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INTRODUCTION |
The opioid system controls pain perception and mood and is
generally implicated in a wide variety of behaviors that are essential in facing threatening situations (1, 2). Opioid receptors also mediate
the strong analgesic and addictive actions of opiate drugs.
Pharmacological studies indicate that the prototypic opiate morphine,
the main clinically useful opiates such as fentanyl or methadone, and
the closely related drug of abuse heroin preferably act by activating
the µ-opioid receptor
(MOR)1 rather than
- or
-opioid receptors (3-5). In support of this, gene targeting
experiments have shown the absence of morphine-induced analgesia
(6-10), reward and physical dependence (6), immunosuppression (11,
12), respiratory depression (13), or constipation (14) in MOR-deficient
mice, demonstrating unambiguously that the µ-opioid receptor is a
main molecular target for morphine action in vivo. The
finding of genetic mutations altering the expression or functional activity of MOR is therefore important to understand inter-individual variable responses to the major opioid drugs, both in the clinical management of pain or heroin addiction.
In addition to the direct mediation of opiate-induced euphoria,
tolerance, and dependence, µ-opioid receptors have been shown to
regulate the effects of other substances with high addictive potential
such as cocaine or alcohol (15). As an example, in humans, the
µ-receptor antagonists naloxone and naltrexone have been shown not
only to reverse heroin overdose but also to alter alcohol consumption
(16, 17). Studies using animal models also point at a possible role of
the MOR gene in ethanol use. Recently we have reported that
MOR-deficient mice do not self-administer alcohol (18), suggesting that µ receptors are essential in mediating the reinforcing properties of
this substance. Also a quantitative trait loci (QTL) analysis in
mice showed that oral morphine preference is largely mediated by a
single locus in chromosome 10 which harbors the MOR gene (19). Thus,
among the various genetic components of the opioid system, the
µ-opioid receptor gene is an evident candidate in the search for
genes potentially involved in the susceptibility to drug abuse.
The cloning of the human opioid receptor gene (20, 21) has prompted
studies of DNA sequence variability within the hMOR gene (22-24). We
have recently conducted a comprehensive polymorphic study of the MOR
gene in 250 patients using large scale multiplex DNA sequencing. We
have identified 43 variants within 7-kilobase pair regulatory, exonic,
and intronic sequences (25). We have found six mutations in the coding
region. Five of these mutations modify the encoded protein sequence,
and these include the previously identified A6V and N40D mutations in
the N-terminal region of the receptor (22-24), as well as three yet
unreported mutations. One of these novel mutations is located in the
third transmembrane domain of the receptor and changes an asparagine
residue into an aspartic acid residue (N152D). The two other mutations
are found in the third intracellular loop of the receptor and replace an arginine and a serine by a histidine (R265H) and a proline (S268P)
residue, respectively.
The three latter mutations occur in regions that may be critical for
receptor function, and this has prompted us to determine whether these
natural MOR variants indeed exhibit an altered pharmacological activity
profile. We have constructed the three novel hMOR variants by
site-directed mutagenesis. We also have generated the N40D mutant, some
properties of which have been described earlier (24), to extend our
knowledge on the functional properties of this frequent polymorphic
variant. We have expressed the four mutant receptors in mammalian
cells, and we determined binding affinities for four prototypic opioid
ligands (morphine, DAMGO, diprenorphine, and CTOP) and the main opioid
peptides ([Met]enkephalin,
-endorphin, and dynorphin A). We
also have investigated agonist-induced functional responses of the
receptor using the [35S]GTP
S binding assay, as well as
a reporter gene assay. Finally, we have examined receptor
down-regulation following chronic exposure to the potent µ-agonist
DAMGO. Our results show no obvious modification in receptor binding or
down-regulation. However, the data indicate decreased heterologous
expression for the N152D mutant receptor and a remarkable decrease of
receptor signaling for the S268P variant.
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EXPERIMENTAL PROCEDURES |
Materials--
DAMGO, naloxone, [Met]enkephalin,
-endorphin, dynorphin A, GDP, and GTP
S were obtained from Sigma;
CTOP was from Peninsula Laboratories (St. Helens, Merseyside, UK);
morphine was obtained from Cooper Biomedical (Melun, France), and
heroin was from Francopia (Paris, France).
[3H]Diprenorphine (specific activity, 58 Ci/mmol),
[3H]DAMGO (specific activity, 54.6 Ci/mmol), and
[35S]GTP
S (specific activity, 1156 Ci/mmol) were
obtained from PerkinElmer Life Sciences. The hMOR cDNA (a kind gift
from Pr. Lei Yu, Cincinnati, OH) was subcloned into pcDNAI/Amp
(Invitrogen) for site-directed mutagenesis and transient expression in
COS cells. The WT and mutant cDNAs were subcloned into pcDNA3
(Invitrogen) for expression in HEK 293 cells. The carrier plasmid used
in the electroporation procedure (pBluescript) was from Stratagene, and
the Superfect reagent was from Qiagen. G418 was from Life Technologies,
Inc. The reporter plasmid pCRE-SEAP was from
CLONTECH, and the Phosphalite detection kit and
Enhancer solution were from Tropix. Kd, Ki, and Bmax values were
calculated using the EBDA/Ligand program (G. A. McPherson,
Biosoft, UK), and EC50 values were determined using the
Prism software (GraphPad, San Diego, CA).
Site-directed Mutagenesis--
The wild-type human MOR cDNA
was FLAG-tagged at the N terminus by polymerase chain reaction using
elongase enzyme (Elongase kit, Life Technologies, Inc., 15 cycles), a
5' synthetic oligonucleotide of 60 base pairs encoding the antigenic
epitope (FLAG epitope, IBI, New Haven, CT), and a 3'-oligonucleotide of
36 base pairs to introduce a NotI restriction site after the
stop codon. The hMOR cDNA was then cloned into
HindIII-NotI sites of pcDNAI/Amp, and
mutagenesis was performed as described previously (26). Sequences of
the oligonucleotides were 5'-CTT AGA TGG CGA CCT GTC CGA-3', 5'-AGA TTA
CTA TGA CAT GTT CAC-3', 5'-AAG AGT GTC CAC ATG CTC TCT-3', and 5'-CCG
CAT GCT CCC TGG CTC CAA-3' to obtain the following point mutations,
respectively: N40D (AAC to GAC) in the N-terminal portion, N152D (AAC
to GAC) in the third transmembrane domain, R265H (CGC to CAC) and S268P
(TCG to CCC) in the third intracellular loop. Nucleotide sequence of
the mutated cDNAs was confirmed by double-strand DNA sequencing.
Ligand Binding--
WT and mutant receptors were transiently
expressed in COS cells. The cells were transfected using a highly
efficient electroporation procedure (27). Briefly, 2 × 107 COS cells were seeded the night before transfection at
a density of 107 cells/140-mm dish. Cells were washed two
times with PBS, detached by applying 5 ml of trypsin/EDTA (Eurobio) for
5 min at 37 °C, diluted with 5 ml of Dulbecco's modified Eagle's
medium with 10% fetal calf serum, and an aliquot used to determine the
total cell number. Cells were collected by centrifugation for 10 min at
1000 rpm and resuspended at a 108 cell/ml density in EP 1×
buffer (50 mM K2HPO4, 20 mM CH3CO2K, 20 mM KOH,
pH 7.4). Plasmidic DNA (4 µg), prepared using Nucleobond columns
(Macherey Nagel) and a carrier plasmid (pBluescript) up to a final 20 µg of DNA quantity, was diluted into EP 1× buffer to a total volume
of 300 µl. The DNA mixture was then supplemented with 13 µl of 1 M MgSO4 and incubated with 200 µl of cell
suspension for 20 min at room temperature. The cell/DNA mixture was
then transferred to a 0.4-cm cuvette (Bio-Rad) and electroporated using a Gene Pulser apparatus (Bio-Rad) at a capacitance setting of 2000 microfarads and voltage setting of 240 V. Cells were then immediately
transferred into 50 ml of Dulbecco's modified Eagle's medium with
10% fetal calf serum and seeded into two 140-mm dishes. After 72 h growth the cells were harvested for membrane preparation.
Membranes were prepared as follows: transfected cells (four 140-mm
dishes at a 50-100% confluency) were washed with two times PBS,
scraped off the plates in PBS, pelleted by spinning at 1500 rpm for 10 min at 4 °C, frozen at
80 °C for 30 min at least, and thawed in
30 ml of cold 50 mM Tris-HCl, pH 7, 2.5 mM
EDTA, and 0.1 mM phenylmethylsulfonyl fluoride added
extemporaneously. The cell lysate was kept on ice for 15 min,
Dounce-homogenized, and spun at 2500 rpm for 10 min at 4 °C. The
pellet was resuspended in 15 ml of buffer, Dounce-homogenized, and spun
again at 2,500 rpm for 10 min at 4 °C. Both supernatants were pooled
and centrifuged at 40,000 rpm for 30 min at 4 °C. The supernatant
was removed, and the pellet was resuspended in 25 ml of 50 mM Tris-HCl, pH 7, Dounce-homogenized, and spun
again at 40,000 rpm for 30 min at 4 °C. The pellet was then
resuspended in 4 ml of 50 mM Tris-HCl, pH 7.4, 0.32 M sucrose, and the protein concentration was measured using the Bradford assay.
Opioid binding experiments were performed on membrane preparations as
described previously (26). For saturation experiments, 5-10 µg of
membrane proteins were diluted in 50 mM Tris-HCl, pH 7.4, in a final volume of 0.25-0.5 ml and incubated with variable concentrations of [3H]diprenorphine (0.18-5
nM) or [3H]DAMGO (0.05-6 nM) for
1 h at 25 °C. Nonspecific binding was determined in the
presence of 10 µM naloxone. For competition studies
membrane preparations were incubated for 1 h at 25 °C with 0.5 nM [3H]diprenorphine in the presence of
various concentrations (10
5 to
10
12 M) of competing opioid
ligands (CTOP, morphine, DAMGO,
-endorphin, and dynorphin A). In the
case of [Met]enkephalin, Ki values obtained
from [3H]diprenorphine displacement were extremely low
(35-130 nM). We therefore used [3H]DAMGO (2 nM) as the radiolabeled ligand, and found
Ki values in the nanomolar range (Table II), as
described previously (28). Incubation mixtures were rapidly washed,
using a Brandell cell harvester, with cold 50 mM Tris
buffer on 0.1% polyethyleneimine-presoaked GF/B filters (Whatman).
[35S]GTP
S Binding Assay--
Agonist-stimulated
[35S]GTP
S binding was performed on WT and mutant
receptors as described previously (27). Briefly, COS cells were
transiently transfected, and membranes were prepared as described above
(ligand binding section). 5 µg of membrane proteins were incubated
for 2 h at 4 °C, with and without the agonist
(10
5 to 10
13
M), in assay buffer containing 50 mM Hepes, pH
7.6, 5 mM MgCl2, 100 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, 0.1% bovine
serum albumin, 30 µM GDP, and 0.2 nM
[35S]GTP
S. As for saturation experiments, the
incubation was terminated by rapid filtration through
H2O-presoaked GF/B filters, followed by three washes with
ice-cold 50 mM Tris-HCl, pH 7, 5 mM
MgCl2, 50 mM NaCl. Bound radioactivity was
determined by scintillation counting. Nonspecific binding was
determined in the presence of 10 µM GTP
S, and basal
binding was defined as specific [35S]GTP
S binding in
the absence of agonist.
Reporter Gene Assay--
The day before transfection, HEK 293 cells were plated in 96-well plates (Biocat, Packard Instrument Co.) at
a density 30,000 cells/well. HEK 293 were cotransfected with the
reporter gene pCRE-SEAP (1 µg/well) and hMOR WT or hMOR S268P (0.2 µg/well) using Superfect reagent. 24 h after transfection, cells
were serum-starved for 16-18 h. Cells were then stimulated by 10 µM forskolin and different concentrations of agonist for
6 h in media without serum. 15 µl of supernatant were
transferred into a second 96-well plate, diluted in a dilution buffer
(150 mM NaCl, 40 mM Tris-HCl, pH 7.2), and
heated at 65 °C for 20 min. Plates were cooled to room temperature.
The enzymatic reaction was done by adding an assay buffer (666 mM diethanolamine, 3.3 mM MgCl2,
6.6 mM L-homoarginine, pH 9.8), an Enhancer
solution, and 0.4 mM SEAP chemiluminescent substrate in a
final volume of 150 µl. 15 min after substrate addition, luminescence
was quantified (Trilux, Wallac).
Down-regulation--
Pools of stable HEK 293 cell lines
expressing WT or mutant receptors were produced. HEK 293 cells were
grown in minimum essential medium supplemented with 10% fetal calf
serum. Cells were transfected by a classical calcium phosphate method
(27) and selected with 0.5 mg/ml G418. Neomycin-resistant clones from
100-mm diameter cell culture dishes were selected for 2 weeks. For
down-regulation studies, the pools of stably transfected HEK cells were
cultured for 20 h in the presence or absence of 10 µM DAMGO, washed twice with PBS, once with PBS-2
mM EDTA, and resuspended in 10 mM Tris, 0.32 M sucrose, pH 7.4 (incubation buffer). Binding reactions were performed using 4 × 105 cells in 0.25 ml of
incubation buffer per assay with either 5 nM
[3H]DAMGO or 1.5 nM
[3H]diprenorphine. Under those conditions, most receptor
sites were occupied since Scatchard analysis of [3H]DAMGO
and [3H]diprenorphine binding on WT hMOR-transfected cell
preparations indicates Kd values of 1.36 ± 0.41 and 0.1 ± 0.03 nM, respectively (not shown).
Specific binding was determined by the difference between binding
measured in the absence (total binding) or presence (nonspecific
binding) of 50 µM naloxone. After 1 h of incubation
at 25 °C, binding reaction was terminated by rapid filtration over
Whatman GF/B filters presoaked with 0.1% polyethyleneimine, and bound
radioactivity was counted using a scintillation counter. Data were
compared using unpaired Student's t test (Statview).
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RESULTS |
Expression of Wild-type and Mutants hMOR Receptors in COS
Cells--
To assess the role of natural protein variants encoded by
the hMOR gene, we have substituted the most frequently found
nucleotides in the cloned hMOR receptor (GenBankTM
accession number L29301) by the nucleotide variants originating from
our polymorphism study. Point mutations introduced in the cloned hMOR
receptor replaced two asparagines by an aspartate residue (N40D and
N152D), an arginine by a histidine residue (R265H), and a serine by a
proline residue (S268P). The localization of these mutations is shown
on Fig. 1. The wild-type (WT) and the mutant receptors were transiently expressed in COS cells. We have quantified expression levels of receptors by monitoring the binding of
[3H]diprenorphine, a nonselective alkaloid antagonist, to
the wild-type and mutant receptors (Table
I). Scatchard analysis indicates that WT,
N40D, R265H, and S268P mutant receptors are expressed at comparable
levels, whereas expression of mutant N152D is significantly lower than
WT.

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Fig. 1.
Site-directed mutagenesis of amino acid
residues of hMOR. A schematic representation of the putative seven
transmembrane domain topology of the receptor is shown. The
single-letter amino acid code is used, and numbers next to
mutated residues refer to their positions within the hMOR protein
sequence. Polymorphisms that affect protein sequence are indicated
(22-25), and mutations examined in this study are highlighted
(bold letters, black circle). The N40D and A6V variants are
most frequent.
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Table I
Receptor densities (Bmax) for hMOR WT and mutant receptor
membrane preparations
Expression levels were measured by Scatchard analysis of
[3H]diprenorphine binding on COS cell membrane preparations
expressing hMOR WT or mutant receptors (see "Experimental
Procedures"). Nonspecific binding was determined with 10 µM naloxone. Bmax values are
means ± S.E. from n independent experiments performed
in triplicate. Affinity values are shown in Table II.
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Effect of the Mutations on Opioid Binding--
Saturation
experiments indicate that [3H]diprenorphine shows
identical affinity to WT and N152D receptors and slightly higher affinity to the other mutant receptors. Differences in
Kd values were subtle (Table
II), and from these data we conclude that
the mutations under study do not markedly alter the high affinity
interaction of diprenorphine with the receptor. We then used
[3H]diprenorphine as the radiolabeled ligand in
competition studies to establish whether the mutations modify binding
affinity of other opioids.
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Table II
Binding affinities of opioids to WT and mutant receptors expressed in
COS cells
Competition experiments were performed on transfected COS cell
membranes expressing wild-type and mutant receptors, using variable
concentrations of unlabeled ligands to displace
[3H]diprenorphine or [3H]DAMGO (for Met-enkephalin,
see "Experimental Procedures"). Experiments were repeated two to
four times in duplicate for each compound, and the table shows affinity
values expressed in nM. Affinity values are
Ki values for all ligands, except for diprenorphine
(Kd).
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We tested three prototypic µ-opioid ligands with distinct structural
(alkaloid or peptide) and functional (agonist or antagonist) properties. These include the prototypic alkaloid agonist morphine, the
peptidic enkephalin-derived agonist DAMGO, and the peptidic somatostatin-derived antagonist CTOP. Again, none of the mutations appeared to modify strongly the binding affinities for these ligands (Table II, top). Ki values tended to be lower at
mutant receptors compared with WT but, as for
[3H]diprenorphine binding, changes in
Ki values between mutant and WT receptors were
minimal and never exceeded a 3-fold difference. There was an exception
for morphine at the N152D mutant and DAMGO at the R265H mutant, where
Ki values were 3.8- and 3.7-fold lower,
respectively. In the latter case, however, we were able to use
[3H]DAMGO, available as a radiolabeled ligand, to get a
direct measurement of DAMGO affinity in a saturation experiment.
Scatchard analysis of [3H]DAMGO binding showed no
differences in Kd values between WT and mutant
receptors (not shown), suggesting that the small modification of
binding affinity that was measured from the competition experiment may
not represent a real affinity change. Altogether, these data suggest
that the polymorphic mutations under study have no major incidence on
opioid binding, independently from the chemical nature of the tested compound.
Heroin showed Ki values ranging from 286 (WT) to 466 nM (N40D), confirming the preservation of binding affinity
for morphinic compounds at the mutant receptors (not shown). Those affinity values, which are much lower than morphine values, are in
accordance with previous data (29, 30) suggesting that heroin
metabolites, rather than heroin itself, act in vivo.
Because polymorphic mutations may influence the physiology of the
endogenous opioid system in humans, we also tested the major endogenous
peptides,
-endorphin, [Met]enkephalin, and dynorphin A. All
affinity values were in the nanomolar range (Table II, bottom), as
previously reported for hMOR WT (28). Again binding at mutant receptors
slightly differed from WT, but variations were even more subtle than
for exogenous or synthetic opioids, and changes in
Ki values barely exceeded 2-fold. This suggests that
natural sequence variations in the coding region of the MOR gene are
unlikely to affect significantly the receptor occupancy by endogenous
opioids. Of note is the previous report by Bond et al. (24)
showing a 3-fold higher affinity of
-endorphin for the N40D mutant.
Our experiments do not show this difference, either from
[3H]diprenorphine displacement (Table II) or
[3H]DAMGO displacement (not shown), perhaps due to
different host cells used for receptor expression. Otherwise, both
studies showed no marked difference in binding affinities for all other
tested opioids at WT and N40D receptors. We cannot exclude that the
polymorphic mutations may have a more perceptible influence on opioid
binding in a neuronal cellular context.
Receptor Signaling: Effect of the Mutations on Basal or
Agonist-induced [35S]GTP
S Binding--
To measure
agonist-induced stimulation of wild-type and mutant receptors at the G
protein level, we used the [35S]GTP
S binding assay. We
first tested the two prototypic agonists DAMGO and morphine on membrane
preparations expressing the wild-type receptor. We compared
agonist-induced stimulation of [35S]GTP
S binding at
saturating ligand concentrations (10
5
M for both morphine and DAMGO). In these preliminary assays
DAMGO activated the wild-type receptor up to 273 ± 25% above
basal level, whereas maximal activation obtained with morphine was
197 ± 18% (not shown), indicating that DAMGO is a full agonist,
and morphine is a partial agonist under our expression conditions. This
is in accordance with other studies that have used either native (31)
or recombinant µ receptors (32). We therefore used DAMGO to examine
further the mutant receptors.
In a preliminary experiment, DAMGO-stimulated activation could not be
detected for the N152D receptor (not shown). This is presumably due to
low expression levels, in accordance with our previous studies showing
that functional activity is difficult to quantify when
Bmax values are below 2 pmol/mg protein in this assay (27). DAMGO significantly increased [35S]GTP
S
binding above basal levels for the N40D, R265H, and S268P mutants,
demonstrating that those mutant receptors remain functional (not
shown). Maximal activation levels, however, were different from wild
type, particularly for the S268P receptor. Since the three mutants were
expressed at levels comparable to that of the wild-type receptor,
altered [35S]GTP
S binding was not due to distinct
receptor densities but rather to modifications of agonist potencies or efficacies.
We then established dose-response curves of DAMGO-induced
[35S]GTP
S binding at WT, N40D, R265H, and S268P (Fig.
2). For the N40D mutant receptor, maximal
activation was increased compared with WT receptor (258 ± 4%
versus 228 ± 16% above basal), whereas the
EC50 value was not significantly modified (Table
III). Agonist-induced [35S]GTP
S binding at the R265H mutant was lower than
wild type, with a signal of 207 ± 1% above basal. DAMGO potency
was not significantly reduced for this mutant (Table III). For the
S268P mutant DAMGO efficacy was dramatically reduced. The maximal
activation level was 132 ± 2% above basal, indicating that
DAMGO-induced receptor activation was 25% from WT. For this mutant
also, DAMGO potency was reduced more obviously than for the two other
mutant receptors, with an EC50 value 2.4-fold higher than
wild type (Table III). Altogether, the data suggest that the two
mutations in the third intracellular impair receptor-mediated G protein
signaling, with a prominent effect of the serine to proline mutation at
position 268. In contrast, the N-terminal mutation is not
harmful but may rather favor receptor-G protein
interactions.

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Fig. 2.
Dose responses for DAMGO-stimulated
[35S]GTP S binding at hMOR WT,
N40D, R265H, and S268P mutant receptors. Increasing concentrations
of DAMGO (10 13 to
10 4 M) were used to stimulate
[35S]GTP S binding. Data are means ± S.E. from
three to seven independent [35S]GTP S binding
experiments performed in triplicate. DAMGO efficacy and potency from
curve-fitting of these data are shown in Table III.
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Table III
Efficacy and potency of DAMGO to stimulate of [35S]GTP S
binding at hMOR WT and mutant receptors
This table displays maximal activation levels and EC50 values
determined from experiment shown in Fig. 2. 100% is defined as
specific [35S]GTP S binding in the absence of DAMGO. Values
from these experiments are means ± S.E. from n
independent [35S]GTP S binding experiments performed in
triplicate.
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We examined whether the strong impairment of DAMGO-induced
[35S]GTP
S binding observed in the S268P mutant
receptor was also detectable for other agonists. We compared maximal
activation levels using DAMGO,
-endorphin, and morphine at
saturating concentrations (10
5
M). In this set of experiments [35S]GTP
S
binding was 172.3 ± 8.0, 141.7 ± 8.6, and 117.6 ± 4.6% of basal level for DAMGO (n = 12),
-endorphin
(n = 7), and morphine (n = 7),
respectively, in membranes expressing WT receptors. In membranes with
S268P receptors, agonists were ineffective in stimulating [35S]GTP
S binding. Maximal levels were 106.9 ± 5.6% of basal for DAMGO (n = 10), 109.0 ± 6.4%
for
-endorphin (n = 7), and 96.3 ± 5.0% for
morphine (n = 7, not shown). This suggests that the poor ability of S268P receptors to stimulate [35S]GTP
S
binding is not a ligand-dependent phenomenon but rather an
intrinsic property of the mutant receptor.
A final observation from this study is that basal
[35S]GTP
S binding levels in mutant receptor
preparations were comparable (N40D) or lower (R265H and S268P) from WT
(Table III), indicating that these mutations do not induce constitutive
activity at the receptor.
Receptor Signaling: Effect of the S268P Mutation on Agonist-induced
Inhibition of the cAMP Pathway--
We further characterized the
phenotypic deficiency of the S268P mutant. We verified whether
decreased signaling observed at the level of G proteins propagates and
translates into decreased cellular response downstream from the
signaling cascade. To this objective, we used a reporter gene
assay responsive to cAMP levels. In HEK 293 cells, we cotransfected the
receptor-encoding plasmid with another plasmid harvesting the alkaline
phosphatase gene under the control of a cAMP-responsive element
promoter. Cells were stimulated with forskolin in the absence or
presence of an opioid agonist, and levels of reporter gene activity
were compared. Dose-response curves to DAMGO,
-endorphin, and
morphine are shown in Fig. 3. In WT
receptors, all three agonists potently inhibited forskolin-induced
reporter gene activity. Maximal inhibitions were 66.2 ± 3.4, 66.7 ± 3.4, and 71.1 ± 2% for DAMGO,
-endorphin, and
morphine, respectively. This indicates similar efficacy for the three
agonists in decreasing cAMP levels in the cell and shows that the
partial agonist activity of
-endorphin and morphine in the
[35S]GTP
S binding assay is less perceptible when
measuring cAMP-dependent reporter gene activity, probably
due to signal amplification. The rank of order of potencies was DAMGO
(EC50 65.8 ± 0.7 nM) >
-endorphin (EC50 290.8 ± 121.6 nM) > morphine (EC50 623.8 ± 44.4 nM). For the S268P mutant, inhibition of forskolin-induced reporter gene activity was much less important than in WT and for all
three agonists (Fig. 3). Maximal inhibitions were only 30.5 ± 4.8% for DAMGO, 41.5 ± 6.5% for
-endorphin, and 43.9 ± 4.8% for morphine. Also, agonist potencies were significantly shifted
to the right (358.6 ± 24.4 nM for DAMGO, 1325 ± 478 nM for
-endorphin, and 1426 ± 209 nM for morphine). We otherwise verified that the WT and the
mutant receptors were expressed at comparable levels under the
experimental conditions of the assay (not shown). These results further
confirm that the S268P receptor is signaling-deficient and that this
property of the mutant receptor is agonist-independent. We show that
the impairment of receptor signaling is observable all along the cAMP
signaling cascade.

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Fig. 3.
Dose responses for opioid agonist activity at
hMOR WT and S268P mutant receptors using the reporter gene assay.
The receptor cDNA and the cAMP-responsive element-alkaline
phosphatase reporter gene were cotransfected in HEK 293 cells. Cells
were incubated with forskolin in the absence or presence of various
concentrations (10 9 to 10 5
M) of agonist. Reporter gene activity was measured by
chemiluminescence, and results were normalized to forskolin-stimulated
reporter gene activity in the absence of agonist (100%). Data are
means ± S.E. from 5 to 6 independent experiments performed in
triplicate. Agonist potency and efficacy values are indicated in the
text.
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Effect of the Mutations on Agonist-induced Regulation of Receptor
Activity--
Agonist stimulation of G protein-coupled receptors
triggers a number of regulatory processes that limit the cellular
response over time. These include short term events occurring at the
cytoplasmic face of the receptor, such as phosphorylation, uncoupling,
and internalization, followed by longer term regulatory processes that
ultimately translate into receptor down-regulation (33, 34). Two
polymorphic mutations (R265H and S268P) are located in the third
intracellular domain that interacts with intracellular factors, and
these mutations may therefore influence adaptive responses to receptor
activation. Furthermore, studies of polymorphic variants of the
2-adrenergic receptor have demonstrated alterations of
agonist-promoted down-regulation caused by mutations located in the
extracellular N-terminal domain of the receptor (35), indicating that
mutations in this domain can also influence the regulation of receptor
activity. We investigated the influence of opioid treatment on the four
hMOR variants, and we chose to examine receptor down-regulation as a
prominent and well described regulatory response to chronic opiates.
Also the long term response is likely to be most relevant in the
context of substance abuse, opioid substitution therapy, or chronic
pain treatment.
DAMGO was previously reported to be most efficient in promoting
receptor down-regulation in transfected Chinese hamster ovary cells
(36) and neuroblastoma (31) cells. In accordance, our stable HEK 293 cells stably expressing the WT receptor underwent 75% down-regulation
following 20 h of treatment (Table
IV), a time duration that allows complete
down-regulation (36). The number of remaining binding sites was
identical (25%), independently whether receptors were labeled using
the hydrophilic peptidic ligand [3H]DAMGO or the
hydrophobic alkaloid compound [3H]diprenorphine. This
suggests that the missing receptors have entered the lysosomic pathway
and undergone protein degradation, as previously reported (36-38).
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Table IV
DAMGO-induced down-regulation of hMOR WT and mutant receptors
Cells were treated with 10 µM DAMGO for 20 h.
Receptor sites were subsequently measured on whole cells using either
[3H]diprenorphine (1.5 nM) or [3H]DAMGO
(5 nM) in the absence (total binding) or presence
(nonspecific binding) of 2 µM naloxone. Data are
expressed as percent remaining receptor sites, defined as specific
binding in DAMGO-treated cells/specific binding in untreated cells × 100. Values from these experiments are means ± S.E. from at
least three independent DAMGO treatments and n independent
binding experiments performed in triplicate. Statistical analysis
comparing WT with each mutant receptors shows no significant difference
(p values >0.05).
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Significantly, mutant receptors did not down-regulate differently from
WT receptors (Table IV), indicating that no major modification of the
various processes that lead to receptor down-regulation has occurred. A
tendency to increased down-regulation was observed for the N40D
receptor, but the statistical analysis showed no difference from WT.
Identical results were obtained when binding sites were labeled with
the radiolabeled ligands at nonsaturating concentrations (not shown).
This suggests that interindividual variations in hMOR sequence may have
no significant consequences on long term responses to chronic opiates.
We cannot exclude, however, that some short term regulatory processes
could be modified in those mutants.
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DISCUSSION |
The detection of genetic variation affecting the opioid system is
an interesting approach to understand the origin of inter-individual differences in response to opiates, in opioid-associated
pathophysiology or in diseases of substance dependence. Vulnerability
to heroin abuse is thought to result from polygenic influences. It was
shown associated with a polymorphism in the D4 dopamine receptor gene (39) or with a mutation in the
-opioid receptor gene, in one of two
studies (40, 41). The µ-opioid receptor has been the focus of several
polymorphism studies because this receptor type is the major target for
opiate drugs and also is a main modulator of reward pathways in the
brain. A number of single nucleotide polymorphisms have now been
described for the µ-opioid receptor gene. Two mutations that modify
the N-terminal sequence of the encoded protein (A6V and N40D) have been
found repeatedly. N40D, the most frequent mutation, occurs at an
allelic frequency of 10-20% depending on the study (23, 24), whereas
the A6V mutation seems less frequent (22-24). Our recent large scale
sequence analysis of the µ-opioid receptor gene in 250 patients has
not only confirmed the occurrence of the A6V and N40D mutations but
also has led to the identification of novel polymorphic variants (25).
The present study shows that some of these natural mutations, which alter the protein sequence of the encoded receptor, may have functional consequences on receptor activity.
The N40D Mutant, Subtle Changes in Receptor Functionality--
The
N40D is the most frequent mutation, and several association studies
have been conducted. The analysis of alcohol dependence has led to
variable results, depending on the populations under study. Although no
association was found in two studies (23, 42), an increased risk factor
for the WT allele or genotype was described in another study (43),
suggesting a possible protective effect of the mutation. In a group of
alcoholics under acute withdrawal, increased dopaminergic sensitivity
was found for the variant genotype compared with the WT genotype (44).
Recently, a higher frequency of the mutant allele was reported in
idiopathic absence epilepsy patients (45), indicating that the N40D
µ-opioid receptor variant may not only contribute to the etiology of
alcoholism but may be implicated in neuronal excitability. A first step
toward understanding the molecular mechanisms underlying associations
between this mutation and complex behaviors is to determine whether the
change in protein sequence alters receptor function.
The N40D mutation is located in the N-terminal region of the receptor
and results in the loss of a putative glycosylation site. One possible
consequence is an alteration of the glycosylation status of the
receptor that could potentially modify receptor expression (46).
However, this was not observed; our data show that receptor densities
do not significantly differ from WT when the N40D receptor is
transiently expressed in COS cells. This is in accordance with previous
studies that have shown that the lack of N-terminal tail (hence the
lack of glycosylation sites) does not prevent expression of the
receptor in heterologous host cells (47-51).
Modifications of pharmacological properties were hardly detectable for
the N40D receptor. First, binding affinities of a large set of opioid
ligands were highly similar for N40D and WT receptors. Second, maximal
activation of the N40D mutant by the agonist DAMGO was slightly
enhanced, but no significant change in potency was observed. Third,
down-regulation tended to be more prominent for the N40D receptor, but
again the difference was not statistically different from WT.
Collectively, the data demonstrate that the N40D polymorphism does not
impair receptor activity. On the contrary, we may speculate that,
although our expression conditions may not have been optimal to reveal
marked modifications of N40D functionality, these subtle differences
altogether could contribute in enhancing N40D receptor responsivity
under specific circumstances. This would be in agreement with the
association studies mentioned earlier (24, 43). These authors have
suggested that a potentially enhanced N40D receptor function could
translate into a hyperactivity of the endogenous opioid system that
would contribute to increase the activity of the
hypothalamic-pituitary-adrenal axis and diminish vulnerability to
opioid dependence (24) or alcoholism (43).
N152D, Decreased Receptor Expression--
The N152D polymorphism
was not described previously and represents a rare mutation (2/250
individuals). In our study this mutant was expressed at lower receptor
density compared with wild-type receptor. Another mutation in the
transmembrane domain III of the
-opioid receptor was previously
shown to alter receptor expression (26), suggesting that structural
integrity of transmembrane domain III, believed to be the most buried
transmembrane domain within the helical bundle, is important for
receptor stability. Otherwise, no obvious affinity change was observed
for all the opioid ligands that we have tested. Therefore, together the
data suggest that the general conformation of the N152D receptor is maintained. Whether expression of this receptor variant is modified in vivo remains to be determined.
S268P, a Loss-of-Function Mutation--
In this paper we have
studied two mutations that occur in the third intracellular domain
(i3), a key region for GPR activity. The amphipathic structures of the
membrane-proximal regions of i3 have been shown to be critical for
productive interaction of GPRs with the
-subunit of G proteins and
therefore receptor signaling (see Ref. 52). This loop is highly
conserved across opioid receptor subtypes (53), is involved in their
coupling to Go/Gi subunits (54), and may
interact with other proteins, as was reported for calmodulin and the
µ-receptor (55). This receptor domain also harbors putative
phosphorylation sites that could be involved in the regulation of
receptor activity (34). The amino acid residue Ser-268 itself
represents a putative phosphorylation site for
Ca2+/calmodulin-dependent protein kinase II
(56). We therefore expected that mutations within this region would
alter receptor function.
The R265H mutation has some influence on hMOR activity, which leads to
decreased signaling. This modification, as well as changes in opioid
binding, remains minor presumably because of the conservative nature of
the mutation. Otherwise receptor down-regulation was unchanged. It is
therefore unlikely that the R265H polymorphism (rare: 1/250) would
drastically alter MOR activity, at least for receptor responses that we
have investigated.
In contrast, the S268P mutation strongly impairs receptor signaling,
independently from the agonist or the functional assays that are used.
Site-directed mutagenesis of this serine residue into alanine was
described previously in the rat µ-opioid receptor (56). The authors
found no impairment of agonist-induced inhibition of adenylyl cyclase
when a double mutant receptor (S261A/S266A) was expressed in HEK cells,
as well as no impairment of agonist-evoked increase in inward
K+ currents using the Xenopus oocyte expression
system. Very recently, the same authors investigated the human S268P
mutant and reported a strong impairment of signaling (57), as we do in
this study. A likely explanation for the distinct consequences of the
alanine or proline mutations is that the alanine mutation leaves the
general structure of the loop intact, and therefore functional coupling to the G protein is maintained. On the contrary, the proline residue of
the polymorphic mutant disrupts the tertiary structure of this functionally critical loop, thus severely compromising signal transduction. Down-regulation of hMOR S268P was otherwise unaltered, suggesting that the absence of this putative phosphorylation site has
no obvious consequences on long term agonist-induced regulation. We
cannot exclude, however, that there may be an impairment of rapid
agonist-induced desensitization, as was shown for the S261A/S266A mutant in the rat (56) or recently suggested for the human S268P mutant
(57).
Many polymorphic variants have been identified among GPR genes, and
some of these mutations account for inherited disorders and diseases
(for a review see Ref. 58). Most mutations are loss-of-function
mutations, and here we provide an additional example for such a
mutation (S268P). Like the R265H mutation described here, the S268P
mutation was found in one individual only (1/250). The individual is
heterozygous for the mutation, suggesting that he may produce an intact
version of the receptor from the WT allele. Although rare and
monoallelic, this polymorphic mutation is of considerable interest
because the consequence of amino acid replacement strongly impairs
receptor signaling. Consequently, the level of fully functional
receptors in this individual is most probably close to half of that
from most individuals, as observed in heterozygous MOR-deficient mice
with one allele inactivated (6). Therefore, future studies will aim at
enlarging the DNA sampling to find more examples of the S268P
polymorphism. In a preliminary study, we have investigated the possible
presence of the mutation in other members of the patient's family, and
DNA sequencing has revealed the existence of the same S268P mutation in
one of the siblings (one allele also, not shown). An expanded study of
this particular mutation should indicate whether individuals homozygous for the mutation do exist. Finally, the clinical examination of patients expressing the S268P polymorphic receptor from one or the two
alleles may provide interesting insights into the functional consequences of decreased µ-opioid receptor function in humans.
Aknowledgements--
We thank J. L. Mandel and W. Berrettini
for helpful comments. We are grateful to F. Pattus and P. Chambon for
continuing encouragement. We also thank Valérie Favier for
participation in the work and Lei Yu for the hMOR cDNA. We are
grateful to Philippe Walker, Manon Valiquette, and Thierry Groblewski
(AstraZeneca, Montreal, Canada) for their contributions.