From the Cell Signaling Laboratory, New England Biolabs, Beverly, Massachusetts 01915
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The µ-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the µ-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study µ-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of µ-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for µ-opioid receptor desensitization.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agonists for the µ-opioid receptor are the therapeutic choice in
the management of severe acute and chronic pain despite the potential
development of tolerance, dependence, and addiction. The mechanisms of
tolerance, defined as the diminishing effect of the drug in response to
chronic exposure, are not fully understood. It has been postulated that
receptor desensitization and down-regulation could be associated with
aspects of tolerance in vivo (1). Opioid receptors undergo
homologous desensitization upon continuous or repeated agonist exposure
in a fashion similar to other G protein-coupled receptors
(GPCR)1 (2-4).
Desensitization of the µ-opioid receptor involves
agonist-dependent phosphorylation of the receptor, most
likely by a member of the family of G-coupled receptor protein kinases
(GRKs) (4, 5). According to this model, upon phosphorylation and
uncoupling from the G protein, the receptor is bound to -arrestins
and internalized into endosomes, reducing the number of receptors
available at the cell surface for further agonist binding (6, 7).
Desensitization of the µ-opioid receptor has been mainly described as
the attenuated reduction of forskolin-stimulated cAMP levels in
response to the agonist (2-4). However, other signaling pathways might
also be desensitized upon prolonged agonist exposure. GPCRs can trigger
a G-mediated activation of a phosphoinositide 3-kinase
(PI3K)/Ras-dependent MAPK signaling pathway (8, 9). Opioid
receptors stimulate MAPK activity as well, although the components of
this signaling cascade have not been fully described (10-12). In this
report we have examined the role of the MAPK (Erk1/2) signaling pathway
on µ-opioid receptor desensitization. This pathway, which involves
the activation of a phosphatidylinositol 3-kinase activity as well as
Raf and MEK1/2, is essential for µ-opioid receptor
desensitization.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents-- [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO), dermorphin, morphine, Met-enkephalin, naloxone, D-Pen2,D-Pen5-enkephalin (DPDPE), lysophosphatidic acid (LPA), pertussis toxin, cycloheximide, forskolin, and isobutylmethylxanthine (IBMX) were from Sigma. Wortmannin and LY294002 were from Calbiochem. Antibodies that recognize only the phosphorylated forms of p42 and p44 MAPK (Thr-202/Tyr-204), Elk-1 (Ser-383), MEK1/2 (Ser-217/221), as well as a control (phosphorylation-independent) MAPK antibody, and the MEK inhibitor PD98059 were from New England Biolabs.
Cell Culture-- A stable Chinese hamster ovary cell line expressing the murine µ-opioid receptor cDNA (13) was maintained in DMEM supplemented with 10% fetal bovine serum and G418 (0.5 mg/ml). In a typical MAPK induction experiment cells were grown in 6-well plates for 24 h prior to treatment, washed, and incubated in serum-free medium for 1-2 h prior to agonist stimulation as indicated in the figures. Incubation with vehicle (0.04% dimethyl sulfoxide) or the inhibitors was initiated 1 h (PD98059), 15 min (wortmannin and LY2940092), 5 min (naloxone), or 16 h (pertussis toxin) prior to agonist stimulation. For desensitization experiments, cells were washed and incubated for 1 h in serum-free medium before 100 nM-1 µM of DAMGO or morphine, and different inhibitors were added to the medium for 2 additional hours. The cells were then washed three times in phosphate-buffered saline and subjected to a second agonist stimulus, typically 100 nM DAMGO for 5 min. C6 glioma cells were transfected with DNA constructs expressing the murine µ-opioid receptor and dominant negative forms of MEK (S221A) (14) and Ras (Asn-17) (15), using the FuGENE 6 transfection reagent (Boehringer Mannheim). Forty-eight hours after transfection, the cells were stimulated with 100 nM DAMGO for 5 min prior to extract preparation.
Immunoblotting and MAPK Assays-- For Western blot experiments, cell extracts were prepared using Laemmli sample buffer and subjected to SDS-10% polyacrylamide gel electrophoresis and immunoblotted as described (16). For MAPK activity assays, cells were grown in 10-cm plates for 24 h, treated as indicated, and extracted using a non-denaturing lysis buffer (10 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl), protease, and phosphatase inhibitors. Active MAPK was immunoprecipitated using a phospho-MAPK specific antibody and used in kinase reactions to phosphorylate 1 µg of GST-Elk-1 (307-428) fusion protein as substrate. Elk-1 phosphorylation was then analyzed by immunoblotting using a phospho-Elk-1 specific antibody (16).
cAMP Assays-- Cells were grown in 6-well plates for 24 h prior to experimental treatment, washed, and incubated in serum-free medium for 1 h, followed by 2 h of incubation in the presence of agonists and different inhibitors. The cells were then washed three times with phosphate-buffered saline and 5 µM forskolin, and 0.5 mM IBMX was added with or without 1 µM DAMGO for 10 min. Cells were then extracted using 0.1 N HCl and assayed using the Biotrak enzyme immunoassay system (Amersham Pharmacia Biotech ).
[3H]Naloxone Binding Assay-- Before membrane preparation, we followed the same desensitization procedures described above and in Fig. 4. Plasma membranes were prepared essentially as described (3). Binding of [3H]naloxone (specific activity, 53.7 Ci/mmol, NEN Life Science Products ) using 25 µg of membrane protein was performed according to the previously described method (3, 17). To generate saturation isotherm the ligand concentration was varied from 0.2 nM to 16 nM. The reaction (90 min at 25 °C) was terminated by a rapid filtration followed by three washes with ice-cold buffer using a Brandel harvester. Cold naloxone, 100 nM, was used to define nonspecific binding. Binding data from saturation experiments were analyzed using the GraphPad Prism 2.01 program (GraphPad Software, Inc.).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We first confirmed that selective µ-opioid agonists were capable
of inducing MAPK activity in a Chinese hamster ovary cell line stably
transfected with the murine µ-opioid receptor (13). MAPK activity and
phosphorylation peaked at 5 min after agonist addition, rapidly
decreasing to reach background levels between 30 and 60 min after
stimulation (Fig. 1a).
Increased phosphorylation and enzymatic activity were already
detectable at low levels (10 nM) of the µ-receptor
agonist DAMGO (data not shown). MAPK phosphorylation was not induced by
DPDPE, a selective agonist for the -opioid receptor, and was blocked
by naloxone and pertussis toxin, confirming that MAPK activation in
these cells is specifically mediated by a
Go/Gi-coupled µ-opioid receptor (Fig.
1b). In all experiments, MAPK phosphorylation correlated
with enzymatic activity as measured in Fig. 1a.
Identical results were obtained using morphine or peptide
µ-agonists such as dermorphin and Met-enkephalin (data not shown).
Consistently, DAMGO also induced the phosphorylation of the
MAPK-activating kinase MEK1/2 in a dose-dependent manner (Fig. 2a, upper
panel), which is indicative of Raf kinase activation, and
following a similar time course as shown for MAPK (not shown). DAMGO-induced MAPK phosphorylation was blocked by the specific inhibitor of MEK activation, PD98059 (18), at concentrations as low as
5 µM (Fig. 2a, center panel), and
by expression of dominant negative forms of MEK1/2 and Ras (Fig.
2a, bottom panel). MAPK phosphorylation was also
reduced by the selective inhibitors of PI3K, wortmannin, and LY294002
at concentrations considered to be specific for PI3K inhibition (8)
(Fig. 2b). These results suggest that µ-agonist activation
of the MAPK pathway requires the involvement of a
wortmannin/LY294002-sensitive PI3K, Ras, Raf, and MEK, as has been
shown for other Gi-coupled receptors (8, 9).
|
|
The rapid decline of MAPK activity despite continuous agonist presence
(Fig. 1a) is reminiscent of short-term receptor
desensitization (6, 7). Receptor desensitization has been extensively
studied in the -adrenergic receptor system (6) and has also been
described for opioid receptors mostly in terms of the diminishing
agonist inhibition of cAMP levels induced by forskolin (2-4). We
investigated whether desensitization of the µ-opioid receptor could
be analyzed by monitoring phosphorylation of MAPK. We first measured
the time necessary for cells exposed continuously to 100 nM
DAMGO to lose their capability to respond to a second agonist stimulus.
Fig. 3a shows the progressive
desensitization of the µ-receptor-stimulated MAPK phosphorylation. A
significant decrease (approximately 50-60% of maximum levels) in
phosphorylated MAPK was already detected after 5 min of agonist
exposure corresponding to the time frame of short-term desensitization
(6). No receptor response, in terms of MAPK phosphorylation stimulated
by a second agonist stimulus, could be detected after 2 h of
agonist exposure (Fig. 3a). Identical results were obtained
using morphine instead of DAMGO for the 2-h desensitizing period.
Desensitization of MAPK phosphorylation was also dependent upon agonist
concentration used in the first stimulus (Fig. 3b). The
attenuated receptor response is reversed upon agonist removal (19). We
next measured the time required for the MAPK signal to become
resensitized. The cells recovered their responsiveness to a second
DAMGO stimulus only after 15-30 min of incubation in agonist-free
medium (Fig. 3c). This parallels the resensitization timing
reported for the
-adrenergic receptor (20). Similar levels of MAPK
phosphorylation as those induced in non-desensitized cells are nearly
reached after a 2-h relapse in agonist-free medium (Fig.
3c). The µ-agonist-induced desensitization of the MAPK
response could be homologous (the loss of response to a specific
stimulus) or heterologous (the loss of response to diverse stimuli). To
distinguish between these two possibilities we used LPA, which
stimulates MAPK phosphorylation via an endogenous Gi-coupled receptor (8). Cells preincubated for 2 h
with DAMGO were washed and then exposed to LPA or DAMGO for 5 min (Fig.
3d). Whereas DAMGO pretreatment greatly reduced the response
to a second DAMGO stimulus, it did not affect the levels of MAPK
phosphorylation induced by LPA as compared with those of cells
stimulated with LPA but not pretreated with the µ-agonist (Fig.
3d). This indicates that the MAPK signal desensitization
provoked by a 2-h exposure to a µ-specific agonist had no effect upon
any component of the LPA-induced MAPK pathway. Therefore, the MAPK
pathway induced by µ-opioid receptor agonists is subject to
homologous desensitization in a dose- and time-dependent
manner (Fig. 3).
|
The desensitization process is critical for timing the duration of the
cell response to a particular stimulus. Upon signaling through GPCRs
the regulation of this process can theoretically occur at any level of
the interaction between receptor, G-protein, and effector pathway. The
role of GRKs and -arrestins in receptor phosphorylation, uncoupling,
and internalization has been described (6, 21), as well as the
importance in these processes of GRK-specific interactions with the
G
subunit (22, 23). However, it is possible that other signals
contribute to the desensitization process as well. We hypothesized that
a feedback signal originating at the effector pathway itself, in this
case the MAPK pathway, may help initiate the desensitization mechanism.
This signal could induce the molecules that actively uncouple the
receptor from the downstream effector pathways. We tested whether
blocking the MAPK signal transduction cascade would affect the
desensitization of a µ-opioid receptor stimulus. Cells expressing the
µ-receptor were exposed to 100 nM DAMGO alone or together
with 10 µM naloxone or 20 µM PD98059 for
2 h, the time required for complete desensitization as shown in
Fig. 3a. The cells were then extensively washed to eliminate
agonist and inhibitors and subjected to a second stimulation with 100 nM DAMGO for 5 min before extracts were harvested to assay
MAPK phosphorylation (Fig. 4a,
upper and middle panels) and MAPK activity (Fig.
4a, bottom panel). Cells exposed to DAMGO alone
for 2 h did not respond to a second agonist stimulus. In contrast,
the presence of PD98059 during preincubation with DAMGO prevented the
desensitization of µ-receptor signaling, similar to the expected
effect of the antagonist naloxone. We also obtained identical results
in these experiments using morphine as the first desensitizing
stimulus. At the concentrations used in this study, PD98059 was shown
to be highly specific in preventing the activation of MEK, without
affecting the activity of 18 other tested Ser/Thr kinases (18). Thus
the inhibition of MEK activation and consequently of MAPK caused the
receptor to remain sensitive despite 2 h of agonist exposure. If
MAPK induced a desensitizing feedback signal that acts at the level of
the receptor or G-protein, then inhibition of this signal should also
affect the desensitization of other downstream effectors, such as the
cAMP pathway. We then examined whether desensitization of the
µ-opioid receptor adenylate cyclase signaling could be blocked by
PD98059, as well as by wortmannin and LY294002. These MEK and PI3K
inhibitors block the MAPK pathway (Fig. 2) but do not affect the
cellular cAMP levels induced by forskolin (not shown). As described
previously (13), DAMGO reduced cAMP levels induced by forskolin and the
phosphodiesterase inhibitor IBMX (Fig. 4b). Pre-exposure to
DAMGO for 2 h desensitized the receptor, thereby abolishing this
effect. However, when the cells were preincubated with DAMGO in the
presence of naloxone, PD98059, wortmannin, or LY294002, a second
agonist stimulus reduced the forskolin-stimulated levels of cAMP as in
non-desensitized cells. Thus, multiple inhibitors acting at different
points along the MAPK cascade are capable of blocking the
µ-agonist-induced desensitization of cAMP signaling (Fig.
4b).
|
It has been shown that, except for morphine (24), agonist-induced
µ-opioid receptor desensitization is accompanied by the loss of
membrane receptor (3, 24). Total receptor binding using the opioid
antagonist [3H]naloxone was determined to test whether
inhibition of the MAPK pathway would block not only desensitization but
also the agonist-induced receptor internalization.
[3H]Naloxone binding to membranes of desensitized cells
was performed by following the same experimental protocol described in
Fig. 4; we expose the cells to no agonist (vehicle, 0.04% dimethyl sulfoxide) or to 1 µM DAMGO, 1 µM morphine,
1 µM DAMGO in the presence of 20 µM
PD98059, or to 20 µM PD98059 alone. After 2 h of
treatment the cells were extensively washed, and plasma membranes were
prepared for radioligand binding assays. [3H]Naloxone
bound in a saturable manner to all cell membrane preparations (not
shown). Scatchard analysis of the binding results was performed to
calculate Bmax data for each treatment, and
these values are compared in the histogram shown in Fig.
5. The results revealed a decrease of
approximately 60% in receptor number in cells pretreated with DAMGO
(Bmax, 1217 ± 136 fmol/mg protein,
n = 4) as compared with untreated cells
(Bmax, 2894 ± 144 fmol/mg protein,
n = 4). A similar degree of internalization caused by
agonist exposure was reported for the µ- and -opioid receptors
(24-26). Total receptor number in cells pretreated with morphine shows
no significant difference to that of untreated cells
(Bmax, 2813 ± 123 fmol/mg protein,
n = 3). Pretreatment with the agonist in the presence of PD98059 was able to prevent the loss of µ-opioid receptor density (Bmax, 2753 ± 311 fmol/mg protein,
n = 3), compared with untreated cells, whereas
pretreatment with PD98059 alone had no effect upon µ-opioid receptor
density (Bmax, 2761 ± 98 fmol/mg protein,
n = 3). The Kd measured for the four
treatment groups are: untreated cells (0.9 ± 0.2 nM,
n = 3); desensitized with DAMGO, (0.9 ± 0.2 nM, n = 3); desensitized with morphine
(3.7 ± 0.4); desensitized with DAMGO in the presence of PD98059
(1.6 ± 0.6 nM, n = 3), and PD98059
treatment alone (0.5 ± 0.08 nM, n = 3). Thus, the inhibition of the MAPK pathway at the level of MEK1/2 also impaired the agonist-induced receptor internalization yielding a
number of coupled receptors available sufficient to maintain an intact
signaling response (Fig. 4).
|
Our results suggest that upon agonist activation a feedback signal
emanates from the MAPK cascade to promote the desensitization of
receptor signaling, since blocking this pathway at different points
with different inhibitors prevented the desensitization of two
independent effector cascades (Fig. 4). Moreover, the results of the
binding studies indicate that an active MAPK pathway also contributes
to receptor internalization (Fig. 5). Morphine does not cause receptor
internalization even at concentrations that strongly inhibit adenylate
cyclase and stimulate MAPK (24). Thus, morphine activation of the MAPK
pathway (Fig. 1 and Ref. 12) may not require immediate receptor
internalization as recently reported for the -adrenergic and LPA
receptors (27). Our results also indicate that morphine is capable of
desensitizing the µ-opioid receptor-stimulated MAPK pathway, and this
effect is blocked by PD98059, without affecting internalization (Figs.
4 and 5). This is consistent with reports suggesting that sequestration
is not required for desensitization (20). Our study also supports the notion that different µ-agonists can induce two distinct receptor conformational changes, one necessary for signaling and desensitization and a second required for internalization (7, 24). Whereas morphine
would induce only the former, other opioid agonists would always induce
both.
What then are the potential targets for this desensitizing signal emanating from the MAPK cascade? According to our results (Figs. 4 and 5), particularly the effects of morphine on signaling, desensitization, and internalization, we speculate that this signal should act before internalization is initiated. One possibility is the direct phosphorylation of the receptor by MAPK as was reported for the AT1 receptor (28). Another possibility is the direct or indirect interaction between the MAPK cascade and GRK2, as GRK2 has been recently implicated in agonist-induced phosphorylation and desensitization of opioid receptors (4, 5, 29). We are currently investigating this hypothesis. A feedback signal promoting uncoupling at the level of the G-protein itself is also possible. Such signal could act on either subunit of the heterotrimeric G-protein or affect the function of regulators of G-protein signaling (RGS proteins) (30). Further investigation on how the MAPK pathway affects µ-opioid receptor desensitization will contribute to elucidate the molecular mechanisms of tolerance to opiate drugs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Evans for the cell line expressing the murine µ-opioid receptor, C. Marshall and L. Feig for plasmids, and C. Noren, H. Nastri, J. C. Gutierrez-Ramos, Richard A. Bond, and O. Behar for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DA05706 (to M. J. C.).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: Cell Signaling
Laboratory, New England Biolabs, 32 Tozer Rd., Beverly, MA 01915. Tel.:
978-927-5054; Fax: 978-922-7069; E-mail: polakiew{at}neb.com.
1 The abbreviations used are: GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; GRK, G protein-coupled receptor kinase; PI3K, phosphoinositide 3-kinase; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; DPDPE, D-Pen2,D-Pen5-enkephalin; LPA, lysophosphatidic acid; IBMX, isobutylmethylxanthine; MEK, MAP kinase/ERK kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|