From the Department of Pharmacology, Temple University School of
Medicine, Philadelphia, Pennsylvania 19140 and the
Department of Microbiology and Immunology, Kimmel Cancer
Institute, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agonist-promoted internalization of some G
protein-coupled receptors has been shown to mediate receptor
desensitization, resensitization, and down-regulation. In this study,
we investigated whether opioids induced internalization of the human
and rat Opioid receptors are members of the G protein-coupled receptor
(GPCR)1 family and can be
classified into at least three types, µ, Most GPCRs show an attenuated responsiveness to agonists following
prolonged or repeated activation. Three temporally distinct processes
that occur over a time scale of seconds to days have been implicated:
desensitization (seconds to hours), internalization (minutes to hours),
and down-regulation (hours to days). These processes have been best
studied for the Internalization or sequestration is generally envisioned to be a rapid
agonist-induced movement of the receptor into a cell compartment
distinct from the plasma membrane, where it is unavailable for binding
hydrophilic ligands but remains detectable by hydrophobic ligands
(18-21). GPCR internalization has been widely studied and has been
shown to be important for resensitization of the Agonist-induced internalization of µ and We have previously established a CHO cell line stably transfected with
the human Materials--
[3H]Diprenorphine (58 Ci/mmol) was
purchased from NEN Life Science Products Inc. (Boston, MA).
( Stable Expression of Human and Rat Internalization of the
CHO-hkor cells cultured in 24-well plates were incubated with U50,488H
for the indicated intervals (up to 60 min) at 37 °C. Culture medium
was removed and the cells were washed three times on ice with ice-cold
phosphate-buffered saline (pH 7.0). Total receptor levels were assessed
by binding with 2 nM [3H]diprenorphine in the
presence or absence of 1 µM diprenorphine, while surface
receptors were measured by binding with 2 nM
[3H]diprenorphine in the presence or absence of 1 µM dynorphin A(1-17). Typically, binding was performed
at room temperature for 60 min. Diprenorphine, a hydrophobic ligand,
can bind to both cell surface and intracellular receptors, whereas
dynorphin A(1-17), a hydrophilic ligand, binds only to the cell
surface receptors. Thus, the difference between total receptor binding
and cell surface receptor binding represents binding to the
intracellular receptor pool. An increase in intracellular
[3H]diprenorphine binding over the basal level following
agonist exposure provides a quantitative measure of internalized receptors.
Expression of GRK2, MAP Kinase Phosphorylation--
CHO-hkor or CHO-rkor cells were
transferred to 24-well plates and changed to serum-free medium for
2 h to overnight to reduce basal MAP kinase phosphorylation. Cells
were treated with or without an agonist at 37 °C for 10 min and then
lysed by addition of Laemmli sample buffer. Aliquots of the lysates
were separated on SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. Phosphorylated MAP kinase was
detected by Western blot using phosphospecific MAP kinase antibodies
and enzyme-linked chemiluminescence with the PhosphoPlus p44/42 MAP
Kinase (Thr202/Tyr204) Antibody Kit according
to the manufacturer's instructions.
Statistical Analysis--
For comparison of multiple groups,
data were analyzed by analysis of variance to determine if there were
significant differences among groups. If so, Scheffe F test was
performed to determine whether there were significant differences
between control and treatment groups. For comparison of two groups,
Student's t test was performed. p < 0.05 was the level of significance in all statistical analyses.
Effects of Opioid Agonists on Internalization of the Human Agonist and Species Differences in the
We also examined whether the rat Time Courses of U50,488H-induced Internalization and Recycling of
the Human
To assess whether the internalized receptors could return to the cell
surface, CHO-hkor cells were initially treated with 1 µM
U50,488H at 37 °C for 30 min, washed extensively, and then allowed
to recover at 37 °C in the absence of agonist. Internalized receptors were found to gradually return to the cell surface over a
60-min period (Fig. 3B). The rate of receptor return was
initially rapid with a half-maximum at ~20 min and then gradually
slowed. These results indicate that U50,488H-induced Effects of Naloxone and Pertussis Toxin on Internalization of
the Human
We also examined whether coupling to pertussis toxin-sensitive G
proteins was required for internalization of the Effect of Hypertonic Sucrose Solutions on Internalization of the
Human Effect of GRK2 and GRK2-K220R on Internalization of the Human
The finding that dominant negative GRK2 attenuated U50,488H-induced Effect of
Recent studies by Menard and co-workers (61) have correlated
agonist-promoted internalization of the Effect of Dynamin I and Dynamin I-K44A on Internalization of the
Human
One potentially intriguing aspect of these results was the observation
that Role of
We next investigated whether etorphine, which did not promote
internalization of the human Methods for Assessing GPCR Internalization--
The two major
methods that have been used to examine GPCR internalization are
radiolabeled ligand binding (18-23, 27, 34, 60, 63) and fluorescence
microscopy primarily using either immunofluorescence of epitope-tagged
receptors (32-34, 36-38, 40) or fluorescence of green fluorescent
protein-tagged receptors (64, 65). In the present study, we employed
radiolabeled ligand binding to circumvent the need to express and
characterize an epitope-tagged
In conclusion, we demonstrated that agonist induced internalization of
opioid receptors stably expressed in Chinese hamster ovary
cells, the potential mechanisms involved in this process and its
possible role in activation of mitogen-activated protein (MAP) kinase.
Exposure of the human
receptor to the agonists U50,488H, U69,593,
ethylketocyclazocine, or tifluadom, but not etorphine, promoted
receptor internalization. However, none of these agonists induced
significant internalization of the rat
opioid receptor.
U50,488H-induced human
receptor internalization was time- and
concentration-dependent, with 30-40% of the receptors
internalized following a 30-min exposure to 1 µM
U50,488H. Agonist removal resulted in the receptors gradually returning
to the cell surface over a 60-min period. The antagonist naloxone
blocked U50,488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no
effect on U50,488H-induced internalization. In contrast, incubation
with sucrose (0.4-0.8 M) significantly reduced
U50,488H-induced internalization of the
receptor. While
co-expression of the wild type GRK2,
-arrestin, or dynamin I had no
effect on
receptor internalization, co-expression of the dominant
negative mutants GRK2-K220R,
-arrestin (319-418), or dynamin I-K44A
significantly inhibited receptor internalization. Whether receptor
internalization is critical for MAP kinase activation was next
investigated. Co-expression of dominant negative mutants of
-arrestin or dynamin I, which greatly reduced U50,488H-induced
internalization, did not affect MAP kinase activation by the agonist.
In addition, etorphine, which did not promote human
receptor
internalization, was able to fully activate MAP kinase. Moreover,
U50,488H or etorphine stimulation of the rat
receptor, which did
not undergo internalization, also effectively activated MAP kinase.
Thus, U50,488H-induced internalization of the human
opioid receptor
in Chinese hamster ovary cells occurs via a GRK-,
-arrestin-, and
dynamin I-dependent process that likely involves
clathrin-coated pits. In addition, internalization of the
receptor
is not required for activation of MAP kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, and
, based on
pharmacological (for review, see Ref. 1), anatomical (2), and molecular
analysis (for reviews, see Refs. 3 and 4). Activation of
opioid
receptors produces many effects including analgesia (5, 6), dysphoria
(6, 7), water diuresis (5, 6), hypothermia (8), and modulation of
immune responses (9).
opioid receptors are coupled through G
proteins to affect a variety of effectors, which include adenylate
cyclase, potassium channels, calcium channels (for review, see Ref.
10), and mitogen-activated protein kinase pathways (11).
2-adrenergic receptor (
2-AR) (for reviews, see Refs. 12-14). Chronic use of
opioid agonists causes tolerance (15) that can be partially
accounted for at the receptor level (5, 15-17).
2-AR (18, 22) as well as desensitization of m3 muscarinic receptors (m3AchR)
(23). Agonist-induced
2-AR internalization also appears to be involved in receptor down-regulation (24) and activation of
mitogen-activated protein kinase pathways (25). While the mechanisms
have not been completely elucidated, there appear to be at least two
distinct pathways involved in agonist-induced internalization of GPCRs.
The first one, for which the
2-AR has been best
characterized, is mediated by agonist-promoted phosphorylation of the
receptor by G protein-coupled receptor kinases (GRKs), binding of
-arrestin, binding of the phosphorylated receptor-
-arrestin complex to clathrin, and subsequent endocytosis of the receptor in a
process dependent on dynamin (26-30). The other, for which the
angiotensin II type 1A receptor is prototypic, appears to be
-arrestin and dynamin-independent (29). Some GPCRs, such as the
cholecystokinin receptor, appear to be internalized by both pathways
(31).
opioid receptors has
been extensively studied (20, 32-42). Etorphine and various peptide
agonists promote internalization of both µ and
opioid receptors
to transferrin-containing endosomes, while morphine and levorphanol do
not (32, 33, 37, 39-42). Hypertonic sucrose solutions block
agonist-induced internalization of µ and
opioid receptors (33,
34, 37). Overexpression of GRK2 enhanced receptor phosphorylation by
morphine and facilitated morphine-induced µ opioid receptor
internalization (43, 44), whereas expression of the dominant negative
mutant GRK2-K220M substantially reduced etorphine-induced µ opioid
receptor internalization (43). While co-expression of
-arrestin
enhanced morphine-induced µ opioid receptor internalization (44),
expression of its dominant negative mutant
-arrestin-V53D reduced
etorphine-induced µ opioid receptor internalization (43). Dominant
negative mutants of dynamin also inhibited etorphine-induced
internalization of µ and
opioid receptors (38, 43, 44). While
opioid receptor internalization has not been extensively investigated,
the potent nonselective opioid agonist etorphine did not induce
internalization of the mouse
opioid receptor expressed in HEK293
cells (38).
opioid receptor (CHO-hkor) (45, 46). These cells exhibit
the expected binding affinity and specificity for opioid ligands, and
activation by
opioid agonists enhances [35S]GTP
S
binding to pertussis toxin-sensitive G proteins and inhibits forskolin-stimulated adenylate cyclase (45, 46). In addition, we
observed that after exposure of CHO-hkor cells to U50,488H, a selective
opioid agonist, the human
opioid receptor underwent desensitization and down-regulation (46). A CHO cell line stably expressing the rat
opioid receptor (47) was also established. In
the present study, we investigated whether agonists promoted internalization of the human and rat
opioid receptors in CHO cells
and, if so, whether GRK,
-arrestin, and dynamin I were involved in
this process. In addition, since Daaka et al. (25) demonstrated that
-arrestin- and dynamin-mediated internalization of
the
2-AR and lysophosphatidic acid receptor was
essential for activation of the MAP kinase pathway, we also examined
whether agonist-promoted
receptor internalization played a critical role in activation of MAP kinase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
)-U50,488H and U69,593 were generous gifts from Upjohn Co.
(Kalamazoo, MI). Diprenorphine, etorphine, ethylketocyclazocine, and
tifluadom were provided by the National Institute on Drug Abuse.
Reagents were purchased from the indicated companies: naloxone, RBI
(Natick, MA); dynorphin A (1-17), Peninsula Laboratories (Belmont,
CA); LipofectAMINE, penicillin, and streptomycin, Life Technologies,
Inc. Co. (Manassas, VA); PhosphoPlus p44/42 MAP Kinase Antibody Kit,
New England BioLabs (Beverly, MA); Dulbecco's modified Eagle's
medium/Ham's F-12, pertussis toxin, and sucrose, Sigma; geneticin
(G418 sulfate), Mediatech Co. (Herndon, VA); fetal calf serum, Hyclone
Co. (Logan, UT). Commonly used chemicals were obtained from Sigma.
Clones of dynamin I and dynamin I-K44A (clone pUHD10-3) (48, 49) were
obtained from Drs. S. Schmid and H. Damke and cloned into pcDNA3 as
described (24).
Opioid Receptors in CHO
Cells--
CHO cells were transfected with the hkor cDNA in the
vector pBK-CMV (50) or the rkor cDNA in pcDNA3 (47) and clonal
cell lines stably expressing hkor or rkor were established with
geneticin selection (0.5 mg/ml) as described previously (45). CHO-hkor cells and CHO-rkor cells express approximately 1.2 pmol of the human
receptor and 1.0 pmol of the rat
receptor per mg of membrane
protein, respectively, as determined by [3H]diprenorphine
binding (46). CHO-hkor or CHO-rkor cells were cultured in Dulbecco's
modified Eagle's medium/Ham's F-12 supplemented with 10% fetal calf
serum, 0.1 mg/ml geneticin, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a humidified atmosphere consisting of 5%
CO2 and 95% air at 37 °C.
Receptor following Agonist
Exposure--
Opioid receptor binding on intact cells was conducted
according to Ref. 51. Briefly, binding was performed on CHO-hkor cells cultured in 24-well plates with [3H]diprenorphine in
Krebs-Ringer HEPES buffer solution (110 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 25 mM glucose, 55 mM sucrose, 10 mM HEPES, pH 7.4). Binding of 2 nM [3H]diprenorphine to untreated CHO-hkor
cells in the presence or absence of 1 µM diprenorphine
was compared at three different temperatures: 0 °C, 12 °C, and
room temperature (~22 °C). Binding reached equilibrium in 4 h
at 0 °C, 3 h at 12 °C, and 1 h at room temperature. At
equilibrium, similar binding levels were attained at the three
temperatures. Saturation [3H]diprenorphine binding to
intact cells was performed at room temperature for 1 h and the
Kd of [3H]diprenorphine was determined
to be ~2 nM, which is much higher than the
Kd of [3H]diprenorphine binding to
receptors in membranes conducted in 50 mM Tris/HCl buffer
(~0.2 nM) (45).
-Arrestin, and Dynamin I and Their
Dominant Negative Mutants--
CHO-hkor cells grown in 100-mm dishes
were transiently transfected with 8 µg of bovine GRK2 (52) in
pcDNA3.1 Zeo(+), GRK2-K220R (53) in pcDNA3.1 Zeo(+), bovine
-arrestin (54) in pcDNA 3.1 Zeo(+),
-arrestin (319-418) (55)
in pcDNA3, or dynamin I or dynamin I-K44A (48, 49) in pcDNA3
using LipofectAMINE (50 µl) following the manufacturer's
instructions. Control cells were transfected with pcDNA 3.1 Zeo(+)
or pcDNA3 and exhibited no opioid receptor binding (data not
shown). Following transfection (~18 h) the cells were incubated with
fresh medium and allowed to recover 24-28 h before being reseeded in
24-well dishes and allowed to grow an additional 24 h. The cells
were then analyzed for agonist-induced receptor internalization.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Opioid Receptor--
We first assessed whether exposure to the
selective
agonist U50,488H (1 µM) promoted
internalization of the human
opioid receptor. CHO-hkor cells were
treated with or without U50,488H at 37 °C for 30 min and
internalized receptors were determined by
[3H]diprenorphine binding. Under basal conditions,
10-15% of
receptors were found to be intracellular. An increase
in
receptor internalization was observed at 0.1 µM
U50,488H, and 1 µM U50,488H promoted internalization of
30-40% of the total receptors following a 30-min exposure (Fig. 1, A and B). Three
different equilibrium binding conditions (4 h at 0 °C, 3 h at
12 °C, and 1 h at room temperature) yielded similar results
(Fig. 1A). Typically, binding was performed at room
temperature for 1 h. Note that we have previously demonstrated that these treatment conditions (1 µM U50,488H for 30 min
at 37 °C) desensitizes the
receptor response without having any
effect on receptor down-regulation (46).
View larger version (35K):
[in a new window]
Fig. 1.
Effects of U50,488H on internalization of the
human opioid receptor. CHO-hkor cells
were treated with U50,488H at 37 °C for 30 min and internalized
receptors were determined by [3H]diprenorphine binding as
described under "Experimental Procedures." A, 1 µM U50,488H pretreatment and
[3H]diprenorphine binding at three different
temperatures; B, pretreatment with different concentrations
of U50,488H and [3H]diprenorphine binding at room temp;
Each point represents mean ± S.E. of three independent
experiments. *, p < 0.05 compared with the basal level
by analysis of variance followed by Scheffe F-test.
Opioid Receptor
Internalization--
We next tested whether other full agonists also
promoted internalization of human
opioid receptors. Etorphine, a
potent nonselective opioid agonist, did not induce internalization of the mouse
opioid receptor (38) and indeed it also did not promote
internalization of the human
opioid receptor (Fig.
2A). Since etorphine was
previously shown to promote internalization of µ and
opioid
receptors (33, 38, 40, 41), these results confirm a type-specific
difference among opioid receptors in their response to this agonist
observed by Chu and colleagues (38). In contrast, incubation with 1 µM of several full agonists, U69,593, ethylketocyclazocine, or tifluadom, effectively promoted
receptor internalization (Fig. 2A). Thus, opioid agonists appear to
have differential abilities to promote
receptor internalization. Interestingly, similar observations have been reported for µ and
opioid receptors (32, 33, 39-42). Peptide ligands and etorphine caused
internalization of µ and
receptors in HEK293 cells while morphine
and levorphanol did not (32, 33, 39, 40, 42). These differences have
also been observed in vivo. Etorphine injected intraperitoneally induced µ receptor internalization into endosomes in the guinea pig myenteric plexus (41) and the rat brain (42). However, morphine did not cause detectable internalization, and it
partially inhibited the etorphine-induced µ receptor endocytosis (41,
42).
View larger version (38K):
[in a new window]
Fig. 2.
Agonist and species differences of the
opioid receptor internalization. CHO-hkor
cells and CHO-rkor cells were treated with U50,488H, U69,593,
ethylketocyclazocine (EKC), tifluadom, or etorphine at 1 µM each at 37 °C for 30 min and internalized receptors
were determined by [3H]diprenorphine binding at room
temperature as described under "Experimental Procedures."
A, agonist-induced internalization of human
opioid
receptor; B, agonist-induced internalization of rat
opioid receptor. Each point represents mean ± S.E. of three to
five independent experiments. *, p < 0.05 compared
with the basal level by analysis of variance followed by Scheffe
F-test.
opioid receptor stably expressed
in CHO cells underwent agonist-induced internalization. To our
surprise, in contrast to the human
opioid receptor, the rat
opioid receptor was not internalized to a significant extent following
incubation with U50,488H, U69,593, ethylketocyclazocine, tifluadom, or
etorphine at 1 µM (Fig. 2B). Thus, there
appears to be a species difference between human and rat in
agonist-induced internalization of the
opioid receptor in CHO
cells. Whether the rat
opioid receptor undergoes agonist-induced
internalization has not been examined in vivo. It seems
likely that the inability of agonists to promote internalization of the
rat
opioid receptor may be due to the cell system used.
Interestingly, Murray et al. (56) reported recently that a
truncated
opioid receptor underwent agonist-induced internalization
in HEK293 cells, but not in CHO cells. However, since both rat and
human
opioid receptors were expressed in CHO cells, this
difference provides a unique opportunity for molecular and biochemical
analysis. Comparison of the third intracellular loop and C-terminal
domain sequences of the rat and human
opioid receptors reveals
several differences. One difference, which may be of relevance to
receptor internalization, is that Ser358 in the C-terminal
domain of the human receptor is replaced by Asn358 in the
rat receptor. Serine residues can be phosphorylated, while Asn cannot.
Whether this difference contributes to differential internalization is
being investigated.
Opioid Receptor--
Treatment of CHO-hkor cells with 1 µM U50,488H at 37 °C resulted in a
time-dependent increase in internalized
opioid
receptors (Fig. 3A).
Internalization was initially rapid with a half-maximal increase at
~10 min and then gradually slowed approaching a plateau at ~60
min.
View larger version (17K):
[in a new window]
Fig. 3.
Time courses of (A)
U50,488H-induced internalization of the human opioid receptor and (B) return of
internalized
receptors to the cell
surface. A, CHO-hkor cells were treated with 1 µM U50,488H at 37 °C for different periods of time.
B, CHO-hkor cells were treated with 1 µM
U50,488H at 37 °C for 30 min, washed extensively and allowed to
recover in culture medium at 37 °C for different intervals.
Internalized receptors were determined as described under
"Experimental Procedures." Each point represents mean ± S.E.
of three or four (A) or three to six (B)
independent experiments.
receptor
internalization is time-dependent and reversible after
agonist removal.
Opioid Receptor--
Naloxone (10 µM), a
nonselective opioid antagonist, had no direct effect on receptor
internalization, but effectively blocked U50,488H-induced
internalization (Fig. 4A).
Thus, U50,488H-induced
receptor internalization requires receptor
activation, although in the case of etorphine, receptor activation does
not appear to be sufficient to promote internalization.
View larger version (23K):
[in a new window]
Fig. 4.
Effects of naloxone (A),
pertussis toxin (B), and hypertonic sucrose
(C) solutions on internalization of the human
opioid receptor. A, CHO-hkor cells
were treated with or without 1 µM U50,488H at 37 °C
for 30 min in the presence or absence of 10 µM naloxone.
B, CHO-hkor cells were treated with or without 1 µM U50,488H at 37 °C for 30 min after pretreatment
with pertussis toxin (100 ng/ml) for 18 h. C, CHO-hkor
cells were treated with or without 1 µM U50,488H at
37 °C for 30 min in the presence of different concentrations of
sucrose. Internalized receptors were determined as described under
"Experimental Procedures." Each point represents mean ± S.E.
of three independent experiments. *, p < 0.05 compared
with the basal level by analysis of variance followed by Scheffe
F-test.
receptor. Pretreatment with pertussis toxin (100 ng/ml) for 18 h, which abolished U50,488H-promoted MAP kinase phosphorylation (data not shown), did not affect basal or U50,488H-induced internalization (Fig.
4B). This demonstrates that activation of pertussis
toxin-sensitive G proteins is not necessary for
receptor
internalization. In this regard, the
receptor is similar to the
opioid receptor where DADLE-promoted internalization of the
receptor in Neuro2A cells was not affected by pertussis
toxin treatment (36). In contrast, DAMGO-induced internalization of the µ opioid receptor in Neuro2A cells was completely
abolished by pertussis toxin pretreatment (36).
Opioid Receptor--
Hypertonic solutions, such as sucrose,
have been shown to inhibit receptor-mediated endocytosis by blocking
the formation of clathrin-coated pits (57). Sucrose (0.4-0.8
M) reduced U50,488H-induced internalization of the human
opioid receptor by about 90%, without affecting the basal level
(Fig. 4C). This suggests that clathrin-coated pits are
likely involved in
receptor internalization. Indeed, similar
results have been obtained with the µ and
receptors where
hyperosmolar sucrose inhibited agonist-induced internalization of µ (33, 37) and
opioid receptors (33, 34) expressed in HEK293 cells.
Opioid Receptor--
GRK2-mediated phosphorylation of the
2-AR and the m2AchR has been shown to play an important
role in agonist-induced internalization (27, 58). Thus, we next
investigated whether GRK2 was also involved in human
opioid
receptor internalization. CHO-hkor cells were transiently transfected
with wild type GRK2, the dominant negative mutant GRK2-K220R, or
vector. While expression of GRK2 had no effect on either basal or
agonist-induced internalization, GRK2-K220R expression substantially
reduced U50,488H-induced internalization (Fig.
5). This effect is quite significant
since GRK2-K220R was transiently transfected and thus only ~50% of
the cells are expressing GRK2-K220R, while 100% of the cells are
expressing the stably transfected
receptor. These observations
suggest that GRK-mediated phosphorylation of the
receptor may be
involved in mediating
receptor internalization. While we have not
directly investigated U50,488H-induced phosphorylation of the human
receptor in CHO cells, Appleyard and co-workers (59) recently
demonstrated that the
opioid receptor in guinea pig hippocampal
slices was phosphorylated to a higher degree in U50,488H-tolerant
animals than in control animals.
View larger version (37K):
[in a new window]
Fig. 5.
Effects of GRK2 and GRK2-K220R on
internalization of the human opioid
receptor. CHO-hkor cells were transiently transfected with
expression constructs for GRK2, GRK2-K220R, or vector. Sixty to 72 h later, cells were treated with or without 1 µM U50,488H
at 37 °C for 30 min and internalized receptors were determined as
described under "Experimental Procedures." Each point represents
mean ± S.E. of three independent experiments. *,
p < 0.05 compared with the vector control by analysis
of variance followed by Scheffe F-test.
receptor internalization is similar to results obtained with the
2-AR, m2AchR, and µ opioid receptors. Co-expression of
dominant negative GRK2 mutant was shown to attenuate agonist-promoted phosphorylation and internalization of both the
2-AR
(27) and m2AchR (58, 60) and internalization of the µ opioid receptor (43). That wild type GRK2 had no effect on U50,488H-induced
receptor internalization may be due to the relatively high endogenous level of GRK2 present in CHO cells (61). Indeed, expression of GRK2 had
a minimal effect on agonist-promoted internalization of the wild type
2-AR in HEK 293 cells, which also have high endogenous
levels of GRK2 (61), while having a significant effect on
internalization of
2-AR-Y326A, an
internalization-defective mutant (27). Wild type GRK2 also enhanced
morphine-induced phosphorylation and internalization of the µ opioid
receptor in HEK293 cells (43).
-Arrestin and
-Arrestin (319-418) on
Internalization of the Human
Opioid Receptor--
The non-visual
arrestins,
-arrestin and arrestin3, play an integral role in
agonist-promoted internalization of the
2-AR (28, 30).
This appears to be due to their ability to function as adapter
proteins, being capable of binding to both GRK-phosphorylated receptors
(26) and clathrin (30), the major protein component of clathrin-coated
pits. To assess the potential involvement of
-arrestin in human
opioid receptor internalization, we transiently transfected CHO-hkor
cells with
-arrestin,
-arrestin (319-418), or vector. Wild type
-arrestin had no apparent effect on
receptor internalization
(Fig. 6). However,
-arrestin
(319-418), a dominant negative mutant that inhibits receptor
internalization by binding constitutively to clathrin (55), effectively
reduced both basal and U50,488H-induced internalization (Fig.
6). These results suggest that
-arrestin plays a significant role in U50,488H-induced
internalization of the
opioid receptor in CHO cells. These results
are similar to recent observations on the µ opioid receptor (43,
44).
View larger version (35K):
[in a new window]
Fig. 6.
Effects of -arrestin
and
-arrestin (319-418) on internalization of
the human
opioid receptor. CHO-hkor
cells were transiently transfected with expression constructs for
-arrestin,
-arrestin (319-418), or vector. Sixty to 72 h
later, cells were treated with or without 1 µM U50,488H
at 37 °C for 30 min and internalized receptors were determined as
described under "Experimental Procedures." Each point represents
mean ± S.E. of three independent experiments. *,
p < 0.05 compared with the vector control by analysis
of variance followed by Scheffe F-test.
View larger version (36K):
[in a new window]
Fig. 7.
Effects of dynamin I and dynamin I-K44A on
internalization of the human opioid
receptor. CHO-hkor cells were transiently transfected with
expression constructs for dynamin I, dynamin I-K44A, or vector. Sixty
to 72 h later, cells were treated with or without 1 µM U50,488H at 37 °C for 30 min and internalized
receptors were determined as described under "Experimental
Procedures." Each point represents mean ± S.E. of three
independent experiments. *, p < 0.05 compared with the
vector control by analysis of variance followed by Scheffe
F-test.
2-AR with the
endogenous cellular levels of GRK2 and arrestins. They demonstrated
that in cell lines that have high GRK and arrestin levels, such as HEK293 and CHO, expression of wild type arrestins has a minimal effect
on
2-AR internalization (28, 29, 55, 61). However, these
cells tend to be good models for studying the effects of dominant
negative mutants of GRKs and arrestins. Thus, dominant negative
arrestin mutants such as
-arrestin (319-418) and
-arrestin-V53D, which have a reduced ability to bind to GPCRs, are effective inhibitors of agonist-promoted internalization of the
2-AR in
HEK293 cells (28, 29, 55). Conversely, cells that have lower endogenous GRK and arrestin levels, such as COS, are good for detecting effects of
wild type GRKs and arrestins on receptor internalization but have a
more limited utility for studying dominant negative proteins (30, 55,
61).
Opioid Receptor--
Dynamin I is a GTPase that regulates
the formation of clathrin-coated vesicles (62). Dynamin I mutants that
are defective in GTP binding, such as dynamin I-K44A, effectively block
endocytosis at a stage after the initiation of coat assembly and
preceding the sequestration of ligands into deeply invaginated coated
pits (48). To further investigate the mechanism of
opioid receptor internalization, we transiently transfected CHO-hkor cells with dynamin
I, dynamin I-K44A, or vector. Dynamin I-K44A significantly reduced both
basal and U50,488H-induced internalization, while wild type dynamin I
had no effect on receptor internalization (Fig. 7). These results are
similar to those previously observed for the
2-AR (29)
and µ and
opioid receptors (38, 43), and further implicate a role
for clathrin-coated pits in
opioid receptor internalization.
-arrestin (319-418) and dynamin I-K44A effectively reduced the
basal level of
receptor internalization (Figs. 6 and 7). These
results suggest that the human
receptor may be constitutively
internalized (in the absence of agonist) and recycled back to the cell
surface in a dynamic process that results in a steady state level of
internalized receptor.
-Arrestin (319-418) and dynamin I-K44A
effectively attenuated this process resulting in a lower steady state
level of internalized
receptors while U50,488H effectively
stimulated this process.
Opioid Receptor Internalization in MAP Kinase
Activation--
Activation of human
opioid receptor in CHO-hkor
cells by U50,488H increased MAP kinase phosphorylation in a
dose-dependent manner (Figs.
8 and 9).
Expression of
-arrestin (319-418) or dynamin I-K44A in CHO-hkor
cells, which effectively reduced the
receptor internalization, did
not have any effect on U50,488H-induced increase in MAP kinase
phosphorylation (Fig. 8). This result suggests that internalization of
the human
receptor is not essential for MAP kinase activation.
However, since
-arrestin (319-418) or dynamin I-K44A did not
completely block
receptor internalization, it is possible that the
internalized receptors, although fewer in number, might still be
sufficient to fully activate MAP kinase.
View larger version (40K):
[in a new window]
Fig. 8.
Effects of -arrestin
(319-418) and dynamin I-K44A on U50,488H-induced MAP kinase
phosphorylation in CHO-hkor. CHO-hkor cells were transiently
transfected with expression constructs for
-arrestin (319-418),
dynamin I-K44A, or the vector pcDNA3 and transferred to 24-well
plates 36 to 48 h later. Sixty to 72 h after transfection,
cells were changed to serum-free medium for 2 h and then treated
with or without U50,488H at 37 °C for 10 min. MAP kinase
phosphorylation was determined as described under "Experimental
Procedures."
-Arrestin (319-418) or dynamin I-K44A did not have
significant effects on MAP kinase phosphorylation. These experiments
were performed twice with similar results.
View larger version (47K):
[in a new window]
Fig. 9.
Effects of activation of human
(A) and rat (B) opioid receptors by U50,488H and etorphine on MAP kinase
phosphorylation. CHO-hkor or CHO-rkor cells were changed to
serum-free medium for 2 h or overnight and then treated with or
without U50,488H or etorphine at 37 °C for 10 min. MAP kinase
phosphorylation was determined as described under "Experimental
Procedures." Both U50,488H and etorphine effectively increased MAP
kinase phosphorylation following activation of either the human
(A) or rat (B)
opioid receptor. These
experiments were performed three (A) or two (B)
times with similar results.
receptor, increased MAP kinase phosphorylation. Etorphine was able to stimulate the human
receptor to activate MAP kinase to a similar extent as U50,488H (Fig.
9A). In addition, activation by U50,488 or etorphine of the
rat
receptor, which did not undergo internalization, effectively
activated MAP kinase (Fig. 9B). The finding is similar to
those of Fukuda et al. (11) that activation of the rat
opioid receptor expressed in CHO cells increased MAP kinase
phosphorylation. These results indicate that internalization of the
opioid receptor is not required for activation of MAP kinase. Indeed, a
similar dissociation between receptor internalization and MAP kinase
activation was observed for
2-adrenergic receptors
expressed in COS-1 cells.2
These findings are different from those of Daaka et al. (25) that internalization of
2-adrenergic and
lysophosphatidic acid receptors is essential for MAP kinase activation.
The reason for these differences between the
opioid receptor and
2-adrenergic and lysophosphatidic acid receptors is not
clear. Since the lysophosphatidic acid receptor is a
Gi/Go-coupled receptor, like the
opioid
receptor, the discrepancy cannot be attributed to differences in G
protein coupling. Different cell systems may contribute in part to the observed difference. While Daaka et al. (25) conducted their studies in HEK 293 cells, we did in CHO cells. In addition, the
opioid receptor may activate MAP kinase via a pathway distinct from
those used by
2-adrenergic and lysophosphatidic acid receptors.
opioid receptor. This technique is
dependent on the ability of hydrophobic ligands to freely pass through
the plasma membrane and bind to both cell surface and intracellular receptors, while hydrophilic ligands will only bind to cell surface receptors. While this method is simple and enables an accurate quantitation of cell surface and internalized receptors, it does have
potential limitations. Perhaps the main limitation for most GPCRs is
the availability of effective hydrophobic and hydrophilic ligands.
While such compounds are available to study the
2-AR, mAchRs, and opioid receptors, there are many GPCRs where this method
cannot be used. Another limitation is the requirement for effective
removal of the pretreatment ligand. For ligands that are difficult to
wash out, such as dynorphin peptides and norbinaltorphimine, it is
impossible to determine their effects on receptor internalization using
this method. We nevertheless feel that this is an appropriate method
for the present study. Indeed results obtained with similar binding
methods (18, 19, 22, 27, 34, 63) were often confirmed using
immunofluorescence or fluorescence methods (30, 34, 38, 64, 65).
opioid receptors. There appears to be agonist and species
differences. Exposure of the human
receptor to U50,488H, U69,593,
ethylketocyclazocine, or tifluadom, but not etorphine, promotes a time-
and concentration-dependent increase in intracellular receptors. In contrast, the rat
opioid receptor was not
internalized significantly following exposure to the same agonists.
U50,488H-induced human
receptor internalization is reversible and
requires receptor activation, but not receptor/G protein coupling.
U50,488H-induced internalization of the human
opioid receptor is
mediated by a GRK-,
-arrestin-, and dynamin I-dependent
process that likely involves clathrin-coated pits. Finally,
agonist-induced receptor internalization of the
receptor is not
essential for MAP kinase activation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Drs. S. Schmid and H. Damke of the Scripps Research Institute for cDNA clones of dynamin I and dynamin I-K44A.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DA04745, DA06650, and DA11263 (to L.-Y. L.-C.) and GM44944 and GM47417 (to J. L. B.) and the Adolor Corp. (to L.-Y. L.-C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Established Investigator of the American Heart Association.
¶ To whom correspondence should addressed: Dept. of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4188; Fax: 215-707-7068; E-mail: liuche{at}astro.temple.edu.
2 DeGraff, J. L., Gagnon, A. W., Benovic, J. L., and Orsini, M. J. (1999) J. Biol. Chem., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
hkor, human opioid receptor;
GRK, G
protein-coupled receptor kinase;
CHO, Chinese hamster ovary cells;
CHO-hkor cells, Chinese hamster ovary cells stably transfected with the
cloned human
opioid receptor;
CHO-rkor, CHO cell line stably
transfected with the rat
opioid receptor;
rkor, the rat
opioid
receptor;
2-AR,
2-adrenergic receptor;
mAchR, muscarinic cholinergic receptor;
MAP kinase, mitogen-activated
protein kinase;
U50, 488H,
(
)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide;
GTP
S, guanosine 5'-3-(thio)-triphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|