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The Journal of Neuroscience, January 1, 1999, 19(1):56-63
Requirement of Receptor Internalization for Opioid Stimulation of
Mitogen-Activated Protein Kinase: Biochemical and Immunofluorescence
Confocal Microscopic Evidence
Elena G.
Ignatova,
Mariana M.
Belcheva,
Laura M.
Bohn,
Mark
C.
Neuman, and
Carmine J.
Coscia
E. A. Doisy Department of Biochemistry and Molecular Biology,
St. Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
Previously, we implicated the opioid receptor (OR),
G  subunits, and Ras in the opioid activation of
extracellular signal-regulated protein kinase (ERK), a member of the
mitogen-activated protein (MAP) kinase family involved in mitogenic
signaling. We now report that OR endocytosis also plays a role in the
opioid stimulation of ERK activity. COS-7 and HEK-293 cells were
cotransfected with the cDNA of  -, µ-, or  -OR, dynamin wild-type
(DWT), or the dominant suppressor mutant dynamin
K44A, which blocks receptor endocytosis. The activation of ERK by
opioid agonists in the presence of DWT was detected. In
contrast, parallel ectopic coexpression of the K44A mutant with OR,
followed by agonist treatment, resulted in a time-dependent attenuation
of ERK activation. Immunofluorescence confocal microscopy of -OR and
DWT-cotransfected COS-7 cells revealed that agonist
exposure for 10 min resulted in an ablation of cell surface -OR
immunoreactivity (IR) and an intensification of cytoplasmic (presumably
endosomal) staining as seen in the absence of overexpressed
DWT. After 1 hr of -agonist exposure the cells displayed
substantial internalization of -OR IR. If the cells were
cotransfected with -OR and dynamin mutant K44A, OR IR was retained
on the cell surface even after 1 hr of -agonist treatment. Parallel
immunofluorescence confocal microscopy, using an anti-ERK antibody,
showed that agonist-induced time-dependent ERK IR trafficking into
perinuclear and nuclear loci was impaired in the
internalization-defective cells. Thus, both biochemical and
immunofluorescence confocal microscopic evidence supports the
hypothesis that the opioid activation of ERK requires receptor internalization in transfected mammalian cells.
Key words:
opioid receptor; opioids; MAP kinase; dynamin; ERK; immunofluorescence confocal microscopy
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INTRODUCTION |
Growth factors elicit a diversity of
cell type-specific responses that regulate cell proliferation. Their
signal transduction pathways involve a series of protein
phosphorylations that begin with a receptor tyrosine kinase (RTK) on
the plasma membrane, continue in the cytoplasm, and culminate in the
nucleus. Several receptor-protein interactions, typically via Src
homology domains (SH2/SH3), result in Ras/Raf activation. Raf
phosphorylates mitogen-activated protein kinase kinase (MEK), which
activates the mitogen-activated protein (MAP) kinases. The latter
include extracellular signal-regulated protein kinases (ERKs), Jun
protein kinases (JNKs), and p38mapk isoforms, all of
which phosphorylate different transcription factors in the nucleus
(Cobb and Goldsmith, 1995 ; Segal and Greenberg, 1996; Khokhlatchev et
al., 1998). Thus, the MAP kinase phosphorylation cascade plays a
central role in growth factor signal transduction.
G-protein-coupled receptors (GPCRs) also mediate MAP kinase activation
(Van Biesen et al., 1996; Gutkind, 1998). Diverse GPCR signal
transduction pathways converge with RTK phosphorylation cascades.
Receptors coupled to pertussis toxin-sensitive G-proteins activate ERKs
and JNKs via G subunits, whereas those coupled to
pertussis toxin-insensitive G-proteins typically activate ERK and JNK
by G .
Opioids stimulate ERK activity in brain (Berhow et al., 1996), in
µ-opioid receptor-transfected (OR-transfected) Chinese hamster ovary
(CHO) cells (Li and Chang, 1996), and in -OR-transfected Rat-1
fibroblasts (Burt et al., 1996). Insight into the molecular mechanism
by which opioids activate ERK has been gained. Belcheva et al. (1998)
implicated G subunits of Gi/o proteins and Ras in the opioid agonist modulation of ERK in COS-7 cells transfected with µ-, -, or -ORs.
Receptors internalize via clathrin-coated vesicles, caveolae, or
uncoated vesicles (Haigler et al., 1980; Raposo et al., 1989; Anderson
et al., 1992; von Zastrow and Kobilka, 1992). GPCR endocytosis is a
dynamic process required for agonist-induced turnover and resensitization. In this sequence the receptors are phosphorylated by
GPCR kinases, causing uncoupling from their cognate G-protein, followed
by endocytosis. Initial evidence of GPCR internalization via a
clathrin-mediated mechanism was obtained by the discovery of OR and
 -adrenergic receptor binding in highly purified preparations of
clathrin-coated vesicles (Bennett et al., 1985; Chuang et al., 1986).
Subsequently, supporting evidence was obtained for these GPCRs (von
Zastrow and Kobilka, 1992; Trapaidze et al., 1996; Chu et al., 1997;
Segredo et al., 1997). Clathrin-coated vesicle formation entails ATP-
and GTP-dependent steps, one of the latter being mediated by dynamin.
Dynamin migrates from cytosol to specific sites in clathrin-coated
structures in its GDP-bound form. GTP binding causes the redistribution
of dynamin and its assembly into a collar at the neck of invaginated
coated pits. Subsequent GTP hydrolysis induces a conformational change
that leads to a detachment of the coated vesicle from the plasma
membrane. Mutation of Lys44 to Ala in the amino acid
sequence of dynamin disrupts its GTPase activity and imposes a
defect in endocytosis. Vieira et al. (1996) reported that the
activation of ERK-1/ERK-2 by EGFR is suppressed in cells transfected
with dynamin mutant K44A. Thus, endocytic trafficking of EGFR appears
to be important for the full activation of ERKs. Five mechanistically
distinct inhibitors of clathrin-mediated endocytosis attenuated ERK
activation by GPCR agonists (Luttrell et al., 1997a; Daaka et al.,
1998), indicating that GPCR endocytosis is required for ERK activity.
Here we report biochemical and immunocytochemical evidence for the
requirement of OR internalization for ERK activation by agonists.
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MATERIALS AND METHODS |
Chemicals. Most chemicals were purchased from Sigma (St.
Louis, MO) with the exception of
[D-Pen2,
D-Pen5]enkephalin (DPDPE),
[D-ala-D-Leu]enkephalin (DADLE), and
[D-ala2,mephe4,gly-ol5]
enkephalin (DAMGE), which were obtained from Multiple Peptide Systems
(San Diego, CA); anti-ERK antibody (Ab) that cross-reacts with ERK-1
and ERK-2 from Santa Cruz Biotechnology (Santa Cruz, CA);
FITC-conjugated donkey anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West Grove, PA); [ -32P] ATP (3000 Ci/mmol) from Amersham (Arlington Heights, IL); and U69,593 from NIDA
Drug Supply (Research Triangle Park, NC).
Cell culture and transfection. COS-7 cells were grown at
37°C in a humidified CO2 (5%) incubator in DMEM
and Ham's nutrient mixture F12 containing 10% heat-inactivated calf
serum for six passages after they were transferred from the same medium
with 10% fetal calf serum. COS-7 cells were grown in 10 cm Petri
dishes and seeded routinely in six-well plates 24 hr before
transfection to achieve 50-60% confluency on the day of transfection.
Transient transfections were performed with ERK-1 cDNA (in pcDNA-3
vector; kindly provided by Dr. J. Baldassare, St. Louis University, St. Louis, MO) or with -OR cDNA (in pCI-neo expression vector) in the
presence of DWT or its mutant form K44A (both in pCB1
vector; kindly provided by Dr. Marc Caron, Duke University, Durham,
NC), using the DEAE-dextran method (Belcheva et al., 1998). HEK-293 cells were grown in Minimum Essential Medium (MEM) containing 10%
fetal bovine serum. Cells were grown in six-well plates, and the
transient transfection of rat µ- or -OR cDNA (pCMV-neo expression vector) as well as the plasmids described above was performed with
Lipofectamine Reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's description. Transfection efficiencies were monitored by OR binding assays (Belcheva et al., 1998). Similar levels of -OR in DWT and K44A-transfected COS-7 cells
were detected by immunoblot analysis.
ERK assay. ERK activity was measured by using the
"in-gel" kinase method as described in Belcheva et al. (1998).
Briefly, OR and dynamin wild-type (DWT) or
K44A-transfected COS-7 or HEK-293 cells that were serum-starved
overnight were exposed to type-specific opioids (0.1 µM
DADLE or 1 µM DPDPE for , 1 µM DAMGE for
µ, and 1 µM U69,593 for ) for the indicated time
periods. Then the cells were washed with cold PBS and lysed with
buffer containing (in mM) 20 HEPES, 10 EGTA, 40 -glycerophosphate, 2.5 MgCl2, 2 sodium vanadate,
and 1 PMSF plus 1% Nonidet-40, 20 µg/ml aprotinin, and 20 µg/ml
leupeptin. Lysates were spun at 14,000 × g for 20 min at 4°C, and the protein concentration of supernatants was determined before the enzyme assay by the micro-Bradford assay (Bio-Rad, Hercules,
CA) with bovine serum albumin as a standard. Cell lysates (30-50 µg
of protein/lane) were separated by 10% SDS-PAGE. Resolving gels
contained 0.5 mg/ml myelin basic protein (MBP). To remove SDS after
electrophoresis, we washed the gels twice with 100 ml of 50 mM Tris-HCl, pH 8.0, containing 20% isopropyl alcohol for 30 min. Then a 1 hr wash with 50 mM Tris-HCl, pH 8.0, and 5 mM -mercaptoethanol was followed by protein denaturation
with 100 ml of 6 M guanidine chloride (twice, 30 min each).
Finally, proteins in gels were renatured with buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM -mercaptoethanol,
and 0.04% Tween 40 for 16 hr at 4°C with at least four to five
changes of buffer. Gels were incubated with 40 mM HEPES
buffer, pH 8.0, containing 2 mM DTT and 10 mM MgCl2 at room temperature for 1 hr. The kinase assay was
performed by placing gels in 40 mM HEPES buffer, pH 8.0, containing 0.5 mM EGTA, 10 mM
MgCl2, 2 µM protein kinase inhibitor
(PKI), and 40 µM -32P-ATP (50 µCi) for 3 hr at room temperature. Gels then were rinsed with a solution of 5%
TCA and 1% sodium pyrophosphate to remove noncovalently bound
32P. Kinase activity, as expressed by the degree of
phosphorylation of MBP, was quantified with the Storm PhosphoImager
(Molecular Dynamics, Sunnyvale, CA) and Image Quant software.
Statistical analyses were performed with the Student's t test.
Phosphatidyl inositide (PI) turnover. COS-7 cells were
transfected with -OR and either DWT or dynamin mutant
K44A. After 48 hr of starvation in serum-free MEM, cells were labeled
overnight in the same medium with 1.5 µCi
myo-[2-3H(N)]-inositol (20 Ci/mmol; New
England Nuclear, Boston, MA) per milliliter. The medium was replaced
with fresh medium containing 5 mM LiCl for a 30 min
incubation before DADLE (1 µM, 1 hr) treatment. The cells
were washed twice in cold PBS and collected in PBS-EDTA with scraping.
Inositol phosphates were extracted in a 1:1 ratio of
methanol/chloroform and eluted from Bio-Rad AG1 × 8 columns with
1 M ammonium formate in 0.1 M formic acid, as
described (Barg et al., 1994).
Immunohistochemistry. Polyclonal Abs were generated against
a mouse -OR CTe peptide with the sequence TRERVTACTPSDGPG (amino acids 353-367). Cells transfected with -OR and ERK or
DWT or K44A mutant were grown on coverslips and were
treated with different ligands for the indicated time periods. Cells
then were fixed in 4% paraformaldehyde for 15 min. After three 5 min
washes with TBS (20 mM Tris, pH 7.4, 150 mM
NaCl, and 1 mM CaCl2), the cells were
permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were
washed again three times with TBS and blocked for 30 min in 3% milk
dissolved in 50 mM Tris, pH 7.4, containing 0.1% Triton
X-100. After washing, a primary Ab (Anti-CTe -OR or anti-ERK) was
applied and incubated at 4°C for 18 hr with constant shaking. Then
the cells were washed five times and incubated with secondary Ab
(FITC-conjugated donkey anti-rabbit IgG) for 1 hr at room temperature. Coverslips were washed five times with TBS and twice in H2O
and mounted on slides that used n-propyl gallate as
fluorescence protectant. Slides were examined with a Zeiss LSM 410 scanning laser confocal microscope system (Oberkochen, Germany). The
system uses a Zeiss 135 Axiovert inverted microscope fit with an
Omnicron argon/krypton laser for confocal excitation. The cells,
mounted on coverslips, were viewed with either a 63× magnification,
1.25 numerical aperture oil or water immersion objective. For the FITC
fluorophore, fluorescence was excited with the 488 nm laser line, and
emission was collected in the 510-540 nm region. In experiments on
-OR and DWT or K44A-expressing COS-7 cells, 50 untreated
and 100 agonist-treated cells were quantified for staining to identify
representative micrographs. Of these cells ~30% displayed the -OR
staining patterns shown.
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RESULTS |
Biochemical evidence for the requirement of OR internalization in
the activation of ERK
To determine the effect of overexpression of DWT
and mutant K44A on classical opioid second messenger signaling, we
assayed COS-7 cells, cotransfected with cDNA of -OR and the dynamins for DADLE-stimulated PI turnover. As seen in Figure
1, DADLE significantly increased PI
turnover in cells transfected with DWT or K44A. The same PI
stimulation was observed in cells expressing DWT or K44A. The data are in agreement with a report that overexpression of the
dynamin mutant K44E did not affect opioid inhibition of cAMP accumulation in HEK-293 cells (Chu et al., 1997). They are also consistent with our previous findings that overexpressed -OR (naltrindole binding parameters, KD = 0.7 ± 0.4 nM; Bmax = 1.3-2.3 pmol/mg
protein) in COS-7 cells is functional (Belcheva et al., 1998).
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Figure 1.
Dynamin mutant K44A overexpression does not affect
DADLE-induced PI turnover in COS-7 cells. Cells transfected with -OR
and DWT or mutant K44A were treated for 1 hr with 1 µM DADLE and assayed for inositol phosphate accumulation.
Open bars, Control cells; hatched bars,
DADLE-treated cells. The data are expressed as a percentage of the
control of PI turnover in untreated cells (average counts for controls
in DWT, 6918 dpm/well; in K44A, 9373 dpm/well) and
are represented as the mean of four experiments performed in
duplicate ± SEM; *p < 0.05.
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Because it is established that different agonists have different
efficacies in inducing receptor internalization and that mechanisms of
GPCR-induced ERK activation are cell type-dependent, it was important
to show the effects of the dynamin mutant on at least two cell lines
tested with several opioid agonists. COS-7 cells, cotransfected with
the cDNA of -OR and DWT or mutant K44A, were treated
with DADLE or DPDPE for different time intervals, and the ERK activity
was measured. As seen in Figure 2, a 2.5- to 3.5-fold activation of ERK by 10 min of agonist treatment was detected in the presence of DWT. Although ERK activation by
1 µM DPDPE continued for 60 min, 0.1 µM
DADLE stimulation of the enzyme did not persist as long. In contrast,
parallel ectopic coexpression of the K44A mutant with -OR, followed
by treatment with either agonist, resulted in the inhibition of ERK
stimulation by 33-44% at 10 min.
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Figure 2.
Evidence for the requirement of OR internalization
in the opioid activation of ERK in COS-7 cells. Cells cotransfected
with -OR and DWT or mutant K44A were treated with 1 µM DPDPE (A) or 0.1 µM DADLE (B) for different time
intervals. Top, Representative autoradiograms showing
the phosphorylated MBP bands. Bottom, Quantification of
ERK activity. n = 2-6; *p < 0.05.
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Similar results were gained in time course experiments that used -,
µ-, or -OR-transfected HEK-293 cells (Fig.
3). Agonist-induced ERK activation was
reduced in endocytosis-defective cells for all receptor types and for
the -OR at all time points. DPDPE stimulation of ERK also was
reduced at the three earliest time points that were studied, whereas
DADLE effects were attenuated only at 10 min in the presence of K44A.
These experiments suggest that the inhibition of receptor
internalization attenuated the activation of ERK at early time points,
which coincided with the highest levels of ERK activation by opioid
agonists.
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Figure 3.
Evidence for the requirement of OR internalization
in the opioid activation of ERK in HEK-293 cells. Cells cotransfected
with -, µ-, or -OR, DWT, or mutant K44A were
treated with 1 µM DPDPE (A), 0.1 µM DADLE (B), 1 µM
DAMGE (C), or 1 µM U69,593
(D) for different time intervals.
Top, Representative autoradiograms showing the
phosphorylated MBP bands. Bottom, Quantification of ERK
activity. n = 2-6; *p < 0.05.
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Agonist-induced -OR immunoreactivity (IR) trafficking in
COS-7 cells
To monitor the intracellular migration of -OR IR and ERK IR
under the same conditions as described above, we plated COS-7 cells on
microscope coverslips, treated them with agonists, and subjected them
to immunofluorescence confocal microscopy. The specificity of
anti--OR Ab was examined by using NG108-15 and parental COS-7
cells. NG108-15 cells express relatively large amounts of -OR (0.6 pmol/mg protein) and have been used extensively as a model system for
the study of -opioid action. NG108-15 cells incubated with
anti--OR Ab that had been preadsorbed with the antigenic peptide
showed little or no staining again as seen for preimmune serum
treatment (Fig. 4). Parental COS-7 cells
displayed no staining with anti--OR Ab, and they resembled
-OR-transfected controls treated with preimmune serum (Fig. 4).
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Figure 4.
Specificity of anti--OR Ab. NG108-15 cells
were fixed, permeabilized, and stained with preimmune serum (1:1000),
anti--OR (Anti-DOR1 Ab; 1:1000) preadsorbed with the
antigenic peptide (0.3 mg/ml), and FITC-conjugated secondary Ab or
anti--OR Ab (1:1000 dilution). COS-7 cells overexpressing -OR
were processed as above and stained with preimmune serum or anti--OR
Ab (1.5 µg/ml). Parental COS-7 cells were stained with anti--OR Ab
(1.5 µg/ml). Shown are representative micrographs from two to six
independent experiments.
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COS-7 cells cotransfected with -OR and DWT or mutant
K44A were treated with DADLE for 10 min or 1 hr and stained with
anti--OR Ab. COS-7 cell surface staining of -OR seen in the
untreated DWT control cells dissipated after 10 min of
exposure to agonist; instead, more granular cytoplasmic staining
suggestive of endosomal compartmentation was observed (Fig.
5). Similarly, in dual immunofluorescence confocal microscopy experiments, 10 min of agonist exposure elicited colocalization of -OR IR and transferrin-Texas Red in NG108-15 cells, indicative of endosomal compartmentation (data not shown). After
1 hr of agonist treatment the plasma membrane staining was dissipated
and fluorescence was localized predominantly in perinuclear/nuclear regions of the cell (Fig. 5). Immunofluorescence confocal microscopy experiments on -OR-transfected HEK-293 cells displayed similar receptor endocytosis except that less perinuclear/nuclear staining was
observed (data not shown). However, in cells transfected with the K44A
mutant, -OR IR was retained on the cell surface at all time
intervals in both COS-7 and HEK-293 cells.
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Figure 5.
Confocal microscopy of -OR IR in
agonist-treated COS-7 cells. Cells were cotransfected with -OR and
DWT or mutant K44A. Control and agonist-treated (0.1 µM DADLE for 10 min or 1 hr) cells were fixed,
permeabilized, and immunostained with anti--OR Ab (2 µg/ml) and
FITC-conjugated secondary Ab. Shown are representative micrographs from
four independent experiments.
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Agonist-induced ERK IR trafficking in COS-7 cells
ERK IR also was displaced after DADLE or DPDPE exposure of COS-7
cells cotransfected with -OR and ERK. Untreated controls and acute
(10 min) DADLE- or DPDPE-treated cells showed diffuse cytoplasmic
staining (Fig. 6). After 1 hr of agonist
exposure the staining became less diffuse and more localized in the
perinuclear/nuclear regions of the cell (Fig. 6). This distribution of
ERK IR is consistent with subcellular fractionation data and is typical
of what is seen in other immunohistochemical studies of nuclear import
of ERK [Khokhlatchev et al. (1998) and references cited therein]. Antagonist (1 µM naltrexone) pretreatment 1 hr before
DADLE prevented ERK nuclear import. Naltrexone alone had no effect on
ERK trafficking (data not shown).
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Figure 6.
Confocal microscopy of ERK IR in agonist-treated
COS-7 cells. Cells were cotransfected with ERK-1 and -OR. DADLE (0.1 µM) or DPDPE (1 µM) was added to the medium
for 0 or 10 min or 1 hr. The bottom panel displays the
effect of a 1 hr pretreatment of cells with 1 µM
naltrexone before DADLE exposure. After fixation and permeabilization
the cells were stained with anti-ERK Ab (0.8 µg/ml) and
FITC-conjugated secondary Ab. Shown are representative micrographs from
three to four independent experiments.
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When COS-7 cells were cotransfected with -OR and DWT or
mutant K44A, DADLE initiated a time-dependent ERK IR nuclear import only in DWT-expressing cells (Fig.
7). Again, diffuse cytoplasmic ERK IR
staining was observed after 10 min of agonist exposure. By 30 min of
agonist treatment the ERK IR accumulated in perinuclear/nuclear regions, which persisted for at least 1 hr. Endocytosis-defective cells
displayed only cytoplasmic staining of ERK at all time intervals, resembling that seen for 0 and 10 min in DWT-overexpressing
cells. These results suggest that ERK activation and nuclear import
require receptor internalization.
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Figure 7.
Confocal microscopy of ERK IR in agonist-treated
dynamin-transfected COS-7 cells. Cells were cotransfected with
DWT or mutant K44A and -OR. DADLE (0.1 µM)
was added to the medium for 0, 10, or 30 min or 1 hr. After fixation
and permeabilization the cells were stained with anti-ERK Ab (0.8 µg/ml) and FITC-conjugated secondary Ab. Shown are representative
micrographs from four to six independent experiments.
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DISCUSSION |
By using receptor endocytosis-defective cells, we provide both
biochemical and immunofluorescence confocal microscopic evidence to
demonstrate that receptor internalization is required for ERK activation. The biochemical findings reveal that µ-, -, and
-opioid activation of ERK is attenuated in cells overexpressing
dynamin mutant K44A. In a previous communication on the essential role of GPCR endocytosis for ERK activation, defective -adrenergic sequestration was monitored by cell sorting (Daaka et al., 1998). The
immunohistochemical data shown here reveal that -OR internalization and intracellular migration as well as ERK trafficking to the nucleus
are impeded under the same conditions in receptor endocytosis-defective cells.
The existence of intracellular ORs has been recognized for some time
(Roth et al., 1981); initially, it was thought that internalization was
required only for resensitization and turnover. Although it is clear
that OR endocytosis is not necessary for classical GPCR signaling to
generate cytosolic second messengers, recent evidence suggests that the
internalization of ORs and other GPCRs may play a role in gene
transcription (Belcheva et al., 1993, 1996; Tencheva et al., 1997; Lu
et al., 1998; Ventura et al., 1998). The data gained here support this hypothesis.
ERK IR appears to be in both cytosol and nuclei of untreated COS-7
cells (Figs. 6, 7). This cannot be attributable to overexpression of
ERK in the COS-7 cells, because ERK-containing plasmids were included
only in the initial experiment (Fig. 6), but not the second (Fig. 7).
The data from both experiments indicate that upon agonist treatment a
time lag exists between ERK activation and its nuclear import. ERK is
phosphorylated maximally within 10 min, and some nuclear ERK is found
at 0 and 10 min time points. Nevertheless, ERK IR that is localized in
perinuclear/nuclear regions is observed only after 30 min. The
classical paradigm predicts that ERK is activated in the cytoplasm and
then is translocated into the nucleus [Khokhlatchev et al. (1998) and
references cited therein]. This is consistent with the marked
redistribution of ERK IR that occurs 30 min after opioid agonist
treatment. The smaller amounts of nuclear ERK IR at earlier time points
could be residual phosphorylated and unphosphorylated molecules
persisting there from previous activation [Khokhlatchev et al. (1998)
and references cited therein]. Moreover, phospho-ERK IR has been found in the cytoplasm and nuclei in HEK-293T and HEK-293 cells; within 90 min of agonist exposure, nuclear labeling is displayed (Joneson et al.,
1998) (E. Ignatova, unpublished observations). Another possibility
recently raised is that ERK migration into the nucleus precedes its
activation by MEK. MEK has a nuclear export signal, and there is
evidence to suggest that it enters the nucleus where it could
phosphorylate ERK (Jaaro et al., 1997; Kim and Kahn, 1997). However,
ERK-3, a widely distributed isoform that is restricted to the nucleus,
may be the target of nuclear MEK (Cheng et al., 1996). The results
obtained here are more compatible with the notion that the MEK
activation of ERK precedes trafficking into the nucleus, but carefully
controlled kinetic studies that use ERK subtype specific Abs for both
phosphorylated and unphosphorylated forms must be performed to resolve
this issue.
The requirement of EGF receptor internalization for ERK stimulation has
been reported despite the fact that membrane-delimited "upstream"
effectors in the EGF pathway such as PLC, Shc, and Raf are activated
in endocytosis-defective cells (Vieira et al., 1996; Daaka et al.,
1998). Here we show that opioid activation of PLC, presumably the
-isoform, is also insensitive to endocytosis blockade. The data in
the literature suggest that a multi-adaptor receptor complex containing
components of both GPCR and RTK pathways may come together within the
cell to trigger the activation of ERK via the cytoplasmic kinase, MEK.
Many membrane-delimited intermediates of this pathway are believed to
be arranged on a protein scaffold that serves to assemble different
components of phosphorylation cascades (Levin and Errede, 1995; Wang et
al., 1996; Luttrell et al., 1997b). The scaffold may be the RTK itself
in some cells (Luttrell et al., 1997b), whereas in others caveolin is a
candidate (Mineo et al., 1996; Couet et al., 1997). In fact, EGFR may
undergo endocytosis via clathrin- or caveolin-mediated pathways, both of which can be blocked by the dynamin mutant K44A (Haigler et al.,
1980; Mineo et al., 1996; Couet et al., 1997; Henley et al., 1998; Oh
et al., 1998). In addition, it has been proposed that growth factors
stimulate the release of dynamin from a complex with the adaptor
protein Grb2, thereby promoting receptor endocytosis (Vidal et al.,
1998). Taken together with the above-mentioned evidence for cytoplasmic
phosphorylation of ERK, an attractive hypothesis is that the
internalized receptor or receptors play a direct role in activating ERK
and possibly even in transferring it to the nucleus. The MAP kinase
phosphorylation cascade has been shown to turn the cell cycle in
oocytes on and off in a "switch-like" manner (Ferrell and
Machleder, 1998; Koshland, 1998). Regulation of ERK by receptor
endocytosis may be a part of the "switch" mechanism.
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FOOTNOTES |
Received June 26, 1998; revised Sept. 21, 1998; accepted Oct. 19, 1998.
This work was supported by National Institutes of Health Grant DA05412.
We thank Dr. John Freeman for performing the confocal microscopy, Matt
Mabery for assistance in densitometric analysis, and Myra Kim for
maintaining cell culture and for transfections.
Correspondence should be addressed to Dr. Carmine J. Coscia, Department
of Biochemistry and Molecular Biology, St. Louis University School of
Medicine, St. Louis, MO 63104.
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Top
Abstract
Introduction
