From the Departments of Biochemistry and Molecular Biology, and Neuroscience, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
Received for publication, September 1, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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This study investigated the mechanism of
agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged The pharmacological effects of opioid drugs and the physiological
effects of endogenous opioid peptides are initiated through the binding
and activation of opioid receptors (1), which are members of the G
protein-coupled receptor
(GPCR)1 family (2). GPCRs
comprise a diverse superfamily of integral membrane proteins that
mediate signal transduction across the plasma membrane. All GPCRs are
postulated to have amino termini located on the extracellular side of
the plasma membrane that are linked to seven-transmembrane helices
connected by relatively short intracellular and extracellular loops,
and contain carboxyl termini that face the interior of the cell.
Ligands approach and engage GPCRs from the extracellular space, and
receptor activation results in coupling to heterotrimeric G proteins on
the intracellular face of membrane. The amino termini of nearly all G
protein-coupled receptors contain consensus amino acid sequences for
asparagine-linked glycosylation; two sites for N-linked
glycosylation are present in Three types of opioid receptor, Short-term exposure of opioid receptors to agonists, like most GPCRs,
leads to receptor desensitization, while chronic exposure leads to
receptor down-regulation (reviewed in Refs. 1 and 21). Homologous
desensitization appears to involve phosphorylation of the
agonist-activated GPCR by members of the G protein-coupled receptor
kinase family, and subsequent binding of arrestin proteins, which effectively uncouple the interaction of activated GPCRs from
heterotrimeric G proteins (21). In addition to preventing receptor/G
protein interactions, arrestins initiate receptor endocytosis via
clathrin-coated pits (22, 23). Endosome-associated receptors can be
resensitized by protein phosphatases and recycled back to the plasma
membrane, or be degraded intracellularly. The mechanism for GPCR
proteolysis has been generally assumed to involve fusion of endosomes
with lysosomes, based on studies of epidermal growth factor receptor
down-regulation (24).
Acute agonist-induced desensitization of opioid receptors appears to
involve similar mechanisms as described for the
This study was designed to test the hypothesis that lysosomal proteases
are responsible for opioid receptor down-regulation. Contrary to this
hypothesis, these data implicate a prominent role for the
ubiquitin/proteasome pathway in agonist-induced down-regulation and basal turnover of opioid receptors.
Cell Culture and Transfection--
Human embryonic kidney
(HEK) 293 cells were cultured at 37 °C in a humidified atmosphere
containing 5% CO2 in Dulbecco's modified Eagle's
medium, supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin sulfate. HEK 293 cells were
transfected using electroporation, as described previously (31), with
expression plasmids encoding murine µ or Membrane Preparation and Radioligand Binding
Assays--
HEK 293 cells expressing epitope-tagged µ or
Opioid receptor binding assays were conducted in duplicate on
membrane preparations diluted 20-40-fold in 50 mM Tris-HCl
buffer, pH 7.5. Radioligands that were used included
( Opioid Receptor Down-regulation--
Cells were treated with 1 µM Tyr-D-Ala-Gly-Phe-D-Leu (DADL)
in serum-free media for various time intervals to study the kinetics of
down-regulation. For recovery experiments, cells cultured in serum-free
medium were incubated for 18 h in the absence and presence of 1 µM DADL, medium was aspirated and cells were washed three times, and then replenished with serum-free medium lacking agonist. Cells were harvested at 0, 2, 4, 8, and 24 h following removal of
DADL, and then assayed for receptor specific binding using 3 nM [3H]ethylketocyclazocine. To assess the
effect of pertussis toxin (Ptx, Life Technologies, Gaithersburg, MD),
cells were preincubated with 100 ng/ml Ptx for 3 h in serum-free
media followed by overnight treatment with DADL (1 µM).
To determine the effect of various membrane-permeable protease
inhibitors on agonist-induced receptor down-regulation, cells were
pretreated for 1 h with the inhibitors at a concentration of 25 µM. The following inhibitors were tested: proteasomal
inhibitors, ZLLL
(N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal, Peptides International, Louisville, KY), ALLN
(N-acetyl-L-leucyl-L-leucyl-L-norleucinal, Sigma), lactacystin (Peptides International), and PSI
(N-benzyloxycarbonyl-L-isoleucyl- Immunoblotting and Immunoprecipitation--
For immunoblotting
experiments, cells were grown to near confluence in 60-mm dishes. Cell
extracts were prepared by incubating the cells in 0.2 ml of lysis
buffer consisting of 150 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, 10% glycerol and
protease inhibitor mixture (containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin, Sigma)
for 1 h on ice. Cell debris was pelleted by centrifugation and the
supernatant was used for further analysis. Protein concentrations in
supernatants were determined using the Bio-Rad assay with bovine serum
albumin as standard. Cell extracts containing ~20 µg of protein
were mixed with an equal volume of gel loading buffer (Bio-Rad) and
heated at 40 °C for 5 min. Proteins were resolved using 12 or 15%
SDS-PAGE and transferred to Immobilon PSQ PVDF membranes
(Millipore, Bedford, MA). Membranes were blocked for 1 h in 2.3%
dried milk, 0.5% bovine serum albumin, 0.1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1%
Tween 20, followed by overnight incubation at 4 °C with mouse
anti-FLAG M1 monoclonal antibody (Sigma). Membranes were then washed
and incubated with anti-mouse IgG conjugated with alkaline phosphatase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room
temperature and developed using CDP-Star Western blot
chemiluminescence reagent (PerkinElmer Life Sciences) as
described previously (32). Kodak Biomax MR film was used to capture chemiluminescence.
Receptor glycosylation was investigated by incubating cell lysates in
150 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,
1% Triton X-100, 10% glycerol, and protease inhibitor mixture with or
without N-glycosidase F (40 units/mg of membrane protein,
Roche Molecular Biochemicals, Indianapolis, IN) at 37 °C for 3 h. For immunoblotting, treated and untreated lysates were diluted with
an equal volume of gel loading buffer, proteins were resolved using
10% SDS-PAGE, and assayed by immunoblotting as described above.
For receptor immunoprecipitation, cell extracts were incubated for
2 h at 4 °C with 30 µl of lysis buffer containing a
suspension of anti-FLAG M1 monoclonal antibody conjugated to agarose
(Sigma). Following incubation, antibody gel beads were washed three
times by centrifugation with lysis buffer and incubated for 1 h at
room temperature with 40 µl of elution buffer (50 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1% Triton X-100, 10%
glycerol, and 5 mM EDTA). Following brief centrifugation,
supernatants containing immunopurified receptor protein were mixed with
an equal volume of gel loading dye and heated at 40 °C for 5 min.
Proteins were resolved using 12% SDS-PAGE and transferred to PVDF
membranes. Receptor proteins were detected using anti-FLAG M1
monoclonal antibodies, as described above. Ubiquitinated receptor
proteins were visualized with rabbit anti-ubiquitin antibodies (Dako,
Carpinteria, CA) followed by goat anti-rabbit IgG secondary antibodies
conjugated with alkaline phosphatase (Santa Cruz). Blots were developed
using CDP Star chemiluminescence reagent.
Pulse-Chase Metabolic Labeling--
For metabolic labeling
studies, stably transfected HEK 293 cells expressing FLAG-tagged Agonist-induced Phosphorylation of MAP Kinase--
MAP kinase
assays were conducted as described previously (33). Briefly, HEK 293 cells expressing the FLAG-tagged In Vitro Transcription and Translation of Opioid
Receptors--
[35S]Methionine-labeled opioid receptor
proteins were expressed using a coupled in vitro
transcription and translation system (TNT rabbit reticulocyte extracts,
Promega, Madison, WI). Plasmids encoding µ and Properties of FLAG-tagged Opioid Receptors and Kinetics of
Down-regulation--
As reported recently (32, 33), we have
established stable HEK 293 cell lines that express µ and
SDS-PAGE and immunoblot analysis with the anti-FLAG M1 monoclonal
antibody revealed that the
Prolonged agonist treatment of HEK 293 cells expressing either µ or
The kinetics of
The kinetics of receptor down-regulation was also examined by
immunoblot analysis (Fig. 4). Stable HEK
293 cells expressing epitope-tagged receptors were incubated in
serum-free medium at 37 °C with 1 µM DADL for various
time intervals ranging from 0.5 to 18 h, and cell lysates were
then analyzed by immunoblot analysis using the M1 monoclonal antibody.
The level of
The rate of recovery of Role of Functional G Protein--
To evaluate the role of
Gi and Go heterotrimeric GTP-binding proteins
in the receptor down-regulation pathway, cells expressing FLAG-tagged µ or
Markedly different results were obtained regarding the sensitivity of µ receptor down-regulation to pertussis toxin. Chronic exposure of
HEK 293 cells expressing the µ receptor with agonist led to an 85%
decrease in the level of µ receptors, as expected. Treatment with
pertussis toxin alone increased µ receptor protein by 50% over
control levels. In striking contrast to the results with the
Qualitatively similar results were obtained when the effect of
pertussis toxin on receptor down-regulation was determined by
radioligand saturation analysis (Table I). Prolonged incubation of HEK
293 cells expressing Proteases Involved in Agonist-induced Down-regulation of µ and
Attention was then focused on the proteasome, a large multisubunit
protease complex that degrades the majority of short-lived intracellular proteins (37, 38). Pretreatment with the cell-permeable proteasome inhibitor, ZLLL, under the same conditions as those described above for E64d, yielded strikingly different results. ZLLL
significantly attenuated the extent of
The effect of lactacystin was tested to further support the involvement
of the proteasome in
The proteasome inhibitor, ZLLL, also effectively inhibited
To probe further the activity of ZLLL, experiments were performed to
estimate the lowest effective concentration necessary to block
agonist-induced down-regulation of the
Immunoblot analysis was also utilized to evaluate the effect of the
tripeptide ZLLL on µ and
It should be noted that for both the µ and
One of the downstream effectors regulated by opioid receptor activation
is MAP kinase, which becomes rapidly activated by phosphorylation on
Thr202 and Tyr204 following agonist
treatment (40). It was of interest to determine whether opioid
receptors were still capable of activating the MAP kinase signal
transduction pathway following incubation of cells with proteasome
inhibitors. HEK 293 cells expressing FLAG-tagged
Another series of experiments was performed utilizing immunoblotting to
assess the ability of other protease inhibitors to modulate the extent
of agonist-induced down-regulation of Ubiquitination of Opioid Receptors--
Proteins that are degraded
by the proteasome are first tagged by covalent attachment of the
8.5-kDa protein, ubiquitin, which is recognized by the 19 S regulatory
complex of the proteasome (37, 38). Based on our observations regarding
the effects of proteasome inhibitors on agonist-dependent
opioid receptor down-regulation and basal turnover, we sought evidence
for ubiquitination of µ and
Furthermore, direct evidence for ubiquitination of opioid receptors,
utilizing immunoprecipitation of opioid receptors followed by
immunoblotting with anti-ubiquitin antibodies is provided in Fig.
14. HEK 293 cells expressing
FLAG-tagged
Analysis of µ receptor immunoprecipitates indicated that in untreated
samples, there were readily detectable receptor proteins conjugated to
ubiquitin, which had apparent molecular masses of ~122 and 230 kDa.
No polyubiquitinated receptor was detectable in the DADL-treated,
down-regulated µ receptor immunoprecipitate (lane 2, right
panel). The absence of any signal in this lane highlighted the
lack of nonspecific binding of the anti-ubiquitin antibody under these
conditions. Blank lanes were also observed when wild type HEK 293 cells
that did not express opioid receptors were immunoprecipitated and
immunoblotted using the same procedures as a negative control (data not
shown). In contrast, a spectrum of polyubiquitinated µ receptor
protein bands was clearly evident in samples preincubated with the
proteasome inhibitor ZLLL, corresponding to molecular masses of 79 kDa
and species with molecular masses from 120 kDa and greater that
accumulated at the top of the gel. It seems reasonable to assume that
inhibition of the proteasome stabilized the polyubiquitinated receptors
that, in the absence of inhibitors, would normally be rapidly degraded.
The major conclusions to be drawn from this study are as follows:
1) proteolysis of µ and In eukaryotic cells, a wide variety of proteins with roles in cell
cycle progression, transcriptional control, signal transduction, and
metabolic regulation are degraded by the ubiquitin-proteasome pathway
(reviewed in Refs. 37, 38, and 41). The 26 S proteasome is a 2.5 MDa
complex consisting of a 20 S proteolytic core complex and two 19 S
regulatory complexes, and is capable of cleaving at basic, acidic, and
hydrophobic amino acids within proteins. Proteins are targeted to the
proteasome by covalent ligation to ubiquitin, a highly conserved
76-amino acid protein. Ubiquitin-protein ligation involves the
sequential action of at least two, and often three, enzymes. The
C-terminal glycine of ubiquitin is first activated through formation of
an ATP-dependent thioester linkage with a cysteine in the
E1 activating enzyme. The ubiquitin is next transferred to an active
site cysteine of a ubiquitin-conjugating protein, E2, prior to
formation of an isopeptide bond between the C terminus of ubiquitin,
and an Membrane-permeable inhibitors of the proteasome have contributed
greatly to our understanding of the involvement of the
ubiquitin/proteasome system in protein degradation. The initial
proteasome inhibitors were hydrophobic peptide aldehydes that can enter
mammalian cells and inhibit proteasome function in vivo
(42-45). The most selective proteasome inhibitor is lactacystin, a
Streptomyces metabolite. Lactacystin inhibits the
trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide
hydrolyzing activities of the proteasome by covalent modification of
the amino-terminal threonine of the mammalian proteasome subunit X,
confirming that this residue is essential for catalytic activity (39).
In this study, we have used four different proteasome inhibitors,
including lactacystin, to confirm the role of the proteasome in µ and
In addition to the selective destruction of cytosolic and nuclear
regulatory proteins, it has become apparent that several yeast and
mammalian membrane proteins also undergo ubiquitination and degradation
by the proteasome (41). In mammalian cells, studies using proteasomal
inhibitors have implicated the proteasome in the down-regulation of the
platelet-derived growth factor receptor (46), low density lipoprotein
receptor (47), Met tyrosine kinase receptor (48), mannose phosphate
receptor (49), and growth hormone receptor (50). In Saccaromyces
cerevisiae, Ste2p, which is the G protein-coupled receptor for the
pheromone Regarding early signals that trigger agonist-induced down-regulation,
in this study we found that pretreatment of cells with pertussis toxin
blocked down-regulation of the µ receptor, while Results derived both from direct receptor immunoblots and from
immunoprecipitation of opioid receptors followed by immunoblotting with
ubiquitin antibodies are consistent with the proposal that µ and We do not wish to dispute recent observations that following endocytic
trafficking, opioid receptors bound to endosomes fuse eventually with
lysosomes (29, 30). We interpret our data as suggesting that the
ubiquitin/proteasome pathway operates upstream of trafficking to
lysosomes to initiate receptor down-regulation. A similar proposal has
been made regarding the down-regulation of the platelet-derived growth
factor receptor and µ receptors with agonists caused
a time-dependent decrease in opioid receptor levels assayed
by immunoblotting. Pulse-chase experiments using
[35S]methionine metabolic labeling indicated that
the turnover rate of
receptors was accelerated 5-fold following
agonist stimulation. Inactivation of functional Gi and
Go proteins by pertussis toxin-attenuated down-regulation
of the µ opioid receptor, while down-regulation of the
opioid
receptor was unaffected. Pretreatment of cells with inhibitors of
lysosomal proteases, calpain, and caspases had little effect on µ and
opioid receptor down-regulation. In marked contrast, pretreatment
with proteasome inhibitors attenuated agonist-induced µ and
receptor down-regulation. In addition, incubation of cells with
proteasome inhibitors in the absence of agonists increased
steady-state µ and
opioid receptor levels. Immunoprecipitation of µ and
opioid receptors followed by
immunoblotting with ubiquitin antibodies suggested that preincubation
with proteasome inhibitors promoted accumulation of
polyubiquitinated receptors. These data provide evidence that the
ubiquitin/proteasome pathway plays a role in agonist-induced
down-regulation and basal turnover of opioid receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
receptors, while five are
found in the µ receptor (3).
, µ, and
, have been cloned and
characterized extensively (4-7). Opioid receptors have unique ligand
specificities, anatomical distributions, and physiological functions
(8-15). Opioid receptors exhibit ~60% identity in their amino acid
sequences, however, marked differences in sequence conservation are
evident within receptor subdomains. The amino acid sequences of
putative transmembrane spanning segments and the three intracellular
loops are highly conserved among opioid receptor types, whereas
sequences in the extracellular amino termini, second and third
extracellular loops, and the intracellular carboxyl termini are
considerably more divergent (1). Upon activation, opioid receptors
interact with multiple G proteins to regulate adenylyl cyclase, the MAP
kinase pathway, phosphatidylinositol 3-kinase, Ca2+
channels, and K+ channels (16-20).
2-adrenergic receptor. It has been shown that
and µ opioid receptors are phosphorylated by G protein-coupled receptor
kinases following agonist treatment (25, 26), and are endocytosed in a
dynamin-dependent process via clathrin-coated pits (27).
Overexpression of arrestin or G protein-coupled receptor kinase leads
to enhanced agonist-induced internalization (26, 28).
Opioid
receptors do not appear to recycle to the plasma membrane efficiently,
while coexpressed
2-adrenergic receptors do (29), and
following treatment of cells for several hours with agonist,
opioid
receptors are apparently associated with lysosomes (30).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
opioid receptors tagged
at the amino termini with the FLAG epitope (kindly provided by Dr. M. Van Zastrow). Cells stably expressing epitope-tagged opioid receptors
were selected in media containing 1 mg/ml G418 (Life Technologies,
Gaithersburg, MD), as described (32, 33).
opioid
receptors were grown to near confluence in 100-mm diameter dishes. For membrane preparations, the culture medium was aspirated and cells were
harvested using a cell scraper in 5 ml of 50 mM Tris-HCl buffer, pH 7.5, per 100-mm dish. The cell suspension was homogenized with a Tekmar tissuemizer (Cincinnati, OH), then centrifuged at 35,000 × g for 20 min. The membrane pellet was washed
3 times in Tris buffer and then resuspended by homogenization in 1 ml of 0.32 M sucrose, 50 mM Tris-HCl, pH
7.5, per dish, and the crude membrane preparation was stored at
80 °C.
)-[9-3H]bremazocine (specific activity 26.6 Ci/mmol,
PerkinElmer Life Sciences),
[tyrosyl-3,5-3H]DAMGO (specific activity 48.9 Ci/mmol, PerkinElmer Life Sciences), [3H]ethylketocyclazocine (specific activity 27 Ci/mmol,
NIDA, National Institutes of Health, Rockville, MD), and
[tyrosyl-3,5-3H][D-Ser2,Leu5]enkephalin-Thr
(specific activity, 57 Ci/mmol, NIDA, National Institutes of Health,
Rockville, MD). Binding assays were conducted at 22 °C in a volume
of 0.25 ml (~20-40 µg of protein/ml); 10 µM
cyclazocine was used to define specific binding. Following a 1-h
incubation, assays were terminated by filtration through Whatman GF/B
filters. Filters were soaked in Ecoscint liquid scintillation mixture
(National Diagnostics, Somerville, NJ) prior to determination of filter
bound radioactivity using a Beckman LS 1701 scintillation counter.
Receptor binding data was analyzed by nonlinear regression using Prism
2.0 (GraphPad Software, San Diego, CA). Protein concentrations were
determined with the Bio-Rad protein assay (Hercules, CA), using bovine
serum albumin as the standard.
-t-butyl-L-glutamyl-L-alanyl-L-leucinal, Peptides International); the lysosomal protease and calpain inhibitor, E64d
(2S,3S-t-epoxysuccinyl-L-leucylamido-3-methylbutane
ethyl ester, Peptides International); the lysosomal protease, calpain and trypsin-like protease inhibitor, leupeptin
(N-acetyl-L-leucyl-L-leucyl-L-arginal, Calbiochem); and the caspase inhibitor,
N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-chloromethylketone (caspase-3 inhibitor III, Calbiochem). All protease inhibitors were
dissolved in dimethyl sulfoxide, and control cells lacking inhibitors
were treated with dimethyl sulfoxide vehicle.
opioid receptors were incubated in serum- and methionine-free minimal
essential media containing 90 µCi/ml [35S]methionine
for 3 h at 37 °C, then chased with methionine-containing minimal essential media in the absence and presence of the peptide agonist, DADL (1 µM), for 2, 4, 6, 8, and 18 h. Cell
lysates were immunoprecipitated with anti-FLAG monoclonal antibody
agarose beads, and resolved using SDS-PAGE. Gels were fixed and dried prior to autoradiography.
Receptor bands were quantified by excising the receptor band from the gel followed by liquid
scintillation counting, and by scanning densitometry of
autoradiographic bands using NIH Image software, version 1.61; results
were comparable.
receptor were incubated with or
without 25 µM ZLLL for 18 h, and then treated with
or without DADL (1 µM) for an additional 10 min. Cells
were lysed in 150 mM Tris-HCl, pH 7.5, 300 mM
NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, 10% glycerol, containing mixtures
of phosphatase inhibitors and protease inhibitors (Sigma). Cell debris
was pelleted by centrifugation and protein concentration in the
supernatant was determined using the Bio-Rad protein assay. Equal
amounts of protein from each sample were resolved using 12% SDS-PAGE
and transferred to Immobilon PSQ PVDF membranes. Membranes
were blocked for 1 h in 2.3% dried milk, 0.5% bovine serum
albumin, 0.1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween 20, followed by overnight
incubation with mouse monoclonal anti-phospho-MAPK antibody or rabbit
anti-MAP kinase antibody (New England Biolabs, Beverly, MA). Membranes were then washed and incubated with goat anti-mouse IgG conjugated with
alkaline phosphatase or goat anti-rabbit IgG conjugated with alkaline
phosphatase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at
room temperature. Blots were washed and developed using CDP Star
Western blot chemiluminescence reagent (PerkinElmer Life Sciences) and
Kodak Biomax MR film. Blots were quantified using NIH Image software,
version 1.61.
opioid receptor
cDNAs were transcribed with T7 RNA polymerase and translated with
reticulocyte extracts in the presence of [35S]methionine
according to conditions recommended by the manufacturer. Luciferase
cDNA transcribed by T7 RNA polymerase served as a positive control
reaction. Translated proteins were resolved using 15% SDS-PAGE. Gels
were fixed, dried, and then exposed to Kodak Biomax MR film for autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
opioid
receptors with a FLAG epitope at the amino termini. Using
[3H]bremazocine as radioligand, the
Bmax values for cell lines expressing epitope-tagged receptors used in the present experiments were ~5 and
13 pmol/mg of protein for µ and
opioid receptors, respectively, and apparent dissociation constants (Kd) for
bremazocine were 1.0 and 0.8 nM, respectively (see Table
I).
Effect of pertussis toxin on agonist-induced down-regulation of and µ opioid receptors
or µ opioid receptors were
preincubated at 37 °C for 3 h in serum-free Dulbecco's
modified Eagle's medium with or without 100 ng/ml pertussis toxin
(Ptx), then incubated for 18 h in the absence and presence of 1 µM DAMGO (µ receptors) or DADL (
receptors).
Membrane fractions were prepared and saturation analysis was performed
using [3H]bremazocine as radioligand. Saturation curves were
analyzed by non-linear regression to generate apparent dissociation
constants, KD (nM), and maximum number
of receptors, Bmax (fmol/mg of protein).
Bmax ratios represent quotients × 100 of
agonist/control, Ptx/control, and Ptx + agonist/Ptx. Data
represent mean ± S.E. of three to four experiments conducted in
duplicate.
opioid receptor was expressed as two
major heterogeneous forms, with apparent molecular masses of ~55-65
and 130-145 kilodaltons (kDa) in HEK 293 cells (Fig. 1). As expected due to its larger mass,
the µ opioid receptor migrated more slowly as two major diffuse bands
with apparent molecular masses of ~70-80 and 150-160 kDa. Evidence
that
and µ opioid receptors are glycoproteins was provided by
digestion with N-glycosidase F, an amidase that hydrolyzes
nearly all types of N-glycan chains from the asparagine in
N-linked glycoproteins. N-Glycosidase F treatment
of
and µ opioid receptors increased the mobilities of both bands
to species with apparent molecular masses of 34 and 93 kDa for the
receptor, and 36 and 82 kDa for the µ receptor. The predicted
molecular masses for the FLAG-tagged receptors are ~41 and 45 kDa for
the
and µ opioid receptors, respectively. The reason for the
aberrant electrophoretic mobilities of the receptors, even after
deglycosylation, is not known at present. The immunoreactive bands with
slower mobilities may represent opioid receptor dimers that have been
described recently (34, 35).
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Fig. 1.
And µ opioid receptors expressed in HEK
293 cells are N-linked glycoproteins. Cell
lysates were prepared by extracting monolayers of HEK 293 cells
expressing
or µ opioid receptors in 0.2 ml of lysis buffer
consisting of 150 mM Tris-HCl, pH 7.5, 300 mM
NaCl, 1 mM MgCl2, 1 mM
CaCl2, 1% Triton X-100, 10% glycerol and protease
inhibitor mixture for 1 h on ice. Cellular debris was pelleted by
centrifugation and the supernatants were treated with or without
N-glycosidase F (protease-free, 40 units/mg of membrane
protein) at 37 °C for 3 h, then resolved using 10% SDS-PAGE.
FLAG-tagged receptors were assayed by immunoblotting using the
anti-FLAG M1 monoclonal antibody. The mobilities of molecular mass
standards (in kDa) are indicated to the right.
OR
(gly), glycosylated (untreated)
receptors;
OR
(degly), deglycosylated (treated with
N-glycosidase F)
receptors; µ OR (gly),
glycosylated (untreated) µ receptors; µ OR (degly),
deglycosylated (treated with N-glycosidase F) µ receptors.
N-Glycosidase F treatment of
receptors increased the
mobilities of the 55-65- and 130-145-kDa bands to species with
apparent molecular masses of 34 and 93 kDa, respectively.
N-Glycosidase F treatment of µ receptors shifted the
mobilities of the 70-80- and 150-160-kDa bands to species with
apparent molecular masses of 36 and 82 kDa, respectively. This
experiment was replicated three times with similar results.
opioid receptors resulted in decreased steady-state receptor
levels, as measured by determination of Bmax
using saturation binding analysis or by immunoblotting (see below).
This agonist-induced decrease in receptor number is the process
referred to in this report as receptor down-regulation. We have
recently reported that the magnitude of
receptor down-regulation
was dependent on which agonist was employed (33). In the present study,
it was found that the extent of µ receptor down-regulation was also dependent on the agonist used (Fig. 2).
Overnight treatment with DAMGO resulted in a >80% decrease in the µ receptor Bmax, while morphine induced only a
45% decrease. Neither ligand altered the apparent dissociation
constant of [3H]bremazocine for the µ receptor
(untreated Kd = 1.1 ± 0.2 nM,
DAMGO-treated Kd = 1.2 ± 0.1 nM,
morphine-treated Kd = 0.9 ± 0.2 nM). Overnight treatment with etorphine, DADL, fentanyl,
and methadone decreased the µ receptor Bmax by 70-80%, suggesting that these agonists had an intrinsic efficacy that
was similar to that of DAMGO for inducing down-regulation of the µ receptor (data not shown).
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Fig. 2.
Agonist-dependent down-regulation
of the µ opioid receptor. HEK 293 cells expressing the
FLAG-tagged µ receptor were incubated in serum-free medium at
37 °C with 1 µM DAMGO or morphine for 18 h, and
washed membrane preparations were used for saturation analysis using
[3H]bremazocine (0.1-6.0 nM) as radioligand,
and 10 µM cyclazocine to define nonspecific binding.
Values for apparent dissociation constants (Kd) and
the maximum number of binding sites (Bmax) were
determined by nonlinear regression analysis of the curves using
GraphPad Prism software and were normalized for protein content.
Neither ligand altered the Kd of bremazocine. Data
are expressed as percentages of the control Bmax
from untreated cells (5 pmol/mg of protein), and represent the means
and standard errors of 4-6 independent assays conducted in
duplicate.
receptor down-regulation was investigated by
pulse-chase analysis following metabolic labeling of cells with
[35S]methionine. HEK 293 cells expressing FLAG-tagged
receptors were preincubated at 37 °C with
[35S]methionine in serum- and methionine-free Dulbecco's
modified Eagle's medium for 3 h, and then chased with unlabeled
methionine-containing medium in the absence and presence of 1 µM DADL. Cell lysates were prepared at various times
following the unlabeled methionine chase, immunoprecipitated with the
anti-FLAG M1 monoclonal antibody, and then analyzed by SDS-PAGE and
autoradiography (Fig. 3). In the absence
of agonist, receptor degradation exhibited first-order exponential
decay with a rate constant of 0.08 ± 0.02, corresponding to a
half-life of 8.7 h. In the presence of DADL, the receptor degradation rate was accelerated significantly with a rate constant of
0.42 ± 0.07 and a half-life of 1.6 h.
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Fig. 3.
Pulse-chase analysis of agonist-induced
down-regulation of the receptor. HEK 293 cells expressing
FLAG-tagged
opioid receptors were incubated in serum- and
methionine-free minimal essential media containing 90 µCi/ml
[35S]methionine for 3 h at 37 °C, then chased
with methionine-containing minimal essential media in the absence and
presence of DADL (1 µM) for 2, 4, 8, and 18 h. Cell
lysates were immunoprecipitated with anti-FLAG monoclonal antibody, and
resolved using SDS-PAGE.
Receptor bands were quantified by excising
the receptor band from the gel followed by liquid scintillation
counting and by scanning densitometry of autoradiographic bands using
NIH Image software, version 1.61; results were comparable. The
mobilities of molecular mass standards (in kDa) are indicated to the
left. The receptor degradation rate was accelerated
significantly in the presence of DADL. This experiment was replicated
five times with similar results.
receptor protein remained steady until 2-4 h after
DADL treatment. Levels then decreased progressively at 4, 6, and
18 h after agonist administration to 77, 38, and 4% of zero time
controls, respectively. The time course of µ receptor down-regulation
was similar, although a decrease in receptor protein was already
apparent at the 2-h time point.
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Fig. 4.
Kinetics of agonist-induced and µ opioid receptor down-regulation assayed by immunoblotting. HEK 293 cells expressing either FLAG-tagged
receptors (upper
panel) or µ receptors (lower panel) were incubated in
serum-free medium at 37 °C with 1 µM DADL for 0-18 h,
as indicated, and cell lysates were assayed by immunoblotting using the
anti-FLAG M1 monoclonal antibody. The mobilities of molecular mass
standards (in kDa) are indicated to the right.
Representative immunoblots are shown from one of four experiments with
similar results.
receptor expression following
agonist-induced down-regulation was measured by treating
receptor-expressing HEK 293 cells with 1 µM DADL for
18 h, then assaying receptor binding at various time points
following removal of the agonist from the media. As displayed in Fig.
5, down-regulation was readily reversible
upon removal of agonist. After an 18-h exposure of cells to DADL,
receptor levels were reduced to 13% of untreated control cells. At 2, 4, 8, and 24 h following agonist removal, receptor levels were 31, 50, 69, and 95% of untreated control cells, respectively.
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Fig. 5.
Recovery of agonist-induced opioid
receptor down-regulation assayed by radioligand binding. HEK 293 cells expressing the FLAG-tagged
receptor were incubated in
serum-free medium at 37 °C for 18 h in the absence and presence
of 1 µM DADL, cells were washed three times, and then
replenished with serum-free medium lacking agonist. Cells were
harvested at 0, 2, 4, 8, and 24 h following removal of DADL,
homogenized, and assayed for specific receptor binding using 3 nM [3H]ethylketocyclazocine, and 10 µM cyclazocine to assess nonspecific binding. Data are
expressed as percentages of the binding to homogenates from control
cells not treated with agonist, normalized for protein content, and
represent the means and standard errors of three to five independent
assays conducted in duplicate.
opioid receptors were pretreated with or without 100 ng/ml
pertussis toxin in serum-free media for 3 h at 37 °C, followed
by overnight incubation in the absence and presence of 1 µM DADL. Receptor levels in cell lysates were compared by
immunoblot analysis and quantified using NIH Image software. Marked
differences between µ and
opioid receptors were observed
regarding the effects of pertussis toxin on receptor down-regulation
(Fig. 6). Incubation of cells expressing
the
receptor for 18 h with DADL resulted in a 90% loss of
receptor protein. Pertussis toxin treatment alone in the absence of
agonist resulted in a nearly 2-fold increase in
receptor
expression. The efficacy of DADL toward inducing down-regulation of the
receptor was not changed appreciably by pretreatment with pertussis
toxin: overnight treatment with the peptide still resulted in a 75%
decrease in receptor protein in cells that were preincubated with
pertussis toxin.
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Fig. 6.
Effect of pertussis toxin on agonist-induced
down-regulation of the and µ opioid receptor. HEK 293 cells
expressing FLAG-tagged
receptors (upper panel) or µ receptors (lower panel) were preincubated at 37 °C in the
absence or presence of 100 ng/ml pertussis toxin (Ptx) for 3 h in
serum-free media followed by overnight treatment with or without DADL
(1 µM), as indicated above the immunoblot.
Cell lysates were prepared and resolved by SDS-PAGE, followed by
immunoblotting with the anti-FLAG M1 monoclonal antibody. The
mobilities of 61- and 85-kDa molecular mass standards are indicated to
the right. Pertussis toxin pretreatment had little effect on
the extent of
receptor down-regulation, but completely reversed
agonist-induced down-regulation of the µ receptor. This experiment
was repeated three times with similar results.
receptor, however, preincubation with pertussis toxin completely
blocked DADL-induced down-regulation of the µ opioid receptor (Fig.
6).
and µ opioid receptors with peptide agonists
decreased the number of receptors by 80-90%. Incubation of cells with
pertussis toxin in the absence of agonist resulted in a 2.7- and
1.6-fold increase in Bmax of
and µ receptors, respectively. Pertussis toxin pretreatment had no effect on
the extent of
receptor down-regulation, but reversed significantly the decrease in the Bmax of the µ receptor
induced by DAMGO. Following an 18-h incubation with DAMGO, the µ receptor Bmax was 20% of the untreated sample;
in the presence of pertussis toxin the DAMGO-treated Bmax was 70% of the control level.
Opioid Receptors--
To attempt to identify which proteases were
responsible for agonist-induced down-regulation of opioid receptors,
immunoblotting and radioligand receptor binding assays were performed
using cells that were preincubated with several different
cell-permeable protease inhibitors prior to agonist stimulation. To
test the prevailing hypothesis that receptors are degraded in the
lysosome, the protease inhibitor examined initially was E64d
((2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, loxastatin). E64d readily enters cells and irreversibly inhibits thiol proteases, and is particularly active against lysosomal cathepsins B, H, and L (36). E64d also inhibits effectively the
proteolytic activities of calpains I and II, which are cytosolic, calcium-dependent cysteine proteases. Stable HEK 293 cells
expressing
opioid receptors were pretreated at 37 °C in
serum-free media for 1 h with or without 25 µM E64d,
followed by 18 h treatment with or without 1 µM
DADL. Saturation analysis of [3H]bremazocine binding to
washed membrane preparations indicated that E64d treatment had minimal
effect on DADL-induced down-regulation (Fig.
7A) and had no effect on
steady-state
receptor levels in the absence of agonist (Fig.
7B).
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Fig. 7.
Effect of protease inhibitors on
agonist-induced down-regulation and basal levels of the opioid
receptor. HEK 293 cells expressing the
receptor were
pretreated for 1 h at 37 °C with or without 25 µM
E64d (a lysosomal protease and calpain inhibitor), ZLLL (proteasome
inhibitor), or lactacystin (proteasome inhibitor), then incubated in
the absence and presence of 1 µM DADL for an additional
18 h at 37 °C. Membrane fractions were prepared and washed
thoroughly to remove agonist, and then used for saturation analysis
using [3H]bremazocine as radioligand and cyclazocine to
assess nonspecific binding. The maximum number of binding sites
(Bmax) was determined by nonlinear regression
analysis of the curves using GraphPad Prism software and were
normalized for protein content. A, effect of protease
inhibitors on DADL-induced down-regulation. DADL, cells
incubated for 18 h with agonist without preincubation with a
protease inhibitor; +E64d, cells preincubated with E64d
followed by 18 h DADL treatment; +ZLLL, cells
preincubated with ZLLL followed by 18 h DADL treatment;
+lactacyst, cells preincubated with lactacystin followed by
18-h DADL treatment. B, effect of protease inhibitors on
steady state levels of opioid receptors. Cells were incubated for
19 h with protease inhibitors (in the absence of DADL) as
indicated. Data are expressed as percentages of the control
Bmax in the absence of agonist, and represent
the means and standard errors of three to five independent assays
conducted in duplicate. In A, asterisks indicate significant
differences from DADL treatment alone; in B, asterisks
indicate significant differences from untreated controls (ANOVA,
Tukey's Multiple Comparison Test, p < 0.05).
Proteasome inhibitors attenuated agonist-induced down-regulation and
increased steady state
receptor levels.
receptor down-regulation. Prolonged treatment with DADL induced an 86% decrease in
Bmax, while in the presence of ZLLL there
was only a 45% decrease (Fig. 7A). Incubation with ZLLL
alone also increased steady-state
receptor levels by 70% (Fig.
7B).
receptor turnover. Lactacystin is a
Streptomyces product that acts as a highly selective
irreversible inhibitor of the proteasome (39). The effects of
lactacystin were very similar to those observed using ZLLL. Lactacystin
significantly blocked DADL-induced down-regulation of the
opioid
receptor with an efficacy that was similar to ZLLL (Fig.
7A), and increased steady-state levels of the
receptor
by 85% (Fig. 7B).
opioid
receptor down-regulation in neuroblastoma × glioma NG108-15 cells
that endogenously express the
opioid receptor gene (Table II). Overnight treatment of NG108-15
cells at 37 °C with 1 µM DADL in serum-free medium
resulted in a 91% decrease in the receptor Bmax, based on saturation analysis with
[3H]bremazocine as radioligand
(Bmax ratio was 9.1%). In the NG108-15 cell
line, incubation with the proteasome inhibitor alone did not increase
the receptor density. This suggests that the proteasome is not involved
in basal turnover of the
receptor in this cell line, unlike its
activity in HEK 293 cells (Fig. 7B, and see below). When
NG108-15 cells were preincubated at 37 °C for 1 h with 25 µM ZLLL prior to agonist treatment, the
Bmax ratio increased to 39% (Table II),
indicating that the extent of DADL-induced down-regulation was reduced
significantly by the proteasome inhibitor (ANOVA, Tukey's Multiple
Comparison Test, p < 0.05).
Effect of the proteasome inhibitor, ZLLL, on agonist-induced
down-regulation of opioid receptors in NG108 cells
opioid receptor and increase
steady-state receptor levels in HEK 293 cells. Using saturation
analysis with [3H]bremazocine to determine
Bmax, it was observed that 2.5 µM
ZLLL in the cell culture medium was sufficient for significant
attenuation of
receptor down-regulation and to inhibit basal
turnover (Fig. 8). The effects did not
reach statistical significance using the lowest concentration of ZLLL
tested (250 nM). As a further control for specificity, the
effect of the related dipeptide aldehyde, ZLL, at 25 µM
was evaluated. This reagent, unlike the tripeptide analog,
displays little inhibitory activity toward the proteasome, but does
inhibit calpain and lysosomal thiol proteases (38). Pretreatment with
the dipeptide aldehyde ZLL had no effect on agonist-induced
down-regulation of opioid receptors (data not shown).
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Fig. 8.
Dose-response analysis of the effect of
proteasome inhibitor ZLLL on agonist-induced down-regulation and basal
levels of the opioid receptor. HEK 293 cells expressing the
receptor were pretreated for 1 h at 37 °C in serum-free
medium with or without 0.25, 2.5, or 25 µM ZLLL then
incubated in the absence and presence of 1 µM DADL for an
additional 18 h. Washed membrane preparations were used for
saturation analysis using [3H]bremazocine as described in
the legend to Fig. 7. A, effect of ZLLL on DADL-induced
down-regulation. DADL, cells incubated for 18 h with
agonist without preincubation with ZLLL; DADL, 0.25 Z, cells
preincubated with 0.25 µM ZLLL followed by 18 h DADL
treatment; DADL, 2.5 Z, cells preincubated with 2.5 µM ZLLL followed by 18 h DADL treatment;
DADL, 25 Z, cells preincubated with 25 µM ZLLL
followed by 18 h DADL treatment. B, effect of various
concentrations of ZLLL (as in panel A) on steady state
opioid receptor levels, in the absence of agonist. In A,
asterisks indicate significant differences from DADL treatment
alone; in B, asterisks indicate significant differences from
untreated controls (ANOVA, Tukey's Multiple Comparison Test,
p < 0.05).
opioid receptor down-regulation and
steady-state levels in HEK 293 cells. As illustrated in the left
panel of Fig. 9, chronic DADL
treatment caused a 70% decrease in the level of µ opioid receptor
protein. DADL-induced down-regulation of the µ receptor was
completely blocked in the presence of the proteasome inhibitor, and
ZLLL treatment alone (in the absence of agonist) increased the
steady-state level of µ receptor protein. Similar results were
observed in immunoblots of the
opioid receptor (Fig. 9, right
panel). The DADL-induced decrease in the level of
receptor
protein was completely reversed by preincubation with ZLLL, and
treatment with ZLLL alone increased steady-state
receptor levels.
Thus, the results obtained utilizing immunoblot analysis agreed well
with radioligand binding data regarding the ability of the proteasome
inhibitor to block agonist-induced down-regulation and to elevate
steady-state receptor levels in HEK 293 cells.
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Fig. 9.
Immunoblot analysis of the inhibitory effect
of ZLLL on agonist-induced down-regulation of µ and opioid
receptors. HEK 293 cells expressing FLAG-tagged µ receptors (µ OR, left panel) or
receptors (
OR, right
panel) were pretreated for 1 h at 37 °C in serum-free
medium with or without 25 µM ZLLL then incubated in the
absence and presence of 1 µM DADL for 18 h, as
indicated above the immunoblots. Cell lysates were prepared
and resolved by SDS-PAGE then immunoblotted with the anti-FLAG M1
monoclonal antibody. The mobilities of molecular mass standards (in
kDa) are indicated to the left. The proteasome inhibitor
completely blocked agonist-induced down-regulation of µ and
opioid receptors, and elevated steady state receptor levels in the
absence of agonist. This experiment has been replicated numerous times
with similar results.
receptor, the
proteasome inhibitor induced the appearance or increased the levels of
several immunoreactive receptor bands compared with untreated samples
(Fig. 9). It was particularly evident that higher molecular weight
species of receptors were induced in cells treated with proteasome
inhibitors. In addition, lower molecular weight species in µ and
receptor samples were stabilized in cells treated with ZLLL.
receptors were
incubated at 37 °C in serum-free media for 18 h in the absence
and presence of 25 µM ZLLL, and then subsequently treated
with and without 1 µM DADL for 10 min. Protein extracts were resolved by SDS-PAGE then assayed by immunoblotting with antibodies specific for the phosphorylated form of MAP kinase, or with
antibodies that recognize total MAP kinase protein as a control. As
shown in Fig. 10, DADL induced
phosphorylation of MAP kinase to a similar extent in both untreated and
ZLLL-treated cells, indicating that disruption of opioid receptor
turnover did not have a significant impact on
agonist-dependent signal transduction.
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Fig. 10.
Agonist-induced phosphorylation of MAP
kinase following pretreatment with a proteasome inhibitor. HEK 293 cells expressing the FLAG-tagged receptor were incubated at
37 °C in serum-free medium with or without 25 µM ZLLL
for 18 h, and then stimulated with or without DADL (1 µM) for an additional 10 min, as indicated
above the immunoblot. Cell lysates were resolved using 12%
SDS-PAGE and immunoblotted with mouse monoclonal anti-phospho-MAPK
antibody or rabbit anti-MAP kinase antibody. The mobilities of 50- and
37-kDa molecular mass standards are indicated to the right.
P-MAPK, phospho-MAP kinase immunoreactivity;
MAPK, total MAP kinase immunoreactivity. This experiment has
been repeated three times with similar results.
and µ receptors. It was
consistently observed that proteasome inhibitors, but not lysosomal
protease inhibitors, calpain inhibitors, or caspase inhibitors, were
effective at blocking agonist-induced down-regulation of both
and µ receptors (Figs. 11 and
12, respectively). As discussed earlier
using ZLLL (Fig. 9), all of the additional proteasome inhibitors
tested, including lactacystin, PSI, and ALLN, increased steady-state
and µ receptor levels when cells were preincubated with the
proteasome inhibitors in the absence of agonist (Figs. 11 and 12).
Accumulation of higher molecular weight receptor species was also
observed with all proteasome inhibitors, although this is not evident
in the cropped figures. As shown in Figs. 11 and 12, preincubation of
HEK 293 cells with lactacystin, PSI, and ALLN completely blocked
DADL-induced down-regulation of FLAG-tagged
and µ receptors. In
contrast, inhibition of lysosomal cathepsins and thiol proteases (by
E64d and leupeptin), trypsin-like serine proteases (by leupeptin),
cytoplasmic calpain (by E64d and leupeptin), and caspases (by caspase-3
inhibitor III) had little or no effect on the extent of agonist-induced
down-regulation of the
opioid receptor (Fig. 11) or µ opioid
receptor (Fig. 12). Our observation that leupeptin did not block opioid
receptor down-regulation differed from a previous study in which it was
reported that high concentrations of leupeptin (100 µg/µl)
attenuated down-regulation of the
receptor (29). We have observed
recently, however, that leupeptin inhibits agonist binding to µ and
receptors in vitro at concentrations greater than 100 µM.2
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Fig. 11.
Immunoblot analysis of the effect of
protease inhibitors on agonist-induced down-regulation of opioid
receptors. HEK 293 cells expressing FLAG-tagged
receptors were
pretreated at 37 °C in serum-free medium for 1 h with or
without 25 µM lactacystin, PSI, ALLN, E64d, leupeptin, or
caspase-3 inhibitor III (Cas-Inh), then incubated in the
absence and presence of 1 µM DADL for 18 h, as
indicated above each immunoblot. Cell lysates were prepared,
resolved by SDS-PAGE, and immunoblotted with the anti-FLAG M1
monoclonal antibody. The mobilities of 75-, 50-, and 37-kDa molecular
mass standards are indicated to the right of each
immunoblot. Proteasome inhibitors, but not lysosome, calpain, or
caspase protease inhibitors, blocked agonist-induced down-regulation of
the
receptor. The effects of each protease inhibitor have been
tested at least three times with similar results.
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Fig. 12.
Immunoblot analysis of the effect of
protease inhibitors on agonist-induced down-regulation of µ opioid
receptors. HEK 293 cells expressing FLAG-tagged µ receptors were
pretreated at 37 °C in serum-free medium for 1 h with or
without 25 µM lactacystin, PSI, ALLN, E64d, leupeptin, or
caspase-3 inhibitor III (Cas-Inh), as indicated then
incubated in the absence and presence of 1 µM DADL for
18 h, as indicated above each immunoblot. Cell lysates
were resolved by SDS-PAGE and immunoblotted with the anti-FLAG
monoclonal antibody. The mobilities of 75-, 61-, and 50-kDa molecular
mass standards are indicated to the right of each
immunoblot. Proteasome inhibitors, but not lysosome, calpain, or
caspase protease inhibitors, blocked agonist-induced down-regulation of
the µ receptor. The effects of each protease inhibitor have been
tested at least three times with similar results.
receptors. Suggestive evidence that
opioid receptors were ubiquitinated came from experiments in which
FLAG-tagged µ and
receptor cDNAs were transcribed and
translated in vitro using rabbit reticulocyte lysates, in
comparison with transcription and translation of luciferase cDNA as
a control. Following SDS-PAGE and autoradiography of the
[35S]methionine-labeled translation products, luciferase
migrated as a sharp band of 61 kDa, along with several minor bands with slightly greater mobilities (Fig. 13).
It is important to note that no bands were evident in the luciferase
sample that corresponded to molecular weights greater than the expected
luciferase protein of 61,000. In contrast, translation of the µ receptor yielded a major band migrating with an apparent molecular mass
of 42 kDa, but also included a diffuse ladder of bands with greater
molecular masses that spanned the length of the gel (Fig. 13).
Similar results were obtained when the
receptor was translated. The
predominant band migrated with an apparent molecular mass of 37 kDa,
however, also evident was a spectrum of bands migrating with slower
mobilities extending the entire length of the gel lane. Rabbit
reticulocyte lysates contain ubiquitin and the enzymes required for
conjugation of ubiquitin to proteins (37, 38), and close scrutiny of
the autoradiograms of in vitro translated opioid receptors
revealed that many bands were spaced at intervals of ~7-10 kDa,
consistent with the molecular mass of ubiquitin. Although alternative
explanations exist, it was plausible that the higher molecular weight
receptor species evident following in vitro translation or
that accumulate in cells incubated with proteasome inhibitors represent
polyubiquitinated receptors.
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Fig. 13.
In vitro transcription and
translation of µ and opioid receptors. Plasmids containing µ opioid receptor (µ OR) or
opioid receptor (
OR) cDNAs
were transcribed with T7 RNA polymerase and translated with rabbit
reticulocyte extracts in the presence of [35S]methionine
according to conditions recommended by the manufacturer. Luciferase
cDNA was transcribed by T7 RNA polymerase to serve as a control.
Translated proteins were resolved using 15% SDS-PAGE, gels were fixed,
dried, and exposed to Kodak Biomax MR film for autoradiography. The
mobilities of molecular mass standards (in kDa) are indicated to the
left. In vitro translated µ and
receptors
migrated with apparent molecular masses of 42 and 37 kDa, respectively,
however, unlike the luciferase control, a spectrum of bands migrating
with slower mobilities was also present, some of which are labeled to
the right of the lanes (<). This experiment was replicated
three times with similar results.
receptors (Fig. 14, left panel) or µ receptors (Fig. 14, right panel) were pretreated with or
without 25 µM ZLLL for 1 h at 37 °C in serum-free
medium, then incubated in the absence and presence of 1 µM DADL for 18 h. Protein extracts were prepared
using lysis buffer containing a mixture of protease inhibitors. Lysates
were immunoprecipitated with anti-FLAG monoclonal antibodies conjugated
to agarose beads, resolved by SDS-PAGE, and then transferred to PVDF
membranes, as described under "Experimental Procedures." To
validate the immunoprecipitation procedure, the blotted PVDF membranes
were initially probed with anti-FLAG M1 monoclonal antibodies to verify the presence of FLAG-tagged opioid receptors. The results (data not
shown) were very similar to those shown in Fig. 9, which correspond to
cell lysates assayed directly by immunoblotting, indicating that opioid
receptors were efficiently immunoprecipitated using the conditions
employed. When the four
receptor immunoprecipitated samples were
blotted with anti-ubiquitin antibodies, untreated (
ZLLL,
DADL) and
DADL-treated (
ZLLL, +DADL) samples displayed very low levels of
polyubiquitinated receptors (Fig. 14, left panel). In
contrast, it was clearly evident that receptors were polyubiquitinated in samples preincubated with the proteasome inhibitor, ZLLL, either in
the absence and presence of DADL (+ZLLL,
DADL and +ZLLL, +DADL, respectively, Fig. 14, left panel). In both of these
samples, polyubiquitinated receptor proteins were evident with apparent
molecular masses ranging from 42 kDa to greater than 187 kDa.
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Fig. 14.
Evidence for ubiquitination of opioid
receptors obtained by immunoprecipitation. HEK 293 cells
expressing FLAG-tagged receptors (
OR, left panel) or µ receptors (µ OR, right panel) were pretreated with or
without 25 µM ZLLL for 1 h at 37 °C in serum-free
medium, then incubated in the absence and presence of 1 µM DADL for 18 h, as indicated above the
immunoblots. Opioid receptors were immunoprecipitated by incubating
cell extracts for 2 h at 4 °C with 30 µl of anti-FLAG M1
monoclonal antibody agarose gel. Immunoprecipitated receptors were
resolved on 12% SDS-PAGE and transferred to PVDF membranes.
Ubiquitinated receptor proteins were detected with rabbit
anti-ubiquitin antibody followed by goat anti-rabbit IgG conjugated
with alkaline phosphatase. Blots were developed using CDP Star
chemiluminescence reagent. The mobilities of molecular mass standards
(in kDa) are indicated to the right of the immunoblots. This
experiment has been replicated three times with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
opioid receptor protein is evident 2-4 h
following DADL treatment and there is a near complete loss of receptor
protein following 18 h of exposure. 2) Based on pulse-chase experiments, the
receptor degradation rate is accelerated 5-fold by
agonist treatment. 3) Functional pertussis toxin-sensitive G proteins
are required for agonist-induced down-regulation of the µ opioid
receptor but not for
opioid receptor down-regulation. 4)
Agonist-induced down-regulation of opioid receptors can be blocked by
proteasome inhibitors but not by lysosomal, calpain, or caspase
protease inhibitors. 5) Proteasome inhibitors, but not lysosomal
protease inhibitors, also increase steady-state levels of µ and
opioid receptors. 6) Ubiquitinated µ and
opioid receptors
accumulate in cells treated with proteasome inhibitors.
-amino group of a lysine within the target protein. Target
protein recognition is often mediated by E3 ubiquitin-ligases. After
the linkage of ubiquitin to the substrate protein, a polyubiquitin chain is usually formed, in which the C terminus of ubiquitin becomes
linked to one of several lysine residues in the previous ubiquitin. The
polyubiquitinated proteins are recognized by the 19 S regulatory
subunits of the 26 S proteasome, the ubiquitin moieties are recycled
through the action of ubiquitin hydrolases, and the targeted protein
substrates are degraded by the 20 S catalytic core complex.
opioid receptor turnover and agonist-induced down-regulation.
mating factor, is ubiquitinated in a
ligand-dependent manner while the receptor resides in the
plasma membrane, and ubiquitination is required for endocytosis (51).
In this case, the receptor is monoubiquitinated rather than being
polyubiquitinated, and the monoubiquitin serves as an internalization
signal but does not target the receptor to the proteasome (52), which
generally requires a ubiquitin chain at least four units in length for
recognition (53). Regarding mammalian G protein-coupled receptors, it
has been reported that rhodopsin and its G protein, transducin, are ubiquitinated and subject to ubiquitin-dependent
proteolysis in vertebrate rod outer segments (54). Evidence has also
been presented that another component of GPCR signaling, namely the G
protein-coupled receptor kinase 2, is degraded by the
ubiquitin/proteasome pathway (55).
receptor
down-regulation remained unaltered. These results are in agreement with
the previous studies (56) using Neuro2A cells expressing cloned opioid
receptors, but contrast with another report in which it was stated that
in C6 cells, down-regulation of the expressed µ receptor by full
agonists was independent of G protein coupling (57). The reason for the
discrepant results is not known, but it may be related to the use of
different cell lines to express opioid receptors. The insensitivity of
receptor down-regulation to pertussis toxin may indicate that
receptor proteolysis is independent of G protein coupling, or that the agonist-stimulated receptor couples to Ptx-insensitive G proteins that
trigger pathways involved in receptor proteolysis. Indeed, evidence in
support of
receptor coupling to G
q and
G
z has been reported (58).
receptor proteins are ubiquitinated directly. It appears that the
majority of ubiquitin-immunoreactive bands present in receptor
immunoprecipitates are also recognized by the FLAG monoclonal antibody.
Ubiquitination sites on the receptors are currently being investigated
by substituting lysine residues with arginine. It is also possible,
although less likely, that the ubiquitin-immunoreactive bands in Fig.
14 represent other proteins that are tightly associated with the opioid
receptors. The protein-protein interactions giving rise to such
complexes would obviously have to withstand the relatively harsh
conditions of detergent extraction, immunoprecipitation, and SDS-PAGE.
It is also possible that opioid receptors are not targeted directly to
the proteasome complex. For example, it is conceivable that inhibition
of receptor down-regulation by proteasome inhibitors is due to
stabilization of a protein which acts to prevent receptor
down-regulation, such as a protease inhibitor, that is degraded by the
ubiquitin/proteasome pathway.
: after ligand stimulation the receptor is
polyubiquitinated, degraded by the proteasome, and resultant peptide
fragments are delivered to and further degraded in lysosomes (46). The
intracellular location where the proteasome-mediated proteolysis of the
receptors is occurring is not known at present. It is of interest to
note that mutations have been made in the
receptor that block
internalization but do not block down-regulation (34, 59). It was also
reported recently that the Src kinase inhibitor, PP1, inhibited
receptor internalization but not receptor down-regulation (60). In
addition, morphine induces down-regulation of the µ receptor, albeit
with less efficacy than DAMGO, but does not trigger receptor
internalization (61, 62). Taken together, these observations suggest
that opioid receptor proteolysis may be occurring at the plasma
membrane. In accord with this proposal, it has been reported recently
that down-regulation of the
2-adrenergic receptor does
not require endocytosis and does not involve the lysosomal degradation
pathway (63). In summary, results of this study implicate a prominent role for the ubiquitin/proteasome pathway in agonist-induced
down-regulation and basal turnover of opioid receptors.
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FOOTNOTES |
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* This work was supported by Grant DA09113 from the National Institute on Drug Abuse.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom reprint requests should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Medicine and
Dentistry of New Jersey, New Jersey Medical School, 185 South Orange
Ave., Newark, NJ 07103. Tel.: 973-972-5652; Fax: 973-972-5594; E-mail: howells@umdnj.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008054200
2 A. Khokhar, K. Chaturvedi, and R. D. Howells, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G
protein-coupled receptor;
ALLN, N-acetyl-L-leucyl-L-leucyl-L-norleucinal;
DADL, Tyr-D-Ala-Gly-Phe-D-Leu;
E64d, (2S,3S)-t-epoxysuccinyl-L-leucylamido-3-methylbutane
ethyl ester;
HEK, human embryonic kidney;
PSI, N-benzyloxycarbonyl-L-isoleucyl--t-butyl-L-glutamyl-L-alanyl-L-leucinal;
Ptx, pertussis toxin;
ZLLL, N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal;
MAP kinase, mitogen-activated protein kinase;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide gel electrophoresis;
DAMGO, [D-Ala2, N-Me-Phe4,Gly5-ol]enkephalin;
E1, ubiquitin activating enzyme;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein isopeptide ligase.
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