
, and µ subtypes. Interaction of opioid receptors on the surface of neurons
in the central nervous system with endogenous opioid peptides and
synthetic alkaloids produces strong analgesic effect, but chronic use
of opioid drug results in drug tolerance and dependence. The molecular
mechanisms of regulation of the receptor responsiveness and opioid
tolerance and dependence are not well understood, although
desensitization of opioid receptor has been implicated as one of the
major mechanisms.
The responsiveness of opioid receptor reduces upon exposure to opioid
agonist, and this agonist-dependent desensitization is
defined as homologous desensitization of the opioid receptor. Several
mechanisms contribute to desensitization of opioid receptors. It has
been demonstrated that, following stimulation of opioid agonist, the
opioid receptor becomes phosphorylated rapidly (1-4), the
phosphorylated receptor uncouples from G proteins and binds to
-arrestins (5, 6), the receptor is subsequently sequestered in an
intracellular compartment (7), and even the expression of the opioid
receptor is down-regulated (8). Phosphorylation of opioid receptors is
the initial step in opioid receptor desensitization, and
phosphorylation of
,
, and µ subtypes of opioid receptors in
response to agonist stimulation has been demonstrated by other laboratories as well as our own (1-4, 9). Experimental results indicate that GPCR kinase (GRK), not PKC and PKA, is the primary protein kinase involved in homologous phosphorylation of opioid receptors stimulated by opioid agonist and plays an important role in
agonist-induced homologous desensitization of opioid receptors (1, 6,
9, 10).
In addition to agonist-specific receptor desensitization,
functions of GPCRs can be regulated by agonist-independent mechanisms, namely, heterologous desensitization. Second
messenger-dependent protein kinases such as PKA and PKC
mediate receptor phosphorylation, and this has been implicated in
heterologous regulation of activities of a number of GPCRs recently
(11, 12). Accumulating evidence suggest that the sensitivity of
opioid receptor in response to neural signals is also regulated by
nonopioid pathways. Animal experiments and clinical studies showed
that NMDA antagonist potentiates morphine-induced analgesia and
prevents opioid tolerance (13). Our previous study demonstrated that
activation of NMDA receptor attenuates opioid receptor-mediated
cellular signaling, and this is mediated by PKC (14). Ohsawa and
colleagues (15) showed that preadministration of insulin inhibits the
antinociceptive effect of (D-Ala2,
N-Me-Phe4,Gly5-ol)enkephalin,
a specific agonist of µ-opioid receptor in mice, and their results
suggest that the effect is mediated by activation of PKC and tyrosine
kinase. Increasing Ca2+ concentration in neurons and
synaptosomes antagonizes opioid-induced antinociception (16,
17). Suppression of µ,
, and
opioid receptor agonist-induced
analgesia by PKC activators in animals has also been shown by many
laboratories (18-21). It has also been observed that chronic opiate
treatment strongly increases PKC activity in specific brain regions,
and inhibition of PKC activity attenuates the development of opioid
tolerance and dependence (22, 23). These data strongly suggest that
opioid signaling is regulated heterologously by agonist-independent
pathways in vivo and PKC is likely an important mediator.
Research indicates that phorbol esters, activators of PKC, reduce
opioid-induced inhibition on cAMP production (24) and potentiate
desensitization of opioid receptor-activated K+ current (2,
25). Furthermore, research from other laboratories and our groups
demonstrated that phorbol ester PMA treatment could induce
phosphorylation of
and µ opioid receptors in the absence of
agonist stimulation (1, 2, 9, 26). These data indicate that
PKC-mediated heterologous phosphorylation of opioid receptor may
contribute to desensitization of opioid receptor in neurons. However,
there is so far no report on the effect of physiological activation of
PKC on opioid receptor phosphorylation and signal transduction, and the
mechanism and functional impact of PKC activation on opioid
receptor-mediated signaling are not known. In the current study, we
identified a PKC-mediated phosphorylation site in the
-opioid
receptor (DOR) and demonstrated that activation of PKC by stimulation
of other types of GPCR or increase in intracellular Ca2+
concentration in HEK 293 cells induces heterologous phosphorylation of
DOR. Our results further established that DOR phosphorylation at
Ser-344 by PKC results in internalization of DOR in HEK 293 cells
through a
-arrestin- and clathrin-mediated mechanism.
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EXPERIMENTAL PROCEDURES |
Materials--
[D-Pen2,D-Pen5]Enkephalin
(DPDPE), forskolin, ionomycin, 1-methyl-3-isobutylxanthine, PD98059,
and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma
Chemical Co. (St. Louis, MO).
[32Pi]Orthophosphate (5000 Ci/mmol) were
purchased from Amersham Pharmacia Biotech.
[1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester] (BAPTA/AM), benzamidine-HCl, cantharidin, KN-62, staurosporine were obtained from Calbiochem (La
Jolla, CA). A61603, GF109203X, and WB4101 were supplied by Tocris
(United Kingdom). HEK 293 cells were obtained from the American Type
Culture Collection (Rockville, MD). Modified Eagle's medium (MEM),
fetal bovine serum, and phosphate-free Dulbecco's MEM were purchased
from Life Technologies, Inc. (Grand Island, NY). Protein A-Sepharose
was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). 12CA5
mouse monoclonal antibody, recognizing influenza hemagglutinin (HA)
epitope, was supplied by Roche Molecular Biochemicals. FITC-conjugated
goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West
Grove, PA).
Plasmid Construction--
Plasmids encoding HA-tagged mouse wild
type
opioid receptor (WT), the C-terminal 31 residues truncated DOR
(
31), and the DOR mutants with the C-terminal serine or threonine
residue substituted were constructed in pcDNA3 as described
previously (1, 9). DOR mutant S344G (m1), T352A (m2), and T353A (m3)
with HA-tag at the N terminus were constructed by exchanging the
NotI/XbaI fragment of DOR with the corresponding
fragment in FLAG-tagged mutant DOR (8). The HA-tagged DOR mutant
T358A/T361A/S363G (m4/5/6), S344D, T358D, T361D, and S363D were
constructed by PCR mutagenesis, and authenticity of sequence was
confirmed by DNA sequencing. The human
-arrestin 1 cDNA
construct was as constructed as described previously (27).
1V53D (a dominant inhibitory mutant of
-arrestin 1 carrying a
V53D substitution) cDNA clone was constructed in pcDNA3 by PCR mutagenesis.
Cell Culture and Transfection--
Human embryonic kidney (HEK)
293 cells cultured in MEM containing 10% fetal bovine serum were
seeded in 60-mm tissue culture dish at 1 × 106/dish
20 h before transfection. Plasmids (3-4 µg each) were
transiently transfected in the cells using the calcium phosphate
method. The expression of opioid receptors was measured by a
radioligand binding assay 44 h after transfection as described
preciously (5), and the levels of the opioid receptors expressed were
2-3 pmol/mg of protein.
Receptor Phosphorylation--
Measurement of opioid receptor
phosphorylation was carried out as described previously (1, 9).
Briefly, the cells were labeled at 37 °C for 60 min with
[32Pi] at 60 µCi/dish in phosphate-free
Dulbecco's MEM 44 h after transfection. Following treatment with
various pharmacological agents, cells were put on ice and solubilized
for 1.5 h in radioimmune precipitation-plus buffer (1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 5 mM EDTA, with 10 mM NaF, 10 mM disodium pyrophosphate, and 5 µM cantharidin as phosphatases inhibitors and 10 µg/ml
aprotinin, 10 µg/ml benzamidine, and 0.2 mM
phenylmethylsulfonyl fluoride as protease inhibitors). After
centrifugation at 80,000 × g for 15 min at 4 °C,
the supernatants were immunoprecipitated for 2 h at 4 °C with 1 µg of 12CA5 monoclonal antibody and protein A-Sepharose beads. After
washing in radioimmune precipitation-plus buffer, the absorbed
complexes were removed from the beads by heating for 20 min at 50 °C
in reducing SDS-polyacrylamide gel electrophoresis sample buffer and
analyzed on 10% polyacrylamide gels. The gels were subjected to
quantitative analysis using a PhosphorImager (Molecular Dynamics) after drying.
Receptor Internalization--
Measurement of internalization was
carried out as described previously (27). Briefly, the cells were
challenged with the indicated chemicals in MEM at 37 °C for 30 min.
After treatment, cells were placed on ice and washed twice with
phosphate-buffered saline (PBS). The cells were incubated with 12CA5 (5 µg/ml) in PBS containing 1% bovine serum albumin and then with
FITC-conjugated goat anti-mouse IgG. The samples were analyzed on a
FACSCalibur flow cytometer (Becton Dickinson, Mountain View,
CA). Basal cell fluorescence intensity was determined with cells
stained with the secondary antibody alone.
cAMP Assay--
Cells were challenged with agonist in the
presence of 10 µM forskolin (Sigma) and 500 µM 1-methyl-3-isobutylxantine (Sigma) at 37 °C for 15 min. The reactions were terminated with 1 N perchloric acid
and neutralized with 2 N K2CO3. The
cAMP level of each sample was determined using radioimmunoassay as
described previously (27). Data were averaged from triplicate samples
and calculated as 100 × [cAMPfor+Agonist
cAMPbasal]/[cAMPfor
cAMPbasal], where cAMPfor+Agonist is cAMP
accumulation in the presence of forskolin and agonist,
cAMPbasal is cAMP in the absence of forskolin and agonist,
and cAMPfor is cAMP in the presence of forskolin alone.
Statistical Analysis--
Data were analyzed using Student's
t test for comparison of independent means with pooled
estimates of common variances.
 |
RESULTS |
As shown in Fig. 1, incubation of
PMA with the HEK 293 cells transiently expressing DOR induced DOR
phosphorylation. Phosphorylation of DOR in these cells was detected in
3 min and reached a peak level (400% of the basal level) in 5 min of
PMA exposure (Fig. 1A). PMA-induced DOR phosphorylation was
concentration-dependent. DOR phosphorylation was detectable
at 100 nM PMA and approached the plateau at 1 µM PMA (Fig. 1B).

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Fig. 1.
PMA-stimulated phosphorylation of DOR in
time- and concentration-dependent activities.
32Pi-Labeled HEK 293 cells transiently
expressing DOR were stimulated with 1 µM PMA at 37 °C
for the indicated times (A) or stimulated with indicated
concentrations of PMA at 37 °C for 10 min (B). DORs were
immunoprecipitated with 12CA5, and receptor phosphorylation was
analyzed. The representative autoradiograms are shown on the
upper panels, and the quantitation of the results from two
separate experiments, analyzed with a PhosphorImager and expressed as
-fold values of the basal level of receptor phosphorylation, is shown
in the lower panels.
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The role of PKC in PMA-stimulated DOR phosphorylation was explored
next. Fig. 2 shows that PKC inhibitors
staurosporine and GF109203X (28) abolished PMA-induced DOR
phosphorylation completely, whereas MEK (MAPK/ERK kinase) inhibitor
PD98059 had no significant effect under the same conditions. In
addition, the PKA activator forskolin did not stimulate DOR
phosphorylation (Fig. 2). These data indicate that PMA-stimulated DOR
phosphorylation is mediated by PKC, whereas other protein kinases such
as PKA and MAPK are not critically involved.

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Fig. 2.
PMA-stimulated DOR phosphorylation was
mediated by PKC. HEK 293 cells transiently expressing DOR were
labeled with 32Pi and challenged with or
without 0.2 µM staurosporine (Stau), 2 µM GF109203X (GF), or 20 µM
PD98059 (PD) for 20 min prior to incubation with 1 µM PMA, 10 µM forskolin, or PBS
(Ctrl) for 10 min. DORs were then immunoprecipitated and
receptor phosphorylation was analyzed. The figure is representative of
three independent experiments performed.
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Our previous research (9) has demonstrated that the C-terminal domain
of DOR plays a critical role in PMA-induced DOR phosphorylation, implicating that PKC-mediated DOR phosphorylation occurs at the C-terminal domain of the receptor. To identify the PKC-mediated DOR
phosphorylation site, DOR mutants S344G (m1), T352A (m2), T353A (m3),
and T358A/T361A/S363G (m4/5/6), with one or more potential Ser/Thr
phosphorylation sites in the C terminus of DOR substituted and the
truncation mutant lacking the 31 residues containing six Ser/Thr
potential phosphorylation sites (designated as
31), were constructed. Results showed that surface expression, ligand binding, and G protein coupling of the above mutant receptors were comparable to
the wild-type DOR (WT) (data not shown). As shown in Fig.
3, removing the C-terminal domain
containing all six potential phosphorylation sites blocked PMA-induced
DOR phosphorylation completely, whereas eliminating Thr-358, Thr-361,
and Ser-363, the three potential phosphorylation residues proximal to
the C terminus, by substitution of the three Ser/Thr residues with
neutral amino acid had no detectable effect on the phosphorylation
level of DOR, indicating that the PKC-mediated DOR phosphorylation site
is likely located among Ser-344, Thr-352, and Thr-353 in the receptor
cytoplasmic tail.

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Fig. 3.
PMA-stimulated phosphorylation of DOR at
Ser-344. The HEK 293 cells transiently expressing WT, 31,
m4/5/6, m1, m2, or m3 were labeled with 32Pi
and stimulated with or without 1 µM PMA for 10 min. DORs
were immunoprecipitated, and receptor phosphorylation was analyzed. The
figures are representative of three independent experiments
performed.
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Analysis of amino acid sequences flanking the three Ser/Thr residues
predicates Ser-344 and Thr-352 as putative PKC phosphorylation sites
(29). To further examine the role of Ser-344, Thr-352, and Thr-353 in
PMA-induced DOR phosphorylation, phosphorylation levels of DOR mutants
m1, m2, and m3 stimulated by PMA were determined. As shown in Fig. 3,
following stimulation with PMA, the extents of phosphorylation of m2
and m3 mutants were comparable to that of the wild type DOR (WT), but
in contrast, phosphorylation of m1 DOR was not detectable under the
same conditions. These data indicate that Ser-344 in the cytoplasmic
tail of DOR is the phosphorylation site in PKC-mediated DOR
phosphorylation. We have demonstrated that PKA and MAPK are not
involved in PKC-mediated DOR phosphorylation. Furthermore, the Ser-344
flanking sequence does not resemble phosphorylation consensus sequences
for PKA, MAPK, or calcium/calmodulin-dependent protein
kinase II, but Ser-344 is located in a typical PKC phosphorylation motif. Therefore, Ser-344 is very likely the in vivo
phosphorylation site of PKC in PMA-stimulated DOR phosphorylation.
PKC is one of the most important signal molecules in cells and its
activity is regulated through a number of different pathways. Phosphorylation status of opioid receptors could be therefore regulated, via PKC, by activation of one or more signal molecules or
signal transduction cascades other than opioid pathway. To explore the
potential physiological significance of PKC-stimulated opioid receptor
phosphorylation, we examined the effect of activation of Gq
protein-coupled receptor,
1A-adrenergic receptor
(
1A-AR), on the phosphorylation of DOR. As shown in Fig.
4A, stimulation of HEK 293 cells transiently coexpressing DOR and
1A-AR with A61603, a selective agonist of
1A-AR, resulted in strong
phosphorylation of DOR at a level comparable to that of stimulated by
PMA. A61603-stimulated DOR phosphorylation was abolished by
preincubation with either the selective
1A-AR antagonist
WB4101 or the PKC inhibitors staurosporine and GF109203X (Fig.
4A). The mutant DOR lacking PKC phosphorylation site Ser-344
failed to become phosphorylated following stimulation with A61603 under
the same conditions (Fig. 4A). These data indicate that
heterologous activation of PKC through activation of
1A-AR, a receptor coupled to Gq protein,
results in DOR phosphorylation at PKC site Ser-344. Furthermore,
ionomycin, a Ca2+ ionophore, elevated
Ca2+concentration in HEK 293 cells (data not shown) and
stimulated DOR phosphorylation at Ser-344, and the effect of ionomycin
could be blocked by inhibitors of PKC but not by an inhibitor of
calcium/calmodulin-dependent protein kinase II, KN-62 (Fig.
4B). In addition to ionomycin, A23187, another
Ca2+ ionophore, and ATP, a natural ligand of
purinoceptor, increased intracellular Ca2+
concentration and stimulated DOR phosphorylation in HEK 293 cells (data
not shown). BAPTA/AM, a Ca2+ chelator, abolished
PKC-mediated DOR phosphorylation stimulated by PMA, A61603, and
ionomycin (Fig. 4C).

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Fig. 4.
PKC-mediated phosphorylation of DOR was
Ca2+-dependent. The transfected HEK 293 cells were labeled with 32Pi and treated as
indicated. DORs were immunoprecipitated, and receptor phosphorylation
was analyzed. The figures are representative of three independent
experiments performed. A, the cells coexpressing
1A-AR and WT or m1 were incubated with or without 2 µM WB4101 (WB) for 30 min, 0.2 µM staurosporine for 20 min, or 2 µM
GF109203X for 20 min prior to stimulation with 1 µM
A61603 (A6), 1 µM PMA, or PBS for 10 min.
B, the cells expressing WT or m1 were incubated with or
without 0.2 µM staurosporine for 20 min, 2 µM GF109203X for 20 min, 10 µM KN-62
(KN) for 20 min prior to stimulation of 1 µM
ionomycin (Iono), 1 µM PMA, or PBS for 10 min.
C, the cells expressing DOR or coexpressing DOR and
1A-AR (for A61603 treatment) were incubated with or
without 50 µM BAPTA/AM (BAP) for 20 min prior
to stimulation of 1 µM PMA, 1 µM ionomycin,
1 µM A61603, or PBS for 10 min.
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To investigate the functional consequence of PKC-mediated DOR
phosphorylation, DOR internalization in response to PKC activation induced by PMA, ionomycin, and A61603 was measured using flow cytometry. As shown in Fig. 5, a 30-min
PMA treatment resulted in a significant reduction in cell surface
fluorescence in the cells expressing the wild type DOR, indicating a
considerable loss of the DOR from the cell surface (40-50%), which
was similar to the extent of DOR internalization induced by the DOR
agonist DPDPE (Fig. 6). In contrast, the
same PMA treatment caused no significant change in the surface
immunofluorescence in the cells expressing DOR mutant m1 lacking PKC
site Ser-344 in the receptor C-tail (Fig. 5). However, m1 mutant DOR
internalized rapidly in response to DOR-selective agonist DPDPE (data
not shown). Similarly, activation of PKC by stimulation of
1A-AR or mobilization of Ca2+ induced
internalization of the wild type DOR but had no significant effect on
the surface density of DOR mutant m1 (Fig. 5C). Similar to
agonist pretreatment, incubation of PMA with the HEK 293 cells transiently expressing DOR strongly attenuated DPDPE-induced inhibition on cAMP accumulation; However, substituting Ser-344 to alanine did not
block the PMA-induced desensitization of DOR-mediated inhibition of
cAMP accumulation (Fig. 5D). These results are consistent with the previous studies that PKC is capable of phosphorylating G
i (13, 30) and adenylyl cyclase V (31), which couple
with opioid receptors (32) and suggest that PKC may regulate opioid signal transduction at levels of G protein and/or cyclase in addition to phosphorylation of opioid receptor. Our results indicate that induction of DOR internalization is one of the functional consequences of agonist-independent phosphorylation of DOR following activation of
PKC via a heterologous signaling pathway (stimulation of another GPCR
or mobilizing Ca2+), whereas PKC-mediated reaction
targeting molecules downstream from the receptor may also play
important roles in desensitization of opioid-induced cellular
responses.

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Fig. 5.
Activation of PKC by PMA, A61603, and
ionomycin induced DOR desensitization. A-C, the HEK
293 cells expressing WT or m1 (and coexpressing 1A-AR
for A61603 stimulation) were challenged with PBS, 1 µM
PMA, 1 µM A61603, or 1 µM ionomycin at
37 °C for 30 min, and the surface receptors were stained with 12CA5
and FITC-conjugated anti-mouse IgG. Representative flow cytometry
profiles of WT (A), m1 (B), and quantitative
analysis of internalization data (C) are shown.
Ctrl, stained only with FITC-conjugated secondary antibody
(basal fluorescence of the cells). The basal fluorescence of the cells
was subtracted, and the receptor internalization is indicated by
percent reduction in mean surface fluorescence in the cells measured as
compared with the untreated cells. *, p < 0.01, as
compared with WT. D, the cells expressing WT or m1 were
incubated with PBS (none), 1 µM DPDPE, or 1 µM PMA for 15 min prior to treatment of 1 µM DPDPE. The cellular cAMP levels were determined and
are expressed as percentages of forskolin-stimulated values in the
absence of agonist. Data are from at least three experiments and
presented as means ± S.E. *, p < 0.01; #,
p < 0.05, as compared with the cells pretreated with
PBS.
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Fig. 6.
Requirements of
-arrestin and clathrin for PKC-stimulated DOR
internalization. A, the HEK 293 cells expressing DOR
were incubated with 1 µM PMA or 1 µM DPDPE
for 30 min at 37 °C, and the cell surface receptors at different
time points following removal of stimuli by washing were assessed by
flow cytometry. Data are plotted as percentage of total cell surface
fluorescence measured in untreated cells. B, the cells
cotransfected with DOR and pcDNA3, -arrestin 1 ( arr1), or 1V53D were incubated with 1 µM PMA or 1 µM DPDPE for 30 min at
37 °C. C, the cells expressing DOR were incubated with or
without 0.4 M sucrose for 15 min at 37 °C before
stimulated with 1 µM DPDPE or 1 µM PMA.
Data are normalized to DOR internalization induced by DPDPE in the
cells cotransfected with DOR and pcDNA3 or the cells not treated
with sucrose and presented as means ± S.E. of three independent
experiments. *, p < 0.01; #, p < 0.05, as compared with that from the cells transfected with pcDNA3
or not treated with sucrose.
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The mechanisms of PKC-mediated DOR internalization were examined next.
As shown in Fig. 6A, flow cytometric analysis of surface receptor indicates that PMA-induced DOR internalization is reversible: The surface DOR fluorescence gradually recovered 1 h after PMA was
removed. Studies showed that opioid-induced receptor internalization involves
-arrestins and clathrin-coated pits (33, 34). Hence, the
effects of coexpression of the wild-type
-arrestin 1 or
1V53D, a
dominant negative mutant of
-arrestin 1, on PKC-mediated DOR internalization were assessed. Overexpression of
-arrestin 1 enhanced PMA-stimulated DOR internalization and overexpression of
1V53D blocked this effect (Fig. 6B). It has been shown
that
-arrestins function in agonist-induced GPCR internalization as an adapter of clathrin in formation of clathrin-coated pits (35). To
examine the role of clathrin in PKC-stimulated DOR internalization, HEK
293 cells transiently expressing DOR were incubated with PMA following
0.4 M sucrose pretreatment, which blocks formation of clathrin-coated pits (36). Analysis of cell surface DOR fluorescence indicates that exposure of the cells to hypertonic sucrose completely blocked PMA-induced DOR internalization (Fig. 6C). These
data clearly indicate that, like agonist-dependent
homologous DOR internalization, the PKC-mediated heterologous
internalization of DOR requires
-arrestin and occurs via
clathrin-coated pits.
As shown in Fig. 7A, PKC
inhibitors staurosporine and GF109203X did not block
DPDPE-stimulated DOR phosphorylation, indicating that PKC is not
required for agonist-dependent DOR phosphorylation (also
called homologous phosphorylation). Our previous studies indicated that
agonist-dependent DOR phosphorylation occurs in the
C-terminal region of DOR and is mediated by GRKs (1, 9). Removal of the
C-terminal 31 residues of DOR containing six potential Ser/Thr
phosphorylation sites (mutant
31) abolished both DPDPE- and
PMA-induced DOR phosphorylation (Figs. 3 and 7B), whereas substitution of PKC phosphorylation site Ser-344 (mutant m1) blocked PKC-mediated receptor phosphorylation but had no significant effect on
agonist-stimulated DOR phosphorylation (Figs. 3, 7B, and
8A). Substituting the last
three Ser/Thr residues (mutant m4/5/6) abolished DPDPE-induced receptor
phosphorylation completely but left PMA-stimulated DOR phosphorylation
intact (Figs. 3, 7B, and 8A). These data
demonstrate that agonist-dependent GRK-mediated homologous
phosphorylation and agonist-independent PKC-mediated heterologous
phosphorylation both occur at DOR cytoplasmic tail at comparable levels
but at clearly different sites and suggest that
responsiveness and phosphorylation of opioid receptor are
regulated by both PKC and GRK through agonist-dependent (homologous) and agonist-independent (heterologous) mechanisms.

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Fig. 7.
Agonist-induced DOR phosphorylation did not
require PKC nor Ser-344. The transfected HEK 293 cells were
labeled with 32Pi and treated as indicated.
DORs were immunoprecipitated, and receptor phosphorylation was
analyzed. The figures are representative of three independent
experiments performed. A, the cells expressing DOR were
challenged with or without 0.2 µM staurosporine or 2 µM GF109203X for 20 min prior to incubation with 1 µM DPDPE (DP) or no agent for 10 min.
B, the cells expressing DOR, 31, or m4/5/6 were incubated
with or without 1 µM DPDPE for 10 min at 37 °C.
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Fig. 8.
Comparison of DOR phosphorylation and
internalization induced by DPDPE and PMA. A-D, the
transfected HEK 293 cells were labeled with
32Pi and treated as indicated. DORs were
immunoprecipitated, and receptor phosphorylation was analyzed. The
figures are representative of three independent experiments performed.
Quantitative data are represented as fold values of the basal receptor
phosphorylation of untreated cells and means ± S.E. of at least
three separate experiments. *, p < 0.01. The cells
expressing WT, m1, or m4/5/6 were incubated with PBS, 1 µM PMA, 1 µM DPDPE, or 1 µM
PMA plus 1 µM DPDPE for 10 min (A). The cells
expressing WT, T358D, T361D, or S363D were incubated for 10 min in the
absence or presence of 1 µM PMA (B). The cells
expressing WT or S344D were incubated with or without 1 µM DPDPE for 10 min (C). D, the
cells expressing WT were stimulated for 7 min with none, 1 µM PMA, 1 µM DPDPE, or 1 µM
PMA followed by 7-min incubation with 1 µM DPDPE
(PMA then DP), or 1 µM DPDPE followed by 7-min
incubation with 1 µM PMA (DP then PMA). Data
are normalized to WT phosphorylation induced by DPDPE alone and
presented as means ± S.E. of three independent experiments.
E, the cells expressing WT were stimulated for 15 min with
none, 1 µM PMA, 1 µM DPDPE, or 1 µM PMA followed by 15-min incubation with 1 µM DPDPE (PMA then DP), or 1 µM
DPDPE followed by 15-min incubation with 1 µM PMA
(DP then PMA), and data are normalized to WT internalization
induced by DPDPE alone and presented as means ± S.E. of three
independent experiments. *, p < 0.01; #,
p < 0.05, as compared with DPDPE-treated
control.
|
|
Although both the PKC-mediated heterologous and GRK-mediated homologous
DOR phosphorylation could occur in the absence of the other at
comparable levels (~4-fold over the basal level), costimulation with
PMA and DPDPE caused no considerable increase in DOR phosphorylation,
as compared with the level of receptor phosphorylation stimulated with
either PMA or DPDPE alone (Fig. 8A). The phosphorylation
level of the wild type DOR (~4.3-fold over basal) was not
significantly different from that of DOR mutant m1 or m4/5/6 following
costimulation of PMA and DPDPE (Fig. 8A). This is unlikely
due to a limitation in the labeling or detection system, because, under
similar conditions, the level of DOR phosphorylation increased to
~9-fold of the basal level following GRK coexpression (data not
shown). These data argue that DOR phosphorylation is regulated by both
PKC- and GRK-mediated mechanisms, but it seems that only one type of
phosphorylation could occur if the receptor is exposed to activated PKC
and GRK at the same time. This could be a result of the inhibitory
effect brought by receptor phosphorylation at one site.
In an effort to estimate the impact of the initial phosphorylation
event on the subsequent GRK- or PKC-mediated receptor phosphorylation, DOR mutants T358D, T361D, and S363D, mimicking GRK-phosphorylated receptors, and S344D, to imitate the PKC-induced phosphorylation state,
were constructed. As shown in Fig. 8B, T358D, T361D, and S363D phosphorylated poorly following PMA stimulation as compared with
wild type DOR, whereas agonist-stimulated S344D phosphorylation was at
a level similar to that of the wild type DOR (Fig. 8C). This
result suggests that the negative charges brought by agonist-stimulated phosphorylation may strongly inhibit PKC-mediated heterologous phosphorylation of DOR at Ser-344, and this is consistent with the
results shown in Fig. 8A. To verify this, receptor
phosphorylation and internalization levels following sequential PMA and
DPDPE treatments were determined. The results show that incubation of PMA followed by DPDPE treatment increased DOR phosphorylation to
~2.5-fold of phosphorylation stimulated by DPDPE or PMA alone (~10-fold of the basal level), whereas receptor phosphorylation in
response to incubation with DPDPE followed by PMA stimulation was not
significantly different from that stimulated by DPDPE or PMA alone
(Fig. 8D). These data are in agreement with our results obtained with phosphorylation state receptor mimics. Furthermore, the
effects of sequential treatment of PMA and DPDPE on DOR internalization were similar to those observed in DOR phosphorylation (Fig.
8E). These results suggest further that, although
phosphorylation induced by either homologous agonist stimulation or
heterologous PKC activation alone is sufficient to bring about changes
in responsiveness of the receptors, opioid signaling is still
regulatable by GRK following PKC-mediated DOR phosphorylation.
 |
DISCUSSION |
Opioid receptor desensitization plays an important role in opioid
drug-induced analgesia, tolerance, and dependence. Chronic opiate
treatment strongly increases GRK levels and PKC activity in specific
brain regions, and inhibition of PKC activity attenuates the
development of morphine tolerance (37-39).
-Arrestin 2 knocking-out mice with impaired opioid receptor desensitization exhibit
enhanced morphine analgesia (40). Phosphorylation of opioid receptors in response to agonist or phorbol ester stimulation has been observed by a number of laboratories, including our own (1-4, 9). Studies revealed that overexpression of GRK2 enhances
agonist-dependent receptor phosphorylation and causes
desensitization and overexpression of a dominant-negative mutant of
GRK2 or inhibition of GRK activity blocks desensitization of opioid
receptors (1, 6, 7, 10). PKC inhibitors attenuate homologous
desensitization of some of opioid-mediated responses (41) but fail to
block agonist-stimulated opioid receptor phosphorylation (1, 2, 9, 26).
Accumulating evidence indicates that GRK is the primary mediator in
agonist-induced opioid receptor phosphorylation and desensitization and
that GRK-catalyzed opioid receptor phosphorylation is an initial step
and important mechanism for opioid agonist-dependent,
homologous regulation of the receptor function (1, 6, 7, 9, 10, 42). However, the mechanism of regulation of responsiveness of opioid receptor by PKC is not clear. In this study, we have demonstrated, for
the first time, that activation of PKC through physiological means,
stimulating another class of neurotransmitter receptor, or increasing
the intracellular Ca2+ concentration, induces
phosphorylation of opioid receptor in an agonist-independent manner.
Our data showed that the PKC-mediated heterologous opioid receptor
phosphorylation occurs at a site distinctly different from that of GRK
catalyzed phosphorylation (4) on the receptor cytoplasmic tail and that
phosphorylation of DOR by PKC results in
-arrestin- and
clathrin-mediated internalization of the receptor. Our study
indicates that PKC-mediated opioid receptor phosphorylation is the
molecular basis of PKC-mediated receptor desensitization, thus
uncovering a molecular mechanism for agonist-independent, heterologous
regulation of opioid receptor-mediated signal transduction.
We have demonstrated in the present study that PMA-stimulated DOR
phosphorylation is mediated by PKC. The enzymatic pathway involved in
PKC-mediated heterologous DOR phosphorylation could involve either
direct phosphorylation of the receptor by PKC or activation of other
type of kinase by PKC. The PKC-mediated phosphorylation site identified
is Ser-344, which is located in an typical PKC (S/T)X(K/R)
consensus sequence (29). Therefore, direct phosphorylation of DOR by
PKC is very likely, although we can not exclude the possibility of
phosphorylation of the receptor by another PKC-activated kinase.
PKC-catalyzed phosphorylation has been shown to regulate activity of
certain GRKs (43, 44). But our results obtained from overexpression of
GRK in HEK 293 cells show that Ser-344 is not a site targeted by GRK
(data not shown), and the current study indicates that PKC-mediated DOR
phosphorylation occurs in the absence of agonist stimulation and at a
site distinct from agonist-stimulated phosphorylation sites. These data
argue that PKC-mediated DOR phosphorylation is unlikely a result of GRK
activation. Our results show that DOR phosphorylation by PKC was
stimulated by activation of
1A-AR coupled to PKC
,
, and
but not
(45) or ionomycin, an ionophore facilitating
Ca2+ uptake by cells, blocked by BAPTA/AM, a
Ca2+ chelator, or GF109203X, an inhibitor of
Ca2+-dependent PKC
,
I,
II, and
(28). These results suggest that the PKC mediating heterologous
phosphorylation and desensitization of DOR may belong to
Ca2+-dependent PKC (c-PKC).
Many neurotransmitters communicate with the cell interior via PKC.
However, little is known about substrates and functional impacts of PKC
activated by these heterologous pathways. We demonstrated that, at
least one mechanism, receptor internalization, is accountable for
PKC-mediated heterologous desensitization of opioid receptor. We have
shown here that activation of PKC promotes receptor phosphorylation and
internalization, and the effects of PKC on receptor phosphorylation and
internalization are linked. Mutation of the PKC site in DOR abolished
PKC-stimulated receptor internalization, indicating that
phosphorylation of Ser-344 in DOR by PKC is required for sequestration
of the receptor. Receptor phosphorylation and internalization may occur
in sequential steps. Experiments utilizing the wild type and a dominant
negative mutant
-arrestin and hypertonic sucrose demonstrated
clearly that PKC-mediated phosphorylation of Ser-344 leads DOR
internalization through clathrin-coated pits in a
-arrestin-dependent manner. The cytoplasmic tail of DOR contains multiple Ser/Thr residues (six in the last 31 amino acids), and there are two consensus PKC phosphorylation sites (Ser-344 and
Ser-352). Removal of all Ser/Thr residues in the C-tail (truncated mutant
31) abolished both agonist- and PKC-induced receptor
desensitization, indicating that the agonist-dependent, GRK
phosphorylation site and agonist-independent PKC site are both located
in the C terminus of DOR. Phosphorylation of the receptor C-tail is
involved in both the homologous and heterologous desensitization of
DOR. We have demonstrated that agonist-independent PKC-mediated
phosphorylation of DOR occurs at Ser-344, whereas Thr-358, Thr-361, and
Ser-363 contribute to agonist-induced receptor phosphorylation. Our
results showed that the agonist-induced homologous receptor
phosphorylation/internalization and PKC-mediated heterologous receptor
phosphorylation/internalization are additive to each other. These data
indicate that agonist-stimulated phosphorylation and PKC-catalyzed
phosphorylation occur at distinctly different sites at the DOR C-tail
and suggest that DOR phosphorylation via the two different mechanisms
plays a complementary role in down-regulation of opioid receptor functions.
Studies have demonstrated the cross-regulation between receptors
coupled to different signal transduction pathways. Recent studies
indicated that, through PKC, a number of receptors cross-talk to other
receptors on the membrane of the cell (11, 13). We have shown in the
current study that, under physiological conditions, stimulation of
1A-AR and purinoceptor could heterologously activate PKC
and result in receptor phosphorylation and desensitization. This study
reveals a molecular basis for the observed agonist-independent regulation of opioid receptor desensitization. These observations may
have important implications for our understanding of both opioid
pharmacology and pathophysiological changes associated with drug
tolerance and addiction. The regulation of opioid receptor function in
the central nervous system via heterologous activation of PKC is likely
to be of substantial physiological importance. Our previous research
showed that activation of the NMDA receptor attenuates opioid
receptor-mediated signaling (14), and clinical study showed that
coadministration of ketamine, an antagonist of NMDA receptor,
potentiates morphine's analgesic effect (13). Insulin and phorbol
ester have been also shown to attenuate the analgesic effect induced by
morphine (15, 20, 21) Ohsawa et al. (17) demonstrated that
the analgesic effect of morphine is attenuated in diabetic mice and
involvement of PKC has been implicated. In this study, we have shown
that the agonist-independent, heterologous activation of PKC induces
desensitization of opioid receptors and this is a molecular mechanism
of regulation of opioid signal transduction. Taken together, these data
suggest that coadministration of opioid analgesics with other medicine
able to activate PKC or elevate intracellular Ca2+ may
reduce the effect of opioid drugs. The basal PKC activity and basal
Ca2+ level in cytoplasm could affect the analgesic effect
of opioid drugs.
 |
ACKNOWLEDGEMENTS |
We thank Kun Ling, Haoqun Qiu, Zhijie Cheng,
Jian Zhao, and Shunmei Xin for technical assistance and helpful
discussions, and Dr. Lakshmi A. Devi for FLAG-tagged cDNA
constructs of m1, m2, and m3 DORs.
 |
FOOTNOTES |
*
This work was supported by grants from the National Natural
Science Foundation of China (39825110 and 39625015) and the Ministry of
Science and Technology (G1999054003 and G1999053907) and by the
Ministry of Education and the Shanghai Municipal Commission of Science
and Technology.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: National
Laboratory of Medical Neurobiology, Fudan University Medical Center, 138 Yi Xue Yuan Rd., Shanghai 200032, P. R. China. Tel.:
86-21-6404-1900 (ext. 2522); Fax: 86-21-6471-8563; E-mail:
lanma@shmu.edu.cn.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M006187200
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G
protein-coupled receptor;
DOR,
-opioid receptor;
GRK, G
protein-coupled receptor kinase;
PKC, protein kinase C;
PKA, protein
kinase A;
PMA, phorbol 12-myristate 13-acetate;
HEK, human embryonic
kidney;
DPDPE, [D-Pen2,D-Pen5]enkephalin;
1A-AR,
1A-adrenergic receptor;
HA, hemagglutinin;
MEM, modified Eagle's medium;
FITC, fluorescein
isothiocyanate;
WT, wild type;
PCR, polymerase chain reaction;
PBS, phosphate-buffered saline;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester;
NMDA, N-methyl-D-aspartic acid..
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