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J. Biol. Chem., Vol. 278, Issue 36, 34631-34640, September 5, 2003
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-Opioid Receptor Produces Desensitization and Internalization*
¶
From the
Departments of Pharmacology and
Anesthesiology, University of Washington School
of Medicine, Seattle, Washington 98195-7280
Received for publication, April 17, 2003 , and in revised form, June 5, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-arrestin mediated desensitization of
the rat 
-opioid receptor (KOR) was previously shown using
Xenopus oocyte expression to require serine 369 within the C terminus
of KOR. To define the effects of phosphorylation of this residue in
desensitization and internalization processes in mammalian expression systems,
wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were
stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp
recording in transfected AtT-20 cells, agonist activation of either
receptor form produced equivalent activation of the intrinsic G-protein-gated
inwardly rectifying potassium channel. Incubation for 60 min with the
agonist U50,488 (100 nM) desensitized the response in cells
expressing wild-type KOR-GFP by 86% but had no effect on
KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected
using a phosphospecific antibody (KOR-P) able to distinguish the
phosphorylated form of the receptor. The agonist-induced increase in KOR-P
labeling was dose-dependent, blocked by co-treatment with the
antagonist norbinaltorphimine, and prevented by co-expression of the dominant
negative form of the G-protein receptor kinase, GRK2(K220R). In contrast,
agonist-induced increase in KOR-P labeling was not evident in KOR(S369A)
expressing cells. Prolonged activation resulted in receptor internalization
that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P
labeling was evident at lower agonist concentrations than required to induce
internalization. Following the removal of agonist, receptor dephosphorylation
detected by loss of KOR-P labeling was complete within 60 min, could be
blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor
internalization. These results demonstrate that GRK-mediated phosphorylation
of serine 369 mediates rat KOR desensitization and internalization. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Direct evidence of -opioid receptor
(KOR)1 phosphorylation
was obtained by immunoprecipitation of 32P-labeled KOR from guinea
pig hippocampal slices treated with a KOR-selective agonist
(3). This increase in KOR
phosphorylation after agonist treatment coincided with an equivalent reduction
in electrophysiological response to the KOR agonist and a significant
reduction in the amount of immunoprecipitated protein detected, possible
examples of agonist-induced receptor desensitization and internalization,
respectively. However, determining the site of 32P incorporation
into the KOR was not possible with this ex vivo model because of the
low abundance of receptor in brain and uncertain stoichiometry of
32P labeling.
Further evidence correlating KOR control mechanisms to agonist-induced
phosphorylation of specific amino acids has come from site-directed
mutagenesis and subsequent expression of receptors lacking potential
phosphorylation sites in cell lines and heterologous gene expression systems,
but this work has yielded contradictory results. For example,
electrophysiological studies of Xenopus oocytes expressing wild-type
rat KOR (rKOR) demonstrated a G-protein receptor kinase (GRK)and
-arrestin-2-mediated desensitization of the agonist response
that was blocked by substitution of KOR(S369A) but not KOR(T363A) or
KOR(S356A,T357A) (4). These
results suggest that rKOR desensitization results from an agonist-induced
phosphorylation of serine 369. Consistent with this finding Joseph and Bidlack
(5) reported that prolonged
exposure to agonists can produced a substantial reduction in KOR
expression in thymoma cells. However, others have reported that
agonist treatment can produce rKOR desensitization without an increase in
phosphorylation or receptor internalization
(6,
7). Taken together, the studies
cited above suggest that the relationship between agonist-induced
phosphorylation of specific sites within KOR and either receptor
desensitization or internalization remains unclear. In addition, the direct
identification of agonist-induced phosphorylation sites responsible for the
desensitization or internalization of KOR has not been achieved.
To address this question, we expressed rat KOR-GFP and KOR(S369A)-GFP
fusion proteins in AtT-20 cells for electrophysiology studies of receptor
desensitization and in HEK293 cells to examine receptor internalization under
confocal microscopy. Moreover, we used immunocytochemistry and a newly
generated antibody (KOR-P) selective for the phosphorylated form of serine 369
in the rKOR to directly determine whether agonist-induced phosphorylation of
rKOR(S369) mediates either receptor desensitization or
internalization.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Peptides and Antibody Production—A phosphorylated peptide corresponding to the carboxyl-terminal domain of KOR designated KOR-P (RVRNTVQDPAS(PO4)MRD; matching amino acids 359–372) was synthesized and purified by PeptidoGenic Research and Co. (Livermore, CA). This peptide sequence was compared with known proteins using the GenBankTM data base, and no sequences in other proteins were found having >50% homology or four identical amino acids in a row. Purified peptide was coupled to keyhole limpet hemocyanin through the N-terminal lysine, and KOR-P antibody was then produced and affinity-purified as described earlier (3). The two antibodies KT2 (raised against the C-terminal tail of the KOR) and KE-4 (raised against the predicted fourth extracellular loop of the rat KOR-(300–312), GSTSHSTAALSSY) were characterized as previously described (3, 8).
Enzyme-linked Immunosorbent Assay—Sterile 96-well plates were coated overnight at 4 °C with 0.5 µg/well of either KOR-P peptide, KOR-Non-P peptide (a nonphosphorylated peptide otherwise identical to KOR-P), or peptides KE-4 or MOR-P (an unrelated peptide from the predicted sequence of the µ-opioid receptor, MOR-(171–184), HPVKALDFRT(PO4)PRNA in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Unbound sites were blocked with 1% nonfat dry milk in PBS for 2 h at 37 °C, and then the wells were washed three times with PBS-T (0.1 M PBS with 0.05% Tween 20). Affinity-purified KOR-P antibody, serially diluted to 0.05 from 10 µg/ml, was added to each well and incubated overnight at 4 °C under gentle agitation. Each concentration was tested in triplicate. Wells were washed three times with PBS-T and then incubated for 2 h in alkaline phosphatase-conjugated anti-rabbit IgG, diluted 1:3000 in PBS plus 1% bovine serum albumin. After washing the wells six times with PBS-T, the enzyme substrate p-nitrophenyl phosphate (1 mg/ml in 10% diethanolamine, 10% MgCl2, and 0.02% sodium azide, pH 9.8) was added and allowed to react for 30 min in the dark. The reaction was stopped with the addition of 2.5 M NaOH, and absorbance was promptly read at 410 nm for each well.
Complementary DNA Clones, Mutagenesis of KOR to Produce KOR
GFP and KOR(S369A)-GFP, and Production of Complementary RNA—
The cDNA for GFP-37 (GenBankTM accession number U87624
[GenBank]
) was subcloned
into the EcoRI site of pcDNA3 (Invitrogen) vector to produce
pcDNA3-GFP37. Restriction sites for KpnI and BstXI were
added to the 5'- and 3'-ends of the rat KOR sequence (thereby
replacing the stop codon with the BstXI site) via PCR amplification.
This KOR cDNA was digested and subcloned into the KpnI and
BstXI sites of the pcDNA3-GFP37 vector with T4 DNA ligase as
described elsewhere (9). The
resultant KOR-GFP construct fused the GFP to the carboxyl-terminal tail of
KOR. Point mutations were made in KOR-GFP to produce KOR(S369A)-GFP, using
techniques described previously
(10). All mutations were
confirmed by DNA sequencing. cDNA encoding for the receptors was used
to make RNA as described previously
(10).
Cell Culture and Construction of Cell Lines Expressing KOR-GFP or KOR(S369A)-GFP—HEK293 cells were grown in Dulbecco's modified Eagle's media/F-12 media supplemented with 10% fetal bovine serum (Invitrogen), and AtT-20 cells were grown in Dulbecco's modified Eagle's media high glucose medium supplemented with 10% horse serum and 2.5% fetal bovine serum (Invitrogen). Stable cell lines expressing KOR-GFP or KOR(S369A)-GFP protein in both types of cells were constructed by transfecting both cells with 10 µg of cDNA coding for KOR-GFP or KOR(S369A)-GFP proteins/well for 2–3 h using Superfect (Qiagen) reagent and the manufacturer's recommended protocol and then placing expressing cultures under selection pressure with G418 (0.4 µg/ml for HEK293 cultures; 1.5 µg/ml for AtT-20 cultures). Colonies expressing GFP were identified under fluorescence, isolated, and maintained under selection pressure for a minimum of 3 more weeks. Additionally, a small set of Xenopus laevis oocytes were microinjected with RNA coding for the wild type rKOR, KOR-GFP, or KOR(S369A)GFP receptors (4 ng/oocyte) along with RNA expressing the heteromeric inwardly rectifying potassium channels, KIR 3.1/3.2 (0.02 ng/oocyte) as described previously (10).
Two-electrode Voltage Clamp and Whole-cell Patch Clamp
Electrophysiology—To confirm the function of KOR-GFP and KOR
(S369A)GFP as -opioid receptors, Xenopus oocytes expressing
either fusion protein or wild type rKOR with KIR 3.1/3.2 were used
in two-electrode voltage clamp recordings as described previously
(10). Otherwise, AtT-20 cells
stably expressing KOR-GFP or KOR(S369A)-GFP were grown on polylysine-coated
coverslips for experimental use. Cells were used in patch clamp experiments in
the whole-cell configuration with a pipette containing 130 mM KCl,
20 mM HEPES, 10 mM EGTA, 5 mM
MgCl2,3mM Na2ATP, and 0.6 mM GTP,
pH 7.25, in an external solution containing 40 mM KCl, 130
mM NaCl, 1 mM CaCl2, 25 mM HEPES,
and 10 mM glucose, pH 7.35. Whole-cell potassium currents were
acquired with an Axopatch 200 and Axotape (Axon Instruments) and then analyzed
with Clampfit software (Axon Instruments). In the whole-cell configuration of
the patch clamp, cells were held at –45 mV and hyperpolarized to
–100 mV for 50 ms every 5 s. Recorded traces represent average current
during the hyperpolarization, plotted over time. Desensitization during
agonist treatment was measured as the percentage change in response to agonist
after a 60-min exposure to 100 nM U50,488.
KOR-labeling Immunocytochemistry and Confocal Microscopy— Cells stably expressing KOR-GFP or KOR(S369A)-GFP were split onto polylysine-coated cover slips in 24-well plates for experimental use. Note that for experiments using the dominant negative mutant of GRK2, GRK(K220R)2, HEK293 cells were transiently transfected with both 10 µg of KOR-GFP and 2 µg of GRK(K220R)2 cDNA, using the Superfect reagent as described above. After 48 h of protein expression, cells were treated at 37 °C as noted and prepared for immunocytochemistry and confocal microscopy as described previously (11). Either KT-2 (15 µg/ml) or KOR-P (4.5 µg/ml) was used as the primary antibody with fixed cells, incubated up to 70 h at 4 °C in 0.3% glycerin/PBS with 0.025% Triton-X. Lissamine-rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) was used as a secondary antibody to label the bound antibody. Both fluorochromes (GFP from the expressed protein and Rhodamine Red-bound KOR-P labeling) were viewed separately or simultaneously using a Bio-Rad confocal microscope (model MRC600) with COMOS 6.01 software.
Data Analysis—To quantify the degree of internalization induced by agonist treatment of HEK293 cells, images collected with the confocal microscope were analyzed with NIH Image version 1.62 software (National Institutes of Health, Bethesda, MD) for the intensity of pixels within the cytoplasm of cells expressing KOR-GFP or KOR(S369A)GFP following medium or agonist treatment. To quantify the ratio of KOR-GFP receptors labeled by the KOR-P antibody, images collected by confocal microscopy were separated into green pixel-only images (showing GFP signaling) and red pixel-only images (showing antibody labeling) using Photoshop software (Adobe Systems Inc., San Jose, CA). Each image was analyzed for number of nonblack pixels, and a ratio of red to green pixels was generated for each original image taken (therefore, a value of 1.0 denotes complete and precise overlap of KOR-P/Rhodamine Red antibody labeling and green GFP signaling). Statistical significance throughout this study was defined by p < 0.05 as determined by Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Moreover, the KOR-GFP expressed in AtT20 cells showed robust receptor
expression and cell surface distribution
(Fig. 1C, a). KOR-GFP
expressed in AtT20 cells that were patch-clamped in high potassium buffer as
described under "Experimental Procedures" produced a strong
potassium current in response to stimulation with the agonist, U50,488
that was antagonized by the -selective antagonist, nor-BNI
(Fig. 1A). Likewise,
activation of KOR(S369A)-GFP produced an equivalent potassium influx in
response to U50,488 and at a similar magnitude to that of the wild-type
receptor (Fig. 1B).
After prolonged agonist incubation (100 nM U50,488, 1 h), the
potassium channel response to 1 µM U50,488 by KOR-GFP was
significantly reduced by 86%, whereas the KOR(S369A)-GFP response following
the same treatment was unchanged (Fig.
1B). This result suggests that the KOR(S369A) residue was
also important for receptor desensitization in a mammalian cell system.
Whereas robust internalization of KOR-GFP was evident following stimulation
with higher concentrations of U50,488 (10 µM, 1 h;
Fig. 1C, c), the lower
concentration (100 nM, 1-h treatment) that produced receptor
desensitization did not increase receptor internalization
(Fig. 1C, b), as
compared with untreated cells (Fig. 1C,
a), suggesting that desensitization was not due to
internalization.
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Development of -Opioid Receptor Antibodies Specific to
the Serine 369-Phosphorylated Receptor—To develop a probe to detect
phosphorylation of serine 369 in KOR, we generated and affinity-purified a
polyclonal antibody (KOR-P) against a 14-amino acid peptide corresponding to
rat KOR-(359–372) containing a phosphorylated serine 369
(Fig. 2A).
Affinity-purified KOR-P antibody reacted up to 80 times more strongly in an
enzyme-linked immunosorbent assay against the phosphopeptide than the
equivalent, nonphosphorylated sequence
(Fig. 2B). The KOR-P
antibody also failed to recognize peptides corresponding to the other regions
of the receptor and unrelated, phosphorylated peptides
(Fig. 2B).
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Demonstration of Immunocytochemical Labeling of KORGFP and
KOR(S369A)-GFP by Two -Opioid Receptor-selective
Antibodies—High levels of background GFP labeling in the cytosol of
KOR-GFP or KOR(S369A)-GFP expressing AtT-20 cells made quantitation of
receptor trafficking difficult. Therefore, HEK293 cells stably expressing
either KOR-GFP or KOR(S369A)-GFP were used in immunocytochemistry experiments
with either KT-2 (not phosphoselective) or KOR-P (phosphoselective) as the
primary antibody. The lower cytosolic background staining evident in
KOR-transfected HEK293 cells facilitated quantitation of receptor trafficking.
Confocal microscopy revealed that KT-2 selectively immunolabeled both KORGFP
and KOR(S369A)-GFP as the red KT-2 antibody labeling overlapped the green GFP
fluorescent signal (Fig. 3,
A–D). Incubation of HEK cells with 10
µM U50,488 produced clear internalization of KOR-GFP but not
KOR(S369A)-GFP, as detected by KT-2 and localization of GFP fluorescence
(Fig. 3, B and
D). In contrast, untreated HEK cells expressing KOR-GFP
were unlabeled by KOR-P (Fig.
4A), whereas KOR-P labeling was evident in cells treated
with U50,488 (0.1–10 µM, 30 min at 37 °C)
(Fig. 4, B–D).
KOR-P labeling remained on the cell surface following treatment (30 min, 37
°C) with 0.1 and 1.0 µM U50,488. However, whereas these
concentrations of U50,488 failed to produce characteristic signs of KOR-GFP
internalization, incubation with 10 µM U50,488
(Fig. 4D) resulted in
punctate structures and internalization of KOR-GFP evident by both KOR-P
antibody labeling and GFP fluorescence. These effects were
activation-dependent, since incubation with the KOR-selective antagonist
nor-BNI did not produce either KOR-P antibody labeling or evidence of receptor
trafficking (Fig. 4E).
In addition, coincubation of nor-BNI with 10 µM U50,488
prevented both receptor trafficking and KOR-P antibody labeling
(Fig. 4F).
Agonist-induced increases in KOR-P labeling and receptor internalization were
also evident after treatment with the -selective opioid peptide,
dynorphin A-(1–13) (Fig.
4G), and labeling was again blocked by coincubation with
nor-BNI (Fig. 4H).
Preabsorbing the KOR-P antibody with the corresponding KOR-P peptide prevented
antibody labeling following 10 µM U50,488
(Fig. 4I). In
addition, HEK293 cells stably expressing KOR(S369A)-GFP failed to demonstrate
KOR-P antibody labeling, either when untreated
(Fig. 4J) or after
incubation with 10 µM U50,488
(Fig. 4K). Finally,
KOR-P antibody labeling was not evident in untransfected HEK293 cells treated
with 10 µM U50,488 (Fig.
4L). The results suggest that KOR-P antibody could
specifically detect the Ser369-phosphorylated form of KOR and that
phosphorylated receptor could be detected at both the plasma membrane and in
cytoplasmic structures following agonist treatment.
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Agonist-induced Phosphorylation of KOR-GFP in HEK293 Cells Is Mediated by a GRK—HEK293 cells stably expressing KOR-GFP incubated with 10 µM U50,488 for 30 min demonstrated robust KOR-GFP internalization and strong KOR-P antibody labeling (Fig. 5A). Neither receptor trafficking nor KOR-P labeling was prevented by pretreatment with either the protein kinase A/protein kinase C inhibitor staurosporine (Fig. 5B), the MEK1/2 inhibitor PD98,059 (Fig. 5D), or the phospholipase C inhibitor U73,122 (Fig. 5E). However, in HEK293 cells transiently expressing both KOR-GFP and the dominant negative mutant GRK2(K220R), U50,488 exposure was unable to induce receptor trafficking, and KOR-P antibody labeling was substantially reduced (Fig. 5C). These results suggest that agonist-induced phosphorylation of KOR-GFP serine 369 was mediated by a GRK in HEK293 cells.
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Agonist-induced Receptor Internalization Is Mediated by KOR Serine-369 in HEK293 Cells—Since previous results suggested that receptor internalization required the serine 369 phosphorylation site (Fig. 4K) and phosphorylation by GRK (Fig 5C), receptor internalization was quantified for both KORGFP (Fig. 6A) and the mutant KOR(S369A)-GFP (Fig. 6C). Treatment with 10 µM U50,488 induced clear receptor trafficking, with punctate, clustered KOR-GFP structures identified on the cell surface and internalized within the cytoplasm (Fig. 6B). Image quantitation showed a significant increase in the cytosolic pixel intensity following agonist treatment of cells expressing KOR-GFP (Fig. 6E). In contrast, KOR(S369A)-GFP did not show agonist-induced receptor trafficking (Fig. 6D), and a uniform distribution of the mutant receptor continued to be primarily localized in the plasma membrane (Fig. 6C). Quantitation of KOR(S369A)-GFP images revealed no increase in the cytoplasmic pixel intensity following U50,488 treatment (Fig. 6E).
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Rapid Reversal of Agonist-induced KOR Serine 369 Phosphorylation Is Mediated by Phosphatases Both in the Cytoplasm and Directly at the Plasma Membrane—Agonist-induced phosphorylation of G-protein-coupled receptors is thought to be quickly reversed upon removal of the agonist by receptor internalization and dephosphorylation (12, 13). To test this, HEK293 cells stably expressing KOR-GFP were treated with 10 µM U50,488 at 37 °C, rinsed free of agonist after 30 min, and then allowed to recover in media for times up to 60 min prior to KOR-P antibody labeling. Similar to previous results (e.g. Figs. 5A and 6B), U50,488-induced increases in KOR-P labeling and KOR-GFP internalization were evident 1 min after agonist removal (Fig. 7A). KOR-P antibody labeling was still evident 10 min after agonist removal, although it was significantly diminished (Fig. 7B). KOR-P antibody labeling of U50,488-treated KOR-GFP/HEK293 cells was barely detectable 30 min after agonist removal (Fig. 7C) and was undetectable 60 min after agonist removal (Fig. 7D). Quantitation of total red and green pixel intensities verified that KOR-P labeling declined steadily during the 60 min following U50,488 washout (Fig. 8, open circles). To determine whether receptor internalization was required for dephosphorylation, cells were treated with 0.4 M sucrose to prevent receptor internalization, and the kinetics of KOR-P labeling decline was assessed following washout of 10 µM U50,488. Inclusion of sucrose did not prevent U50,488-induced increase in KOR-P antibody labeling, although KORGFP internalization was blocked (Fig. 7E). In addition, KORGFP dephosphorylation was not blocked by sucrose, since KOR-P labeling still declined in cell culture media containing 0.4 M sucrose (Fig. 7F). However, the rate of loss of KOR-P labeling was significantly delayed by sucrose treatment, suggesting that dephosphorylation of KOR may be facilitated by receptor internalization (Fig. 8, squares).
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To characterize the phosphatase mediating dephosphorylation, HEK293 cells stably expressing KOR-GFP and stimulated with 10 µM U50,488 were treated with 100 nM okadaic acid to inhibit the protein serine/threonine phosphatases 1 and 2A or with a combination of 0.4 M sucrose and 100 nM okadaic acid. Neither okadaic acid alone nor the combination of sucrose and okadaic acid treatment impaired the U50,488-induced increase in KOR-P antibody labeling observed immediately after the removal of agonist (Fig. 7, G and I, respectively). Okadaic acid alone slowed, but did not prevent, the decline in KOR-P labeling (Figs. 7H and 8 (triangles)). In contrast, the combination of sucrose and okadaic acid prevented the loss of KOR-P antibody labeling as long as 60 min after agonist removal (Figs. 7J and 8 (diamonds)). Together, these results suggest that receptor internalization may accelerate the dephosphorylation of KOR serine 369 and that receptor dephosphorylation by either protein phosphatase 1 or 2A may occur at the plasma membrane without internalization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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opioid receptor
was desensitized by phosphorylation of serine 369 by a GRK-dependent
mechanism. Receptor phosphorylation was detected by a novel phosphospecific
antibody, KOR-P that enabled immunocytochemical localization of the
-opioid receptor. Substitution of alanine for serine 369 blocked both
receptor uncoupling and receptor internalization, two processes that underlie
desensitization. Moreover, KOR dephosphorylation was facilitated by receptor
internalization but could occur at the plasma membrane. This study directly
demonstrated phosphorylation at a specific site within the opioid receptor and
helps to resolve the key steps in the desensitization and resensitization
process regulating KOR functioning in mammalian cells. These findings are
consistent with other reports implicating an agonist-induced phosphorylation
event underlying the desensitization of human KOR
(7) and µ-opioid receptors
(14–16). Agonist-induced receptor desensitization is thought to be a necessary prerequisite to the internalization and down-regulation of opioid receptors (17, 18). However, the correlation between agonist-induced KOR desensitization and internalization is at best unclear. For example, a chronic incubation of cultured rat spinal cord-dorsal root ganglia neurons with 10 µM U50,488 produced complete desensitization and KOR-induced inhibition of adenylyl cyclase activity (19). A subsequent study demonstrated that a 24-h pretreatment with 10 µM U50,488 or 1 µM etorphine failed to produce a down-regulation of KOR number in dorsal root ganglia cultures (20). Likewise, epitopetagged KOR expressed in HEK293 cells failed to internalize after acute treatment with 10 mM etorphine (21). In contrast, 0.1 µM U50,488 treatment produced a 50% reduction in KOR number on R1.1 mouse thymoma cells without significant receptor desensitization (5). Curiously, in another study using epitope-tagged KOR expressed in Chinese hamster ovary cells, dynorphin peptides readily induced KOR internalization and down-regulation, whereas nonpeptidic agonists such as U50,488 did not (22). Subsequent work with Chinese hamster ovary cells expressing epitope-tagged KOR demonstrated that 1 µM U50,488 induced an increase in 32P incorporation that corresponded with desensitization of the human, but not rat, KOR (7). A parallel study demonstrated that 1 µM U50,488 induced internalization of the human, but not rat, KOR (23), although the involvement of KOR phosphorylation in agonistinduced internalization was not directly demonstrated. The authors suggested that the lack of agonist-induced rKOR regulation was due to the presence of Asn358 in the cytoplasmic tail of the rKOR, preventing agonist-induced phosphorylation in Chinese hamster ovary cells (7, 23). However, direct 32P incorporation into specific phosphorylation sites was not demonstrated, and the substitution of rKOR(N358S) did not restore an agonist-induced desensitization as might be predicted, suggesting that another phosphorylation site may underlie receptor desensitization and down-regulation.
The results of the present study show reasonable agreement with the
majority of studies on the topic; pretreatment with lower (
1
µM) concentrations of U50,488 failed to produce KOR
internalization but still induced significant wild type receptor
desensitization in the intact cell. Only higher concentrations of U50,488 (10
µM) induced KOR internalization. Admittedly, these data contain
two important caveats. First, receptor internalization was not quantified in
the agonist exposure experiments, making a direct comparison with previous
studies difficult. Second, the receptor constructs examined in this study were
by necessity GFP tail fusion proteins, which are not conclusively determined
to faithfully mimic the internalization properties of the wild type KOR.
Still, the present study extends previous findings, as the KOR-P antibody
labeling intensity increased in an agonist concentration-dependent manner that
was specific for the GFP-tagged KOR and reversed with receptor recycling to
the cell surface. These results suggest that agonist-induced phosphorylation
of KOR serine 369 underlies agonist-induced receptor regulation events, as
detected by correlative KOR-P antibody labeling, consistent with earlier
observations in cell lines
(17,
18).
The agonist-induced phosphorylation of KOR detected by KOR-P labeling was prevented by coexpression of KOR-GFP with the dominant negative mutant GRK2(K220R), which is thought to impede native GRK in host cells (24). Inhibitors selective for the kinases protein kinase A, protein kinase C, phospholipase C, or MEK1/2 were ineffective in blocking agonist-induced KOR-GFP phosphorylation. Whereas the possible interaction of other kinases with the KOR cannot be discounted, the ability of both overexpression of GRK2(K220R) and substitution of KOR(S369A) to prevent agonist-induced receptor internalization suggests that agonist-induced phosphorylation of KOR-GFP serine 369 in HEK293 cells was mediated by a GRK. These results further support the hypothesis that a GRK-mediated, agonist-induced phosphorylation of KOR(S369) is required for receptor internalization.
Similarly, dephosphorylation of agonist-phosphorylated KOR(S369) was
suggested by the loss of KOR-P labeling correlated with the recycling of
internalized KOR-GFP to the cell surface. Relatively little is known about the
role or rate of receptor dephosphorylation in reversing the internalization of
opioid receptors, perhaps due to the difficulty in specific identification of
dephosphorylated, recycled receptors with 32P incorporation
methods. Studies using transfected MOR indicate that in cell lines,
dephosphorylation occurs quickly following cessation of agonist stimulation
(25). Recycling of
-adrenergic-2 or cannabinoid receptors internalized after short agonist
exposure was shown to require dephosphorylation, since recycling was prevented
by okadaic acid-sensitive phosphatases
(12,
13). The present results
suggest that a similar dephosphorylation event at serine 369 was required for
the recycling of internalized KOR-GFP. Intriguingly, a dephosphorylation of
serine-369 following agonist stimulation of KOR-GFP occurred, although
delayed, even after the prevention of KOR internalization with 0.4
M sucrose treatment, suggesting that serine phosphatase activity
may restore receptor function at the cell membrane.
A major conclusion of this study is that efficient dephosphorylation can
occur without internalization of the KOR. This contrasts with current views on
this issue based on studies of several other G-protein-coupled receptors. For
example, agonist-induced desensitization of -adrenergic-2 receptors was
reversed by rapid receptor sequestration and the return of receptor to the
cell surface within minutes of agonist removal
(26). These events were shown
to be dependent on agonistinduced receptor phosphorylation and thought to
involve subsequent binding with cytosolic proteins, such as the
-arrestins, to induce receptor internalization
(27). Likewise, regulation of
receptor internalization and recycling have been demonstrated to depend on
agonist-induced phosphorylation of specific receptor residues, with receptor
resensitization requiring subsequent receptor dephosphorylation in studies
examining bradykinin receptors
(28), 
-opioid receptors
(29,
30), and µ-opioid receptors
(25,
31–33).
However, at least in the case of the MOR, this effect may prove to be
agonist-dependent, since etorphine, morphine, and the enkephalins were shown
to induce rapid DOR desensitization and internalization, whereas morphine
could not induce rapid MOR internalization
(32). These results may
suggest that alternative mechanisms to receptor internalization exist in the
process of receptor resensitization. Indeed, a recent study with desensitized
neurokinin-1 receptors identified a receptor resensitization that occurred
before receptor recycling
(34), consistent with the
findings described here.
Given the importance of the phenomena of opioid tolerance, insights into
the manifestation of tolerance on a cellular level remain valuable. By
extension of the present study, the agonist-induced phosphorylation of
KOR(S369) would be predicted to lead to the development of opioid tolerance
through the induction of receptor uncoupling and internalization. The elevated
phosphorylation of KOR isolated from U50,488-tolerant guinea pig hippocampus
supports this prediction (3).
Likewise, a study with morphine-tolerant rats reported a 56% increase in MOR
incorporation of 32P that correlated with the desensitization of
MOR function in adenylyl cyclase assays
(15). Additionally, GRK3
knockout mice demonstrate a delayed onset of U50,488 analgesic tolerance,
presumably through the inability to phosphorylate agonist-stimulated
KOR.2 Whereas the
direct demonstration of an agonist-induced phosphorylation of KOR(S369)
underlying -opioid tolerance remains, future studies using the KOR-P
antibody will be directed at examining the phosphorylation of KOR in
opioid-tolerant animals.
The results from the present study demonstrate that agonist-induced phosphorylation of rat KOR serine 369 mediated by a GRK is required for KOR desensitization and internalization in mammalian cell lines. Moreover, dephosphorylation of KOR(S369) is implicated in the recycling of the internalized receptor to the cell surface. Taken together with previous findings, these results suggest that the state of agonist-induced receptor phosphorylation determines the regulatory response mechanism to agonist stimulation that may result in the development of cellular opioid tolerance.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280. Tel.: 206-543-4266; Fax: 206-685-3822; E-mail: cchavkin{at}u.washington.edu.
1 The abbreviations used are: KOR, -opioid receptor; GFP, green
fluorescent protein; KOR-P, affinity-purified rabbit polyclonal antibody
directed to phosphoserine 369 in the rodent -opioid receptor; KT-2,
affinity-purified rabbit anti-rat/mouse -opioid receptor polyclonal
antibody; KE-4, affinity-purified rabbit anti-rat/mouse -opioid
receptor polyclonal antibody; MOR, µ-opioid receptor; GRK, G-protein
receptor kinase; nor-BNI, norbinaltorphimine; U50,488,
(±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide
methane sulfonate hydrate.
2 J. P. McLaughlin and C. Chavkin, submitted for publication.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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). KOR-P antibody was also incubated with
separate peptides KE-4 (
; corresponding to KOR 300–312), and MOR-P
(
; corresponding to MOR-(170–184) and containing a phosphorylated
threonine 180) to further estimate nonspecific KOR-P antibody binding. Samples
were then processed as described under "Experimental Procedures,"
and absorbance was measured at 405 nm. Results displayed are plotted with
background, nonspecific signal (as measured by a lack of antibody) subtracted.
Points represent average absorbance from three experiments, performed in
triplicate.
), 100
nM okadaic acid (
), or sucrose and okadaic acid (
) and
then treated with 10 µM U50,488 and allowed to recover up to 60
min in the same conditions as detailed in the legend to