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Volume 272, Number 46, Issue of November 14, 1997
pp. 28869-28874
(Received for publication, July 24, 1997)
From the ABSTRACT µ opioid receptors are subject to
phosphorylation and desensitization through actions of at least two
distinct biochemical pathways: agonist-dependent µ receptor phosphorylation and desensitization induced by a biochemically
distinct second pathway dependent on protein kinase C activation (1).
To better understand the nature of the agonist-induced µ receptor
phosphorylation events, we have investigated the effects of a variety
of opiate ligands of varying potencies and intrinsic activities on µ receptor phosphorylation and desensitization. Exposure to the potent
full agonists sufentanil, dihydroetorphine, etorphine, etonitazine, and
[D-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) led to
strong receptor phosphorylation, while methadone,
l- INTRODUCTION Opioid receptors are G-protein coupled receptors that mediate the
potent analgesic actions and addictive properties of morphine-derived compounds. Under physiological conditions, these receptors interact with endogenous opioid peptides to modulate pain-controlling pathways and circuits that modulate behaviors including mood and reward (2). µ opioid receptors interact with rapidly acting opioid drugs, such as
heroin, to produce marked euphoria and behavioral reward. They are also
primary targets of the slower and longer acting opioids, such as
methadone and LAAM,1 that
represent the best current substitution therapeutics for opiate
addiction (3).
µ receptors desensitize after repeated stimulation by opioid
agonists, in fashions that display similarities to desensitizing events
noted for other G-protein coupled receptors. Agonist- induced µ receptor desensitization can be correlated with receptor
phosphorylation. µ receptors display naloxone-reversible
phosphorylation and desensitize after morphine or DAMGO treatments (1).
Both of these agonist-induced events are insensitive to pretreatments
with the protein kinase C inhibitor staurosporine, which inhibits
phorbol ester-induced µ receptor phosphorylation and
desensitization.
Many opiates and opioid ligands can recognize µ receptors with high
affinities. Plant-derived alkaloids, synthetic compounds of several
classes, and endogenous opioid peptides can function as agonists,
partial agonists, or antagonists with a range of abilities to induce or
block induction of analgesia and euphoria. However, mutagenesis studies
and studies with receptor chimeras support the idea that different
receptor features could be involved in recognition of these different
ligand classes (4-6). Mutations that change naloxone from a full
antagonist to a partial agonist can leave the intrinsic activity of
opioid peptides unchanged, for example see Claude et al.
(7). These differences raise the possibility that µ receptor
occupancies by opioid drugs of different classes could alter the
conformation of the µ receptor in distinct fashions. Some of these
differences could render the receptor an improved or a worse substrate
for kinases and phosphatases and thus directly confer different
susceptibilities to phosphorylation or dephosphorylation.
Alternatively, selective activation of different G-protein classes by
different µ agonists could trigger different µ receptor
phosphorylation and desensitization events.
The current study thus investigates the effects of opioid ligands of
various classes and varying intrinsic activities on µ receptor
activation, phosphorylation, and desensitization using human µ receptors expressed in CHO cells (hµCHO), and the receptors coexpressed in Xenopus oocytes with a G-protein linked
K+ channel. The results support striking parallels between
opioid efficacies in opening ion channels and inhibiting adenylyl
cyclase activity and their efficacies in agonist-induced µ receptor
phosphorylation and desensitization. The data aso reveal that methadone
and LAAM provide phosphorylation and desensitization disproportionate
to their efficacies in mediating µ receptor-mediated ion channel activation or adenylyl cyclase inhibition, differences that could conceivably contribute to their efficacies as principal current agonist-substitution antiaddiction therapeutics.
MATERIALS AND METHODS Opioid peptides and naloxone were purchased from
Research Biochemicals Inc. (Natick, MA); morphine and buprenorphine
were purchased from Mallinckrodt Chemical Co (St. Louis, MO); and
dihydroetorphine was a gift from Dr. Xiongqi Gong (China). Other opioid
ligands were kindly provided by Dr. Richard Rothman and Dr. Heng Xu,
(National Institute on Drug Abuse-IRP, Baltimore). All other chemicals
and reagents were purchased from Sigma or as indicated in methods specifically.
Phosphorylation of the µ opioid receptor in hµCHO (8) was described as (1). Briefly,
hµCHO and non-transfected Chinese hamster ovary (CHO) cells were
plated at 80% confluence in 6-well plates and grown for 24 h in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were
incubated at 37 °C for 2 h with 300 µCi/ml of
[32P]orthophosphate (8500 Ci/mmol; NEN Life Science
Products) in phosphate-free Dulbecco's modified Eagle's medium.
Labeled cells were then exposed to various opioid ligands at 1 µM for 20 min or to other treatment times and
concentrations as indicated in the figure legends. Ligands and free
32P were removed from cells by washing with ice-cold
phosphate-buffered saline; subsequent procedures were carried out at
4 °C. Cells were solubilized for 60 min with 0.8 ml of
RIPA+ buffer (1% IGEPAL CA-630, 0.5% Na2
deoxycholate, 0.1% SDS, 5 mM EDTA, 10 mM NaF,
10 mM Na2 pyrophosphate, 1 µM
okadaic acid, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine, 10 µg/ml leupeptin, and 1 µg/ml pepstatin A in
phosphate-buffered saline buffer). Supernatant from a 15 min,
150,000 × g centrifugation was preadsorbed by
incubation with 100 µl of presoaked protein A-Sepharose beads
(Pharmacia Biotech Inc.), followed by microcentrifugation. A sample of
this supernatant was assayed for protein concentration by the Bradford
method (Bio-Rad). For immunoprecipitation, 700 µl of the supernatant
was incubated for 2 h with 100 µl of the protein A-Sepharose
bead slurry and a 1:500 final dilution of an antiserum directed against
the C-terminal 18 amino acids of the µ opioid receptor. Beads were
washed three times by resuspension with 1 ml of RIPA+
followed by microcentrifugation, and immunoprecipitated proteins then
dissociated from beads by extraction with 60 µl of SDS-PAGE gel
loading buffer (4% SDS, 25 mM Tris-HCl, pH 6.8, 5%
glycerol, 0.5% 2-mercaptoethanol, and 0.005% bromphenol blue). 20 µl of the immunoprecipitated proteins were separated on 8% SDS-PAGE gels with prestained molecular mass standards (Amersham), and radiolabeled proteins were identified by autoradiography using Hyperfilm-MP (Amersham) with intensifying screens. Autoradiographic densities of bands of interest were quantified by scanning densitometry and normalized to the amounts of extracted cell protein subjected to
immunoprecipitation.
cDNAs encoding the human
opioid µ receptor (8) and GIRK1 (9) were subcloned into the
expression vector pcDNAI, plasmids linearized with XbaI,
and capped mRNA-sense RNAs prepared by in vitro
transcription using T7 polymerase (mMACHIEN kit; Ambion). RNA quality and sizes were assessed after separations using 1.2% formaldehyde agarose gels, and full-length RNAs were stored in 75%
ethanol at Whole cell currents in
expressing oocytes were measured at 22 °C under 2-electrode voltage
clamped at hµCHO cells were cultured as described above, harvested,
washed with 2.0 mM Tris·HCl, pH 7.4, 2.0 mM
EDTA buffer, and suspended in the same Tris buffer. Cell suspension
corresponding to 30 µg of protein/sample was added on ice to assay
tubes containing 10 µM forskolin, assay buffer (80 mM Tris, pH 7.4, 10 mM theophylline, 1 mM MgSO4, 0.8 mM EGTA, 30 mM NaCl, 0.25 mM ATP, 0.01 mM GTP), and tested drugs. Triplicate samples for each treatment were incubated at 37 °C for 10 min, adenylyl cyclase activity was terminated by
boiling for 2 min, and the amounts of cAMP formed were determined by a
cAMP protein binding assay as described (10, 11). Briefly, 4 nM [3H]cAMP (Amersham, 39 Ci/mmol), and
bovine adrenal binding protein preparations were incubated with samples
at 4 °C for 90 min. Assays were terminated by adding charcoal and
centrifuging, and supernatant radioactivity containing bound cAMP was
assessed by liquid scintillation counting. Dose-response curves were
obtained by nonlinear regression analyses using PRISM (GraphPad
Software, Inc. San Diego, CA).
RESULTS 22 compounds, representing each of the major opiate and opioid
classes, produced substantially different effects on µ receptor phosphorylation when tested in receptor phosphorylation assays at
screening dose concentrations (1 µM) that were
significantly above their previously reported 0.1-10 nM
Ki values (Table I)
(12). When drugs were incubated with hµCHO cells at this concentration for 20 min, dihydroetorphine, sufentanil, etorphine, etonitazine, and DAMGO caused increased µ receptor phosphorylation producing values 6-8-fold above basal levels. Methadone, morphine, meperidine, DADL, Table I.
Inhibition of forskolin-stimulated adenylyl cyclase activity in
hµCHO cells
[View Larger Version of this Image (60K GIF file)]
[View Larger Version of this Image (43K GIF file)]
[View Larger Version of this Image (41K GIF file)]
Since these results indicated that different opioid ligands could
display differential influence on µ receptor phosphorylation, we
selected eight representative opioid ligands for further study. Potencies and efficacies of etorphine, sufentanil, DAMGO, methadone, LAAM, morphine, buprenorphine, and
3-m-hydroxy-5-phenylmorphan were assessed in opening a
G-linked K+ channel coexpressed with the µ receptor in
Xenopus oocytes and in inhibiting adenylyl cyclase in
hµCHO cells. These studies provided assessments of the efficacies of
these compounds, with rank order etorphine > sufentanil > DAMGO > morphine > methadone > LAAM > 3-m-hydroxy-5-phenylmorphan > buprenorphine (Fig.
4, Table I). The profound desensitization
exerted by sufantanil and etorphine did not allow us to accurately
assess their potencies in adenyl cyclase inhibition.
[View Larger Version of this Image (29K GIF file)]
Each of these opioid ligands also exerts a
concentration-dependent effect on receptor phosphorylation
(Fig. 5). Significant agonist-induced µ receptor phosphorylation enhancements were observed at concentrations
as low as 10 pM for etorphine, 100 pM for
sufentanil, more than 10 nM for DAMGO, 100 nM
for methadone and LAAM and more than 100 nM for morphine.
Concentrations of buprenorphine yielding robust receptor
phosphorylation were 100,000 times higher than effective concentrations
of etorphine, whereas even 100 µM phenylmorphan treatments barely altered µ receptor phosphorylation.
[View Larger Version of this Image (33K GIF file)]
Desensitization, examined in both the Xenopus oocyte system
and hµCHO (Fig. 6 and
7), was mediated by etorphine > sufentanil > DAMGO > methadone
[View Larger Version of this Image (35K GIF file)]
[View Larger Version of this Image (28K GIF file)]
DISCUSSION The current findings substantially expand data documenting
relationships among the effects of different opioid ligands on µ opioid receptor activation, phosphorylation, and desensitization (1,
13, 14). The parallels between ligand efficacy and desensitization in
the current data are consistent with the relationships between the
properties of these opioid ligands in neurally derived cell lines that
endogenously express Studies from several laboratories, but not all, have demonstrated
desensitization observed in agonist-treated µ receptor/GIRK coexpressing Xenopus oocytes by treatments that alter levels
of kinases including calcium/calmodulin-dependent protein
kinase and protein kinase C (1, 16, 17). Only one report (1) separated
a kinase-dependent pathway from an agonist-induced pathway. Kovoor et al. (18), however, interpreted data from a similar system to suggest that desensitization may not involve direct receptor
phosphorylation. Although the results from the current study do not
document direct evidence for µ receptor phosphorylation in
Xenopus oocytes, the similar patterns of opioid ligand
efficacies observed in the Xenopus oocyte and the hµCHO
systems are totally consistent with the idea that the mechanisms of
receptor desensitization found in these two distinct test systems are
likely to be similar. Studies of phosphorylation and desensitization
patterns in receptor mutants with systematic alanine substitutions for
individual candidate phosphoacceptor sites will help to add evidence to
the increasingly strong current hypothesis that µ receptor
phosphorylation is targeted by agonist activation and leads to
desensitization.
The parallels between higher ligand efficacy and enhanced receptor
phosphorylation that suggest that an activated receptor conformation
may enhance µ receptor phosphorylation have also been described for
Studies of ligand interactions with mutant µ receptors suggest that
different ligand classes recognize amino acids lying in different µ receptor domains. Receptor occupancies by opioid drugs of different
classes might thus alter µ receptor conformations in distinct
fashions. The ligand-to-ligand differences in µ receptor phosphorylation observed in the present studies could reflect ligand-to-ligand differences in receptor/ligand conformations that
could improve, or worsen its ability to serve as a substrate for
activated kinases or phosphatases. If methadone and LAAM occupancies produced a µ receptor conformation that resulted in better access to
kinases without enhanced efficacy in G-protein activation, for example,
its observed differences from morphine could be explained. Distinct
ligand/µ receptor conformations could also differentially activate
different G-proteins. More prominent activation of selected G-proteins
by µ receptors occupied by methadone or LAAM could also conceivably
yield more strongly enhanced efficacies in activating the kinases that
lead to receptor phosphorylation than those that activate ion channels
or inhibit adenylyl cyclase, providing another explanation for the
observed methadone/morphine differences described here. Further
experiments that test effects of µ receptor mutants and chimeras on
methadone, LAAM, and morphine affinities and efficacies, as well as
association and dissociation rates for receptor binding, may provide
further evidence to support the relative contributions of each of these
mechanisms.
Desensitization could represent the inability of µ receptors still
expressed on cell surfaces to activate appropriate G-proteins when they
bind agonist ligands. Removal of µ receptors from cell surfaces to
sites where they can no longer transduce ligand recognition to
G-protein activation could also contribute to the observed patterns of
desensitization. There is some correlation between desensitization
results and the loss of membrane receptors in some expressing cell
systems (22). Ligand-dependent opioid receptor internalization has been reported (23). µ receptor internalization in
CHO cells can be mediated by opiate agonists in the rank order etorphine > DAMGO > methadone in initial
studies.2 However, morphine treatments provoke
no internalization at concentrations up to 10 µM.2 The time courses for agonist-induced µ receptor phosphorylation are also faster than the reported losses of
membrane receptors (1, 22). Receptor dephosphorylation and
resensization both take place within minutes of agonist removal (Fig.
2). Each of these lines of evidence suggests significant differences
between the internalization results and the results obtained for
phosphorylation and for desensitization. It thus seems unlikely that
internalization contributes to all of the desensitizing effect observed
in the current studies. Further studies, including those using
phosphoacceptor site mutants, may well document which contributions
internalization may make to agonist-induced desensization and the role
that phosphorylation may play in each event.
G-protein receptor kinases (GRKs) readily phosphorylate the
ligand-activated forms of several G-protein coupled receptors but can
find unoccupied or antagonist-occupied receptors poor substrates (24).
Desensitization of µ receptors, observed in systems including CHO
cells, Xenopus oocytes, SHSY5Y cells, and locus coeruleus
neurons (1, 25-28), appears unlikely to require a kinase of limited
distribution. It is conceivable that a widely distributed member of the
G-linked receptor kinase family, or another broadly-distributed kinase,
could be responsible for agonist-induced µ receptor phosphorylation.
Regulation of dephosphorylation could also play a significant role in
the accumulation of phosphorylated receptor species.
The pharmacological profile of µ opioid receptor efficacy,
phosphorylation, and desensitization that these data define documents that phosphorylation and desensitization are widespread consequences of µ opioid receptor occupancy by agonists that include opioid peptides.
Responses to both exogenous opioids and endogenous opiates are thus
both likely to desensitize through broadly distributed biochemical
mechanisms. Conceivably, these data will also help to explain the
especial therapeutic efficacies of methadone and LAAM through enhanced
abilities to phosphorylate and desensitize the µ opioid receptors
that contribute so substantially to opioid-mediated reward.
FOOTNOTES REFERENCES
µ Opioid Receptor Phosphorylation, Desensitization, and
Ligand Efficacy*
,,,
Department of Pharmaceutical Sciences,
School of Pharmacy, University of Maryland at Baltimore, Baltimore,
Maryland 21201, the § Laboratory of Molecular and Cellular
Neurobiology, National Institute on Alcohol Abuse and Alcoholism,
Bethesda, Maryland 20892-8205, and the ¶ Molecular Neurobiology
Branch, National Institute on Drug Abuse, and Departments of
Neurology and Neuroscience, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21224
-acetylmethadone (LAAM), morphine, meperidine, DADL,
-endorphin(1-31), enkephalins, and dynorphin
A(1-17) produced intermediate effects. The partial agonist
buprenorphine minimally enhanced receptor phosphorylation while
antagonists failed to alter phosphorylation. Buprenorphine and full
antagonists each antagonized the enhanced µ receptor phosphorylation
induced by morphine or DAMGO. The rank order of opiate ligand
efficacies in producing µ receptor-mediated functional
desensitization generally paralleled their rank order of efficacies in
producing receptor phosphorylation. Interestingly, the desensitization
and phosphorylation mediated by methadone and LAAM were
disproportionate to their efficacies in two distinct test systems. This
generally good fit between the efficacies of opiates in µ receptor
activation, phosphorylation, and desensitization supports the idea that
activated receptor/agonist/G-protein complexes and/or receptor
conformational changes induced by agonists are required for
agonist-induced µ receptor phosphorylation. Data for methadone and
LAAM suggest possible contribution from their enhanced desensitizing
abilities to their therapeutic efficacies.
70 °C. Oocytes were isolated from mature female Xenopus laevis (Xenopus I, Ann Arbor, MI),
defolliculated by treatment with 0.2% collagenase A, injected with
16-20 ng of RNAs encoding the µ opioid receptor and GIRK1 in molar
ratios of 3:1, and incubated for 2-3 days at 19-20 °C in ND96
solution (96 mM NaCl, 2 mM KCl, 2.5 mM CaCl2, 1.0 mM MgSO4,
and 5 mM HEPES, pH 7.5) supplemented with 2 mM
sodium pyruvate, 10,000 units/liter penicillin, 10 mg/liter streptomycin, and 0.5 mM theophylline.
70 mV, using a GeneClamp 500 amplifier (Axon Instrument).
Oocytes were placed on a nylon mesh in a 90-µl bath chamber and
continuously superfused at 6 ml/min with either ND96 or "hK" medium
(ND96 medium with 96 mM KCl and 2 mM NaCl).
Oocytes were superfused with ND96 between applications of hK solution
alone, hK solution during which opioid agonists were transiently
applied with or without opioid antagonists, or ND96 containing phorbol
esters (Sigma) in dimethyl sulfoxide (Me2SO) concentrations
less than 0.01%. Values presented are mean ± standard error
(S.E.). Concentration-response curves were obtained using the program
NFIT by fitting data to the logistic equation, y = {(Emax Emin)/(1 + [x/EC50]
n)} + Emin, where × represents concentration, y
represents response, Emax represents the maximal
response, Emin represents the minimal response,
EC50 represents the half-maximal concentration, and n represents the apparent Hill coefficient.
-endorphin(1-31), Met-enkephalin,
Leu-enkephalin, and dynorphin A(1-17) produced
intermediate effects, ~2-5 fold above basal levels. Neither the
partial agonist buprenorphine, the largely 
agonists ketocyclazocine
and -neoendorphin, the mixed agonist/antagonist pentazocine, the
mixed /µ agonist butorphanol, the weak µ agonists meperidine and
3-m-hydroxy-5-phenylmorphan, or the µ antagonists
naloxaonazine and LY255582 had any robust effect on µ receptor
phosphorylation. Each stimulated phosphorylation levels less than twice
basal levels (Fig.
1A-D).
Phosphorylation was reversible. Addition of naloxone to cells incubated
with morphine reduced µ receptor phosphorylation levels to close to
basal values by 30 min (Fig. 2). The
partial agonist properties of buprenorphine extended to its effects on µ receptor phosphorylation. 1 µM buprenorphine blocked µ receptor phosphorylation when coincubated with 1 µM concentrations of the fuller µ agonists morphine or DAMGO (Fig. 3).
Drug
Maximum
inhibition
EC50
nM
DAMGO
88%
20.5 ± 4.9
Morphine
75%
5.3 ± 0.14
Methadone
75%
16.0
± 1.7
LAAM
44%
10.3 ± 4.6
Buprenorphine
42%
0.02 ± 0.01
Fig. 1.
A-D, effects of opioid ligands on µ receptor phosphorylation. hµCHO cells were incubated for 20 min
at 37 °C with 1 µM concentrations of the indicated
opioid ligands during [32P]i metabolic
labeling. Proteins were extracted from the cells, immunoprecipitated
with anti-µ receptor antibodies, separated on SDS-PAGE, and
phosphoproteins identified after 2-day autoradiographic exposure.
Protein size standards derived from prestained markers electrophoresed
in adjacent lanes. The autoradiography depicted displays a result
representative of three independent experiments. Densitometric readings
rise from 606 arbitrary units for basal phosphor µ receptor to 4982 for phosphor µ receptor found following dihydroetorphine
treatment.
Fig. 2.
Naloxone effects on µ receptor
phosphorylation. hµCHO cells were preincubated without
(lane 1) or with 1 µM morphine (lanes
2-6) for 10 min at 37 °C. Naloxone was then added, and the
[32P]i labeling reaction was terminated 2, 5, 10, or 30 min later (lanes 3-6). The autoradiography shows
results representative of two independent experiments.
Fig. 3.
Antagonist effects of buprenorphine on µ receptor phosphorylation induced by morphine and DAMGO. Drugs were
added to cultures as indicated, and experiments were carried out as
noted above. The autoradiography shows results representative of two independent experiments.
Fig. 4.
Concentration-response relationships for
opioid ligand-activated K+ currents in Xenopus
oocytes coexpressing µ opioid receptors and G-protein activated
inward rectifier potassium channels (GIRK1). Coexpressing oocytes
had membrane potentials clamped at 70 mV, drugs were applied, and the
amplitude of each current response amplitude was determined as the
difference between the amplitude of peak current activated by that
opioid ligand in HK and the amplitude of current activated by the HK
vehicle alone. Opioid activated currents are shown as responses
normalized to that of 10 µM morphine. Each data point
represents mean ± S.E. of the recordings from 5-7 oocytes. The
curves shown were fitted to the data using the logistic equation
described in the methods. Some error bars not visible since they are
smaller than the symbol sizes. The order of maximal response obtained
are: etorphine (241 ± 10%, n = 5) > sufentanil
(216 ± 14%, n = 7) > AMGO (157 ± 14%, n = 7) > morphine (100 ± 7%, n = 7) > methodone (80 ± 13%, n = 5) > LAAM
(72 ± 3%) > phenylmorphan (35 ± 3%, n = 6) > buprenorphine (13 ± 2%, n = 7).
Fig. 5.
Concentration-dependent effects
of opioid ligands on µ-receptor phosphorylation. Drugs were
added to expressing cells as noted, and µ receptor phosphorylation
assessed as noted above. The autoradiography shows results
representative of three independent experiments.
LAAM > morphine > buprenorphine (in hµCHO) or
3-m-hydroxy-5-phenylmorphan (in Xenopus oocyte).
The rank order of the desensitizing abilities of these opioid ligands
was thus similar to their rank order for efficacies, with two
exceptions. Methadone and LAAM each led to more dramatic receptor
desensitization than morphine, despite efficacies and potencies in
producing µ receptor-mediated K+ channel opening and
adenylyl cyclase inhibition that were lower than those of morphine.
Interestingly, both LAAM and methadone treatments led to levels of µ receptor phosphorylation greater than those caused by morphine (Fig. 5;
especially compare responses at 1 µM). This response
appeared to represent a general property of µ receptor interactions
with these drugs, and not to depend just on one test system, since
morphine displayed equal efficacy to methadone and greater efficacy
than LAAM in inhibiting adenylyl cyclase from the same hµCHO cell
system in which the µ receptor phosphorylation results were obtained
(Table I).
Fig. 6.
Opioid ligand-induced desensitization of µ opioid receptor mediated responses in Xenopus oocytes.
Oocytes were preincubated for 20 min with indicated drugs in ND96 and
then tested for µ receptor-mediated K+ channel responses
following application of the same ligands. A, records of
currents activated by opioid ligand before and after 20-min agonist
preincubations. Open bar, period of HK solution superfusion;
solid bar, period of opioid ligand application in HK.
B, average decreases in opioid receptor-mediated
K+ channel responses as compared with no preincubation
controls. Each data point represents mean ± S.E. of the
recordings from 5-7 oocytes. Decreases in current amplitude induced
each opioid ligand after preincubation are: etorphine, 79 ± 6%;
sulfentanil, 73 ± 6%; LAAM, 52 ± 4%; DAMGO, 50 ± 7%; methadone, 49 ± 4%; morphine, 19 ± 9%, and
phenylmorphan, 0% of control currents.
Fig. 7.
Opioid ligand-induced desensitization of µ opioid receptor mediated responses in hµCHO cells. hµCHO cells
were preincubated for 20 min with indicated drugs in culture medium;
each drug concentration was 100 times of its EC50 (Table I)
and then was tested for µ receptor mediated adenylyl cyclase
inhibition as described under "Materils and Methods,"
with application of the ligands at the same concentration. Percentile changes in opioid receptor mediated cAMP production as compared with no
preincubation controls are shown. Each data point represents mean ± S.E. of two independent experiments, each performed in triplicate.
opioid receptors (15). Correlations between
the efficacies of these opioid ligands in receptor activation and their
abilities to cause µ receptor phosphorylation, relationships between
receptor desensitization and phosphorylation, and parallels between
time course and dose-response relationships for µ receptor
phosphorylation and desensitization (1) each support the possibility
that causal relationships could exist among agonist efficacy in ion
channel opening, adenylyl cyclase inhibition, receptor phosphorylation
and desensitization.
2 adrenergic receptors (19). The data from methadone and LAAM, on
the other hand, provide highly interesting exceptions to this general
rule. Methadone and LAAM represent some of the current most effective
anti-addiction substitution pharmacotherapeutics (3, 20). Although
their long-acting human pharmacologies have been considered important
for their therapeutic efficacies (21), detailed studies of these
mechanisms of action have not been carried out. More prominent receptor
phosphorylation and desensitization could enhance the abilities of both
methadone and LAAM to block effects of subsequent doses of addictive
abused opiates, possibly contributing to their pharmacological blockade properties and therapeutic efficacies.
To whom correspondence should be addressed: Dept. of
Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, Baltimore, MD 21201. Tel.: 410-706-6868; Fax:
410-706-2973.
1
The abbreviations used are: LAAM,
l--acetylmethadone; DAMGO, [D-Ala2, MePhe4,
Glyol5]enkephalin; CHO, Chinese hamster ovary cells; hµCHO, CHO
cells stably expressing human µ receptor; PAGE, polyacrylamide gel
electrophoresis.
2
C. Evans, personal communication.
Volume 272, Number 46,
Issue of November 14, 1997
pp. 28869-28874
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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E. A. Johnson, S. Oldfield, E. Braksator, A. Gonzalez-Cuello, D. Couch, K. J. Hall, S. J. Mundell, C. P. Bailey, E. Kelly, and G. Henderson Agonist-Selective Mechanisms of {micro}-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells Mol. Pharmacol., August 1, 2006; 70(2): 676 - 685. [Abstract] [Full Text] [PDF]
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 J. R. Ross, J. Riley, C. Quigley, and K. I. Welsh Clinical Pharmacology and Pharmacotherapy of Opioid Switching in Cancer Patients Oncologist, July 1, 2006; 11(7): 765 - 773. [Abstract] [Full Text] [PDF]
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M. R. Dowling, J. M. Willets, D. C. Budd, S. J. Charlton, S. R. Nahorski, and R. A. J. Challiss A Single Point Mutation (N514Y) in the Human M3 Muscarinic Acetylcholine Receptor Reveals Differences in the Properties of Antagonists: Evidence for Differential Inverse Agonism J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1134 - 1142. [Abstract] [Full Text] [PDF]
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H. Haberstock-Debic, K.-A. Kim, Y. J. Yu, and M. von Zastrow Morphine Promotes Rapid, Arrestin-Dependent Endocytosis of {micro}-Opioid Receptors in Striatal Neurons J. Neurosci., August 24, 2005; 25(34): 7847 - 7857. [Abstract] [Full Text] [PDF]
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S. Maudsley, B. Martin, and L. M. Luttrell The Origins of Diversity and Specificity in G Protein-Coupled Receptor Signaling J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 485 - 494. [Abstract] [Full Text] [PDF]
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 M. A. Simmons Functional Selectivity, Ligand-Directed Trafficking, Conformation-Specific Agonism: What's In A Name? Mol. Interv., June 1, 2005; 5(3): 154 - 157. [Abstract] [Full Text] [PDF]
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L. Thielemans, I. Depoortere, J. Perret, P. Robberecht, Y. Liu, T. Thijs, C. Carreras, E. Burgeon, and T. L. Peeters Desensitization of the Human Motilin Receptor by Motilides J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1397 - 1405. [Abstract] [Full Text] [PDF]
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W. Guang, H. Wang, T. Su, I. B. Weinstein, and J. B. Wang Role of mPKCI, a Novel {micro}-Opioid Receptor Interactive Protein, in Receptor Desensitization, Phosphorylation, and Morphine-Induced Analgesia Mol. Pharmacol., November 1, 2004; 66(5): 1285 - 1292. [Abstract] [Full Text] [PDF]
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J. Celver, M. Xu, W. Jin, J. Lowe, and C. Chavkin Distinct Domains of the {micro}-Opioid Receptor Control Uncoupling and Internalization Mol. Pharmacol., March 1, 2004; 65(3): 528 - 537. [Abstract] [Full Text] [PDF]
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C. P. Bailey, D. Couch, E. Johnson, K. Griffiths, E. Kelly, and G. Henderson {micro}-Opioid Receptor Desensitization in Mature Rat Neurons: Lack of Interaction between DAMGO and Morphine J. Neurosci., November 19, 2003; 23(33): 10515 - 10520. [Abstract] [Full Text] [PDF]
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Y. Qiu, P.-Y. Law, and H. H. Loh {micro}-Opioid Receptor Desensitization: ROLE OF RECEPTOR PHOSPHORYLATION, INTERNALIZATION, AND RESENSITIZATION J. Biol. Chem., September 19, 2003; 278(38): 36733 - 36739. [Abstract] [Full Text] [PDF]
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A. C. Barrett, E. S. Smith, and M. J. Picker Use of Irreversible Antagonists to Determine the Relative Efficacy of {micro}-Opioids in a Pigeon Drug Discrimination Procedure: Comparison of {beta}-Funaltrexamine and Clocinnamox J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1061 - 1070. [Abstract] [Full Text] [PDF]
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S. L. Borgland, M. Connor, P. B. Osborne, J. B. Furness, and M. J. Christie Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors J. Biol. Chem., May 23, 2003; 278(21): 18776 - 18784. [Abstract] [Full Text] [PDF]
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B. D. Stout, W. P. Clarke, and K. A. Berg Rapid Desensitization of the Serotonin2C Receptor System: Effector Pathway and Agonist Dependence J. Pharmacol. Exp. Ther., September 1, 2002; 302(3): 957 - 962. [Abstract] [Full Text] [PDF]
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 A. Christopoulos and T. Kenakin G Protein-Coupled Receptor Allosterism and Complexing Pharmacol. Rev., June 1, 2002; 54(2): 323 - 374. [Abstract] [Full Text] [PDF]
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M. Pfeiffer, T. Koch, H. Schroder, M. Laugsch, V. Hollt, and S. Schulz Heterodimerization of Somatostatin and Opioid Receptors Cross-modulates Phosphorylation, Internalization, and Desensitization J. Biol. Chem., May 24, 2002; 277(22): 19762 - 19772. [Abstract] [Full Text] [PDF]
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 H. H. Szeto, Y. Soong, D. Wu, and J. Fasolo Resensitization of Blood Pressure Response to {micro}-Opioid Peptide Agonists After Acute Desensitization Anesth. Analg., September 1, 2001; 93(3): 581 - 586. [Abstract] [Full Text] [PDF]
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C. Chavkin, J. P. McLaughlin, and J. P. Celver Regulation of Opioid Receptor Function by Chronic Agonist Exposure: Constitutive Activity and Desensitization Mol. Pharmacol., July 1, 2001; 60(1): 20 - 25. [Full Text]
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J.-G. Liu and P. L. Prather Chronic Exposure to {micro}-Opioid Agonists Produces Constitutive Activation of {micro}-Opioid Receptors in Direct Proportion to the Efficacy of the Agonist Used for Pretreatment Mol. Pharmacol., July 1, 2001; 60(1): 53 - 62. [Abstract] [Full Text]
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A. Mehta, G. Bot, T. Reisine, and M.-F. Chesselet Endomorphin-1: Induction of Motor Behavior and Lack of Receptor Desensitization J. Neurosci., June 15, 2001; 21(12): 4436 - 4442. [Abstract] [Full Text] [PDF]
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C. Watson, G. Chen, P. Irving, J. Way, W.-J. Chen, and T. Kenakin The Use of Stimulus-Biased Assay Systems to Detect Agonist-Specific Receptor Active States: Implications for the Trafficking of Receptor Stimulus by Agonists Mol. Pharmacol., April 13, 2001; 58(6): 1230 - 1238. [Abstract] [Full Text]
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P. Huang, G. B. Kehner, A. Cowan, and L.-Y. Liu-Chen Comparison of Pharmacological Activities of Buprenorphine and Norbuprenorphine: Norbuprenorphine Is a Potent Opioid Agonist J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 688 - 695. [Abstract] [Full Text]
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M. Waelbroeck Activation of Guanosine 5'-[{gamma}-35S]thio-triphosphate Binding through M1 Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 2. Testing the "Two-States" Model of Receptor Activation Mol. Pharmacol., April 1, 2001; 59(4): 886 - 893. [Abstract] [Full Text]
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 T. KENAKIN Inverse, protean, and ligand-selective agonism: matters of receptor conformation FASEB J, March 1, 2001; 15(3): 598 - 611. [Abstract] [Full Text] [PDF]
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 J. T. Williams, M. J. Christie, and O. Manzoni Cellular and Synaptic Adaptations Mediating Opioid Dependence Physiol Rev, January 1, 2001; 81(1): 299 - 343. [Abstract] [Full Text] [PDF]
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P.-Y. Law, L. J. Erickson, R. El-Kouhen, L. Dicker, J. Solberg, W. Wang, E. Miller, A. L. Burd, and H. H. Loh Receptor Density and Recycling Affect the Rate of Agonist-Induced Desensitization of {micro}-Opioid Receptor Mol. Pharmacol., August 1, 2000; 58(2): 388 - 398. [Abstract] [Full Text]
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F. Noble, M. Szücs, B. Kieffer, and B. P. Roques Overexpression of Dynamin Is Induced by Chronic Stimulation of {micro}- but Not delta -Opioid Receptors: Relationships with {micro}-Related Morphine Dependence Mol. Pharmacol., July 1, 2000; 58(1): 159 - 166. [Abstract] [Full Text]
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A. Hasbi, S. Allouche, F. Sichel, L. Stanasila, D. Massotte, G. Landemore, J. Polastron, and P. Jauzac Internalization and Recycling of delta -Opioid Receptor Are Dependent on a Phosphorylation-Dephosphorylation Mechanism J. Pharmacol. Exp. Ther., April 1, 2000; 293(1): 237 - 247. [Abstract] [Full Text]
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P. A. Zaki, D. E. Keith Jr., G. A. Brine, F. I. Carroll, and C. J. Evans Ligand-Induced Changes in Surface {micro}-Opioid Receptor Number: Relationship to G Protein Activation? J. Pharmacol. Exp. Ther., March 1, 2000; 292(3): 1127 - 1134. [Abstract] [Full Text]
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 W. Xie, G. M. Samoriski, J. P. McLaughlin, V. A. Romoser, A. Smrcka, P. M. Hinkle, J. M. Bidlack, R. A. Gross, H. Jiang, and D. Wu Genetic alteration of phospholipase C beta 3 expression modulates behavioral and cellular responses to {micro} opioids PNAS, August 31, 1999; 96(18): 10385 - 10390. [Abstract] [Full Text] [PDF]
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R. Seifert, U. Gether, K. Wenzel-Seifert, and B. K. Kobilka Effects of Guanine, Inosine, and Xanthine Nucleotides on beta 2-Adrenergic Receptor/Gs Interactions: Evidence for Multiple Receptor Conformations Mol. Pharmacol., August 1, 1999; 56(2): 348 - 358. [Abstract] [Full Text]
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 M. Bunemann and M M. Hosey G-protein coupled receptor kinases as modulators of G-protein signalling J. Physiol., May 15, 1999; 517(1): 5 - 23. [Abstract] [Full Text] [PDF]
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P.-Y. Law and H. H. Loh Regulation of Opioid Receptor Activities J. Pharmacol. Exp. Ther., May 1, 1999; 289(2): 607 - 624. [Abstract] [Full Text]
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R. El Kouhen, O. M.-E. Kouhen, P.-Y. Law, and H. H. Loh The Absence of a Direct Correlation between the Loss of [D-Ala2,MePhe4,Gly5-ol]Enkephalin Inhibition of Adenylyl Cyclase Activity and Agonist-induced µ-Opioid Receptor Phosphorylation J. Biol. Chem., April 2, 1999; 274(14): 9207 - 9215. [Abstract] [Full Text] [PDF]
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A. E. Remmers, M. J. Clark, X. Y. Liu, and F. Medzihradsky Delta Opioid Receptor Down-Regulation Is Independent of Functional G Protein yet Is Dependent on Agonist Efficacy J. Pharmacol. Exp. Ther., November 1, 1998; 287(2): 625 - 632. [Abstract] [Full Text]
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A. Kovoor, J. P. Celver, A. Wu, and C. Chavkin Agonist Induced Homologous Desensitization of µ-Opioid Receptors Mediated by G Protein-Coupled Receptor Kinases Is Dependent on Agonist Efficacy Mol. Pharmacol., October 1, 1998; 54(4): 704 - 711. [Abstract] [Full Text]
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H.-h. Chuang, M. Yu, Y. N. Jan, and L. Y. Jan Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K+ channels PNAS, September 29, 1998; 95(20): 11727 - 11732. [Abstract] [Full Text] [PDF]
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J. L. Whistler and M. von Zastrow Morphine-activated opioid receptors elude desensitization by beta -arrestin PNAS, August 18, 1998; 95(17): 9914 - 9919. [Abstract] [Full Text] [PDF]
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J. Zhang, S. S. G. Ferguson, L. S. Barak, S. R. Bodduluri, S. A. Laporte, P.-Y. Law, and M. G. Caron Role for G protein-coupled receptor kinase in agonist-specific regulation of µ-opioid receptor responsiveness PNAS, June 9, 1998; 95(12): 7157 - 7162. [Abstract] [Full Text] [PDF]
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B. Xiang, G.-H. Yu, J. Guo, L. Chen, W. Hu, G. Pei, and L. Ma Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of delta -Opioid Receptor at Serine 344, Resulting in beta -Arrestin- and Clathrin-mediated Receptor Internalization J. Biol. Chem., February 9, 2001; 276(7): 4709 - 4716. [Abstract] [Full Text] [PDF]
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K. Befort, D. Filliol, F. M. Decaillot, C. Gaveriaux-Ruff, M. R. Hoehe, and B. L. Kieffer A Single Nucleotide Polymorphic Mutation in the Human {micro}-Opioid Receptor Severely Impairs Receptor Signaling J. Biol. Chem., January 26, 2001; 276(5): 3130 - 3137. [Abstract] [Full Text] [PDF]
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O. M.-E. Kouhen, G. Wang, J. Solberg, L. J. Erickson, P.-Y. Law, and H. H. Loh Hierarchical Phosphorylation of delta -Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization J. Biol. Chem., November 17, 2000; 275(47): 36659 - 36664. [Abstract] [Full Text] [PDF]
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J. P. Celver, J. Lowe, A. Kovoor, V. V. Gurevich, and C. Chavkin Threonine 180 Is Required for G-protein-coupled Receptor Kinase 3- and beta -Arrestin 2-mediated Desensitization of the {micro}-Opioid Receptor in Xenopus Oocytes J. Biol. Chem., February 9, 2001; 276(7): 4894 - 4900. [Abstract] [Full Text] [PDF]
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T. Christophe, A. Karlsson, C. Dugave, M.-J. Rabiet, F. Boulay, and C. Dahlgren The Synthetic Peptide Trp-Lys-Tyr-Met-Val-Met-NH2 Specifically Activates Neutrophils through FPRL1/Lipoxin A4 Receptors and Is an Agonist for the Orphan Monocyte-expressed Chemoattractant Receptor FPRL2 J. Biol. Chem., June 8, 2001; 276(24): 21585 - 21593. [Abstract] [Full Text] [PDF]
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R. El Kouhen, A. L. Burd, L. J. Erickson-Herbrandson, C.-Y. Chang, P.-Y. Law, and H. H. Loh Phosphorylation of Ser363, Thr370, and Ser375 Residues within the Carboxyl Tail Differentially Regulates {micro}-Opioid Receptor Internalization J. Biol. Chem., April 13, 2001; 276(16): 12774 - 12780. [Abstract] [Full Text] [PDF]
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J. L. Whistler, P. Tsao, and M. von Zastrow A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes J. Biol. Chem., August 31, 2001; 276(36): 34331 - 34338. [Abstract] [Full Text] [PDF]
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