From the Department of Pharmacology and Toxicology,
Otto-von-Guericke University, 39120 Magdeburg, Germany
The rat µ opioid receptor is alternatively
spliced into two isoforms (MOR1 and MOR1B) which differ in length and
amino acid composition at the carboxyl terminus. When stably expressed
in HEK 293 cells, both splice variants bind the µ receptor agonist [D-Ala2,N-Me-Phe4,-Gly-ol5]enkephalin
(DAMGO) with similar affinity and exhibit functional coupling to
adenylyl cyclase with similar efficiency. However, the shorter isoform,
MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist
withdrawal (20 min). Immunocytochemical analysis revealed that
DAMGO-induced internalization of MOR1B proceeded much faster than that
of MOR1 followed by rapid recycling of the receptor to the cell
surface. In addition, the greater resistance of MOR1B to homologous
desensitization compared with MOR1 as well as MOR1B resensitization was
abolished when receptor reactivation/recycling was blocked with
monensin, an inhibitor of endosomal acidification. It is concluded that
the sequence at the cytoplasmic tail of MOR1B facilitates
clathrin-coated vesicle-mediated endocytosis which, in turn, promotes
accelerated receptor reactivation. Taken together, our findings suggest
that carboxyl-terminal splicing of the rat µ opioid receptor
modulates agonist-induced internalization and receptor
resensitization.
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INTRODUCTION |
Prolonged exposure of G protein-coupled receptors to agonists
results in a rapid decrease of receptor responsiveness. It is now
generally accepted that agonist-induced desensitization involves phosphorylation of intracellular receptor domains. Several kinases have
been implicated in opioid receptor desensitization, including cAMP-dependent protein kinase
(PKA),1 protein kinase C
(PKC), and calcium/calmodulin-dependent protein kinase II
(CaM kinase II) (1-5). Specific phosphorylation sites have been
localized in the third intracellular domain and at the carboxyl
terminus, which play a critical role in homologous desensitization of
the opioid receptor (6-10). Following phosphorylation, the receptor is
being targeted to the endocytotic machinery. A large body of evidence
suggests that the main route of internalization of G protein-coupled
receptors is via clathrin-coated pits and vesicles into early
endosomes. Within the acidic environment of the endosomes, the ligand
is effectively separated from the receptor which becomes
dephosphorylated and thus resensitized. As a final step, the receptor
recycles back to the cell surface (11).
For the µ opioid receptor, desensitization seems to be regulated by
CaM kinase II-mediated phosphorylation of two serine residues (Ser261/Ser266) in the third intracellular loop
(3, 9). Another important phosphorylation site of the µ opioid
receptor is the threonine at position 394 in the carboxyl terminus.
Indeed, we and others have recently observed that site-directed
mutagenesis of Thr394 to alanine profoundly delays
DAMGO-induced desensitization, suggesting that this site may be a
primary target for phosphorylation by GRKs upon agonist binding to the
MOR1 (9, 10).
We have previously shown, that the cytoplasmic tail of the rat µ opioid receptor undergoes alternative splicing giving rise to two
isoforms, MOR1 and MOR1B. The receptor variants share 100% amino acid
sequence identity up to amino acid 386 but differ from residue 387 to
the carboxyl terminus (MOR1,
387LENLEAETAPLP398; MOR1B,
387KIDLF391). Both isoforms exhibit similar
pharmacological profiles, however, MOR1B which lacks Thr394
appears to be more resistant to agonist-induced desensitization than
MOR1 (12).
In the present study, we examined the role of receptor internalization
in the desensitization process of the two µ opioid receptor isoforms.
Evidence will be provided, that the carboxyl-terminal amino acid
sequence of MOR1B contains a putative endocytotic motif that leads to
an enhanced internalization/reactivation rate and, hence, may explain
the delayed desensitization kinetics of MOR1B as compared with
MOR1.
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MATERIALS AND METHODS |
Tissue Culture and Transfections--
MOR1 cDNA subcloned
into pRc/CMV (kindly provided by Dr. L. Yu, Indianapolis, IN), and
MOR1B cDNA subcloned into pcDNA3 expression vectors were used
for transfection of human embryonic kidney HEK 293 cells (ATCC). Cells
were maintained in Dulbecco's modified Eagle medium NuT F-12 medium
supplemented with 10% fetal calf serum in a humidified atmosphere
containing 5% CO2. Transfections were performed using the
calcium phosphate precipitation method as described by Chen and Okayama
(13). Approximately 1.5 × 106 cells were transfected
with 20 µg of plasmid DNA. Cells were selected in the presence of 500 µg/ml G418 (Life Technologies, Inc., Eggenstein, Germany), and the
whole pool of resistant cells was used without selection of individual
clones.
Determination of Receptor Desensitization and Resensitization by
Measurement of cAMP Accumulation--
Transfected cells were seeded at
a density of 1.5 × 105 per well in 22-mm 12-well
dishes. After 24 h, cells were exposed to 1 µM DAMGO
(Bachem, Heidelberg, Germany) for 0, 0.5, 1, 2, or 4 h. When
indicated, cells were preincubated either with 0.4 M sucrose (Sigma, Deisenhofen, Germany), an inhibitor of clathrin-coated vesicle-mediated endocytosis, for 30 min or with 50 µM
monensin (Sigma), an inhibitor of endosomal acidification, for 60 min
and subsequently maintained under these conditions during agonist exposure. For resensitization assays, cells were washed after 4 h
of DAMGO exposure followed by an additional incubation period of 0, 5, 10, or 20 min in the absence of agonist. For the measurement of cAMP
accumulation, medium was removed from individual wells and replaced
with 0.5 ml of serum-free RPMI medium (Seromed, Berlin, Germany)
containing 25 µM forskolin (Biotrend, Köln,
Germany) or 25 µM forskolin plus 1 µM
DAMGO. The cells were incubated at 37 °C for 15 min. The reaction
was terminated by removing the medium and sonicating the cells in 1 ml
of ice-cold HCl/ethanol (1 volume of 1 N HCl/100 volumes of
ethanol). After centrifugation, the supernatant was evaporated, the
residue was dissolved in TE buffer (50 mM Tris-EDTA, pH
7.5), and the cAMP content was determined using a commercially
available radioimmunoassay kit (Amersham, Braunschweig, Germany).
Statistical evaluation of results was performed using ANOVA followed by
the Bonferroni test.
Radioligand Binding Assay--
For whole cell binding,
106 cells treated as described above were incubated with
2.5 nM [3H]DAMGO (NEN, Köln, Germany)
for 40 min at 25 °C in 50 mM Tris-HCl, pH 7.8. Cells
were collected on GF 10 glass-fiber filters and unbound ligand was
removed by extensive washes with 50 mM Tris-HCl, pH 7.8. The radioactivity on the filters was determined by liquid scintillation
counting. Specific binding was calculated by subtracting nonspecific
binding from total binding. Nonspecific binding was determined as
radioactivity bound in the presence of 1 µM unlabeled DAMGO. Results were calculated as fmol bound radioligand per mg of
protein, measured by Lowry (14). The binding characteristics of MOR1
and MOR1B were determined by saturation binding assays on membranes
prepared from transfected HEK 293 cells. The dissociation constant
(KD) and number of [3H]DAMGO binding
sites (Bmax) were calculated by Scatchard
analysis (15) using at least seven concentrations of labeled DAMGO in a
range from 0.25 to 10 nM.
Confocal Microscopy--
HEK 293 cells stably expressing either
MOR1 or MOR1B were grown on poly-L-lysine-treated
coverslips overnight. Cells were then exposed to 1 µM
DAMGO for 0, 10, 30, or 50 min. When indicated, the medium was
supplemented with sucrose or monensin as described above. Cells were
fixed with 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 6.9, for 45 min at room temperature and subsequently washed several times in TPBS (10 mM Tris,
10 mM phosphate buffer, 137 mM NaCl, 0.05%
thimerosal, pH 7.4). After 1 h of preincubation in TPBS containing
0.3% Triton X-100 and 3% normal goat serum, cells were incubated
either with anti-MOR1 (9998) or anti-MOR1B (9636) antibodies at a
dilution of 1:5000 in TPBS containing 0.3% Triton X-100 and 1% normal
goat serum overnight at RT. The antibodies were generated against the
following peptide sequences: LENLEAETAPLP, which corresponds to
residues 387-398 of MOR1, and VDRTNHQKIDLF, which corresponds to
residues 380-391 of MOR1B and have been characterized extensively
(16). Bound primary antibody was detected with biotinylated goat
anti-rabbit IgG (1:400; Vector, Burlingame, CA) followed by
streptavidin-cyanin 3.18 (1:400; Amersham, Germany). Cells were then
dehydrated, cleared in xylol and permanently mounted in DPX (Fluka,
Neu-Ulm, Germany). Specimens were examined using a Leica TCS-NT laser
scanning confocal microscope. Cyanin 3.18 was imaged with 568-nm
excitation and 570-630-nm bandpass emission filters. Confocal
micrographs were taken by a person blinded to the treatments who was
instructed to randomly select one colony of 4-12 cells per
coverslip.
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RESULTS |
Agonist-induced Desensitization of µ Opioid Receptor Isoforms
Expressed in HEK 293 Cells--
The splice variants MOR1 and MOR1B
were stably expressed in HEK 293 cells. First, we compared binding
affinities and functional coupling to adenylyl cyclase of the expressed
receptors. Saturation binding experiments indicated no major difference
between MOR1 and MOR1B with respect to their affinity to
[3H]DAMGO. The maximum percentage inhibition of adenylyl
cyclase activities for MOR1 and MOR1B was also identical. However, the number of binding sites was higher in MOR1-expressing cells than in
MOR1B-expressing cells (Table I).
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Table I
Functional properties of MOR1 and MOR1B opioid receptor types after
stable expression in human embryonic kidney cells
The KD and Bmax for the binding
of [3H]DAMGO to both splice variants were determined by
Scatchard analyses. The effect of 1 µM DAMGO on
forskolin-stimulated cAMP accumulation was determined as described
under "Materials and Methods." Values shown are the mean ± S.E. from at least four experiments.
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Next, we studied the time-course of agonist-induced loss of functional
coupling to adenylyl cyclase for MOR1 and MOR1B. Forskolin treatment
resulted in a five-fold increase in intracellular cAMP levels as
compared with untreated HEK 293 cells. DAMGO inhibited forskolin-stimulated cAMP formation by 40% in both MOR1- and
MOR1B-expressing cells (Table I). When transfected HEK cells were
preincubated with 1 µM DAMGO for extended time periods, a
time-dependent loss of coupling efficiency, which was
complete after 4 h of agonist exposure, was observed for both
receptor isoforms (Fig. 1). However, MOR1
and MOR1B did significantly differ in their time-course of DAMGO-induced desensitization. While the ability of MOR1 to inhibit cAMP accumulation was strongly reduced already after 1 h of DAMGO exposure, the MOR1B-mediated inhibition of cAMP formation was retained
for longer time periods (Fig. 1). In wild-type HEK 293 cells, no
DAMGO-induced inhibition of adenylyl cyclase was observed (data not
shown).

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Fig. 1.
Time course of agonist-induced
desensitization of MOR1 and MOR1B. Transfected HEK 293 cells were
exposed to 1 µM DAMGO for 0, 0.5, 1, 2, or 4 h.
After removal of agonist-containing medium, cells were treated either
with 25 µM forskolin or 25 µM forskolin
plus 1 µM DAMGO for 15 min, and cAMP levels were
determined using a radioimmunoassay. Values represent means ± S.E. of triplicate determinations from four independent experiments.
Asterisk indicates significant difference (p < 0.05) between MOR1 and MOR1B as determined using ANOVA followed by
the Bonferroni test.
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Agonist-induced Loss of Cell Surface Binding--
To examine the
role of the carboxyl-terminal tail in µ opioid receptor
internalization, HEK 293 cells expressing MOR1 or MOR1B were treated
with 1 µM DAMGO for various time periods, and the number
of cell surface binding sites was determined. As depicted in Fig.
2, cell surface binding in
MOR1-expressing cells was only slightly reduced to 80% during the 4-h
agonist exposure. In contrast, in MOR1B-expressing cells the number of
binding sites progressively decreased to 40% within 2 h and
remained at this level during the rest of the treatment period.

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Fig. 2.
Agonist-induced changes of cell surface
binding sites. HEK 293 cells expressing either MOR1 or MOR1B were
incubated in the presence of 1 µM DAMGO for 0 min, 10 min, 30 min, 1 h, 2 h, or 4 h. Agonist-containing medium
was removed, and the number of cell surface binding sites per cell was
determined by incubation with [3H]DAMGO and liquid
scintillation counting. Each data point represents mean ± S.E. of
triplicate determinations from two independent experiments.
Asterisk indicates significant difference (p < 0.05) between MOR1 and MOR1B as determined using ANOVA followed by
the Bonferroni test.
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Direct Observation of Agonist-induced Endocytosis of µ Opioid
Receptor Isoforms--
To test the possibility that the loss of cell
surface binding sites may be due to receptor endocytosis, MOR1- and
MOR1B-expressing HEK 293 cells were exposed to 1 µM DAMGO
for 0, 10, 30, or 50 min. The culture medium was either not
supplemented or supplemented with monensin or sucrose. These cells were
subsequently fixed, permeabilized, and fluorescently labeled with
antibodies specific for either MOR1 or MOR1B. The subcellular
distribution of the receptor proteins was then analyzed by confocal
microscopy. The results are depicted in Figs.
3, 4, and 5. Fig. 3 (upper
panel) shows that, in the absence of DAMGO, MOR1-like
immunoreactivity (Li) was mostly present at the level of the plasma
membrane and to a lesser extent in the cytoplasm. After 30 min of
agonist exposure, a dramatic loss of MOR1-Li from the plasma membrane
with a concomitant accumulation in vesicle-like structures within the
cytoplasm was observed. When exposure to DAMGO was continued, MOR1
began to reappear at the plasma membrane while a significant proportion of the receptor remained in the intracellular compartment. When MOR1-expressing cells were cultured in the presence of monensin, an
inhibitor of endosomal acidification, the receptor progressively accumulated in the cytoplasm and did not redistribute to the plasma membrane during the 50 min of agonist exposure (Fig. 3, middle panel). When sucrose, an inhibitor of clathrin-coated
vesicle-mediated endocytosis, was added to the culture medium, MOR1
internalization was completely blocked (Fig. 3, lower
panel).

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Fig. 3.
Agonist-induced endocytosis of MOR1 in HEK
293 cells. Upper panel, MOR1-expressing HEK 293 cells
were exposed to 1 µM DAMGO for 0, 10, 30, or 50 min.
Middle panel, MOR1-expressing HEK 293 cells were exposed to
1 µM DAMGO in the presence of 50 µM
monensin for the indicated time intervals. Lower panel,
MOR1-expressing HEK 293 cells were exposed to 1 µM DAMGO
in the presence of 0.4 M sucrose for the indicated time
intervals. Cells were subsequently fixed, fluorescently labeled with
anti-MOR1 antibodies, and examined by confocal microscopy. Note that
Figs. 3, 4, and 5 show results from experiments conducted in parallel
with MOR1- and MOR1B-expressing HEK 293 cells. Shown are representative
results from one of three independent experiments performed in
duplicate. Scale bar, 20 µm.
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Fig. 4 compares the rate and extent of
MOR1 and MOR1B internalization in the presence of monensin, which
blocks receptor recycling to the plasma membrane. The results clearly
show that MOR1B internalization proceeds at a faster rate than that of
MOR1. While internalization of MOR1 was not complete before 50 min
(Fig. 3, middle panel and Fig. 4, upper panel),
in most cells, internalization of MOR1B was complete already after 10 min of DAMGO treatment (Fig. 4, lower panel). In addition,
we noted that in MOR1B-expressing cells, a significant proportion of
receptor-Li was localized in the intracellular compartment even in the
absence of DAMGO. This phenomenon was particularly pronounced when
monensin was added to the culture medium (Fig. 4, lower panel,
left micrograph, and Fig. 5). In contrast, MOR1B receptor protein was virtually absent from the cytoplasm when the cells were cultured in the presence of sucrose (Fig.
5, right micrograph). These results suggest that the MOR1B receptors are subject to constitutive internalization in the absence of
agonist. Monensin effectively trapped internalized receptor proteins in
endosomes, resulting in intracellular accumulation of MOR1B-Li. Sucrose
prevented internalization of recycled receptor proteins so that
MOR1B-Li almost completely disappeared from the cytoplasm. Moreover,
binding analyses revealed that addition of the µ opioid receptor
antagonist naloxone (12 h before the binding experiment) led to an
increase of the Bmax of only MOR1B but not MOR1
receptor type, supporting the idea of the constitutive internalization of the MOR1B receptor (Fig. 6).

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Fig. 4.
Comparison of agonist-induced endocytosis of
MOR1 and MOR1B. Upper panel, MOR1-expressing HEK 293 cells were exposed to 1 µM DAMGO for 0, 10, 30, or 50 min
in the presence of 50 µM monensin. Lower
panel, MOR1B-expressing HEK 293 cells were exposed to 1 µM DAMGO for 0, 10, 30, or 50 min in the presence of 50 µM monensin. Cells were subsequently fixed, fluorescently
labeled with antibodies specific for either MOR1 or MOR1B and examined
by confocal microscopy. Note that Figs. 3, 4, and 5 show results from
experiments conducted in parallel with MOR1- and MOR1B-expressing HEK
293 cells. Therefore, the same micrographs are shown in Fig. 3,
middle panel and Fig. 4, upper panel.
Representative results from one of three independent experiments
performed in duplicate. Scale bar, 20 µm.
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Fig. 5.
Constitutive internalization of MOR1B in HEK
293 cells. MOR1B-expressing HEK 293 cells were incubated with
either 50 µM monensin or 0.4 M sucrose for 60 min in the absence of agonist. Cells were subsequently fixed,
fluorescently labeled with anti-MOR1B antibodies, and examined by
confocal microscopy. Note that Figs. 3, 4, and 5 show results from
experiments conducted in parallel with MOR1- and MOR1B-expressing HEK
293 cells. Therefore, the same micrographs are shown in Fig. 4,
lower panel (left micrograph) and Fig. 5
(left micrograph). Shown are representative results from one
of three independent experiments performed in duplicate. Scale bar, 20 µm.
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Fig. 6.
Effect of pretreatment with µ opioid
antagonist naloxone (1 µM for 12 h) on the
Bmax values of MOR1 and MOR1B opioid receptor
types after stable expression in human embryonic kidney cells. The
Bmax for the binding of [3H]DAMGO
to the naloxone-untreated splice variants (Table I), determined by
Scatchard analyses, were defined as 100% for each receptor type.
Values represent means ± S.E. from four separate measurements.
Asterisk indicates significant difference (p < 0.05) between naloxone-treated and -untreated receptor types as
determined using ANOVA followed by the Bonferroni test.
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Resensitization of MOR1 and MOR1B in HEK 293 Cells--
To
investigate whether desensitization of the µ opioid receptor isoforms
is reversible by agonist withdrawal, HEK 293 cells expressing either
MOR1 or MOR1B were treated for 4 h with 1 µM DAMGO,
and medium was removed followed by an additional agonist-free incubation interval of 0, 5, 10, 15, or 20 min and determination of
cAMP accumulation. Fig. 7A
reveals that, after complete receptor desensitization, only MOR1B but
not MOR1 resensitized during the 20 min of DAMGO withdrawal. In
addition, the rapid resensitization of MOR1B was prevented by monensin
(Fig. 7B). These results suggest that the faster rate of
internalization of MOR1B is associated with accelerated receptor
resensitization and that MOR1B reactivation involves redistribution of
receptor proteins to the cell surface.

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Fig. 7.
Resensitization of MOR1 and MOR1B without
(A) and under the influence of 50 µM monensin
(B). Transfected HEK 293 cells were cultured in the
presence of 1 µM DAMGO for 4 h, which results in
complete desensitization of both receptor isoforms. Medium was removed,
and cells were washed extensively followed by an additional incubation
for 0, 5, 10, or 20 min in agonist-free medium. Cells were then treated
either with 25 µM forskolin or 25 µM
forskolin plus 1 µM DAMGO for 15 min and cAMP levels were
determined using a radioimmunoassay. Values represent means ± S.E. of triplicate determinations from two independent experiments.
Asterisk indicates significant difference (p < 0.05) between MOR1 and MOR1B as determined using ANOVA followed by
the Bonferroni test.
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Effects of Monensin on Agonist-induced µ Opioid Receptor
Desensitization--
To determine to what extent the faster receptor
internalization/reactivation of MOR1B contributes to its greater
resistance to homologous desensitization compared with MOR1, we carried
out desensitization assays as described above except that monensin was
included in all incubations. While agonist-induced desensitization of
MOR1 did not dramatically change, MOR1B showed very rapid
desensitization which was already complete after 2 h of DAMGO
exposure (Fig. 8). These findings suggest
that the delayed desensitization of MOR1B involves its rapid targeting
to the endocytotic machinery which, in turn, promotes accelerated
receptor reactivation and recycling.

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Fig. 8.
Effect of monensin on agonist-induced
desensitization of MOR1 and MOR1B. Transfected HEK 293 cells were
preincubated with 50 µM monensin for 60 min and then
exposed to 1 µM DAMGO for 0, 0.5, 1, 2, or 4 h in
the presence of monensin. After removal of agonist-containing medium,
cells were treated either with 25 µM forskolin or 25 µM forskolin plus 1 µM DAMGO for 15 min,
and cAMP levels were determined using a radioimmunoassay. Values
represent means ± S.E. of triplicate determinations from two
independent experiments. Asterisk indicates significant
difference (p < 0.05) between MOR1 and MOR1B as
determined using ANOVA followed by the Bonferroni test.
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DISCUSSION |
The µ opioid receptor splice variants, MOR1 and MOR1B, markedly
differ in their desensitization kinetics when expressed in HEK 293 cells. Both variants share 100% amino acid sequence identity up to
amino acid 386 but differ from residue 387 up to the carboxyl terminus
(MOR1, 387LENLEAETAPLP398; MOR1B,
387KIDLF391). Specifically, MOR1B that lacks
threonine 394 appears to be more resistant to agonist-induced
desensitization than MOR1. It is therefore very tempting to speculate
that MOR1B due to its lack of this threonine may be a bad substrate for
phosphorylation by GRKs and, hence, more resistant to agonist-induced
desensitization. However, an alternative explanation emerges from our
internalization studies showing that MOR1B undergoes faster endocytosis
than MOR1.
After internalization, receptors will be dephosphorylated, separated
from bound ligands and become subject to endosomal sorting either by
passing through a process of receptor degradation or being recycled to
the cell membrane. Our immunocytochemical analysis revealed, that MOR1
internalization in HEK 293 cells reached a maximum at 30 min of DAMGO
preincubation and that after 50 min, receptors seemed to reappear at
the cell membrane. In addition, pretreatment of µ opioid receptor
expressing HEK 293 cells with the recycling blocker monensin led to a
complete internalization of both MOR1B and MOR1 within 50 min of DAMGO
treatment. It can be assumed that the fast process of receptor
reappearance after agonist-induced internalization is due to receptor
recycling and not due to receptor de novo synthesis since
studies with other G-protein-coupled receptors showed that
cycloheximide did not inhibit fast receptor reappearance at the cell
surface (17-20). Previous evidence with the
2-adrenergic receptor suggests that receptor
desensitization and internalization are two distinct and independent
processes (21, 22). In fact, blockade of receptor internalization by
sucrose does not prevent agonist-induced desensitization (23-25),
indicating that, first of all, functional uncoupling depends on
receptor phosphorylation followed by spatial uncoupling involving receptor endocytosis.
We suggest a model whereby rapid endocytosis of MOR1B permits
accelerated resensitization and recycling of the receptor. This enhanced rate of resensitization of MOR1B compared with MOR1, in turn,
confers a greater resistance to agonist-induced desensitization. Several lines of evidence indicate that enhanced resensitization of
MOR1B as compared with MOR1 does indeed contribute to its slower time
course of desensitization. First, after complete receptor desensitization, only MOR1B but not MOR1 resensitized during 20 min of
agonist withdrawal. Second, MOR1B did no longer resensitize when
receptor recycling was blocked with monensin. Third, when desensitization assays were carried out in the presence of monensin, the greater resistance of MOR1B to homologous desensitization compared
with MOR1 was abolished.
Another difference between MOR1 and MOR1B is that in the absence of
agonist a significant proportion of the MOR1B receptor protein seems to
be constitutively internalized and recycled to the cell surface. This
assumption is favored by the fact that MOR1B receptor proteins could be
effectively trapped in endosomes by monensin incubation, whereas
sucrose treatment prevented constitutive MOR1B internalization (Fig.
5). In addition, our binding analysis showed that preincubation with
the antagonist naloxone increased only the receptor number in the
MOR1B-expressing cells (Fig. 6), suggesting that antagonist treatment
inhibits constitutive internalization by stabilizing the receptor in a
conformation that prevents exposing a receptor domain critical to
directing it into the clathrin-dependent endocytotic
pathway.
It is thus conceivable that the carboxyl-terminal tail of MOR1B
contains an endocytotic sequence motif which facilitates targeting of
the receptor protein to the endocytotic machinery. Constitutive internalization has previously been observed with a truncation mutant
of the µ opioid receptor that lacks a carboxyl-terminal Ser/Thr-rich
domain (354Thr-Ser-Ser-Thr357). When the
truncation spared this Ser/Thr-rich domain, constitutive internalization did not occur (26). These findings confirm the complex
role of the carboxyl terminus in µ opioid receptor endocytosis. While
the Ser/Thr-rich domain (354Thr-Ser-Ser-Thr357)
which is shared in both µ opioid receptor splice variants suppresses internalization, the cytoplasmic tail of MOR1B greatly enhances internalization.
Internalization results in a loss of membrane binding sites in intact
cells. The binding analyses revealed that 40% of the MOR1B receptor
sites have disappeared at 30 min of agonist treatment, without markedly
affecting functional activity at this time. The simplest explanation
for this finding is that only 60% of intact MOR1B receptors are
necessary for maximum inhibition of intracellular cAMP formation. The
decreasing pool of receptors in the membrane after agonist treatment
may then be balanced by receptor recycling after internalization.
Surprisingly, after 4 h of DAMGO preincubation, the MOR1 receptor
is completely desensitized, whereas nearly 80% residual MOR1 binding
sites could be detected in the cell membrane. This indicates, that
phosphorylation and uncoupling of the receptor did not automatically
induce massive internalization of the receptor and that only a portion
of the residual receptor binding sites seem to represent active
receptors coupling to intracellular G-proteins.
In summary, we have established that receptor internalization and
resensitization is differentially affected by the divergent cytoplasmic
tails of the µ opioid receptor isoforms, MOR1 and MOR1B. It appears
that the sequence motif at the cytoplasmic tail of MOR1B facilitates
clathrin-coated vesicle-mediated endocytosis and, in turn, promotes
accelerated receptor reactivation and redistribution. The comparison
between MOR1 and MOR1B provides strong evidence that receptor
internalization is a primary rate-limiting step for receptor
resensitization, and that enhanced resensitization also confers
apparent resistance to agonist-induced desensitization. Thus,
alternative carboxyl-terminal splicing of G-protein-coupled receptors
may be a physiologically relevant mechanism that determines rate and
extent of receptor internalization and resensitization.