J Biol Chem, Vol. 274, Issue 43, 30636-30643, October 22, 1999
Regulated Migration of Epidermal Growth Factor Receptor from
Caveolae*
Chieko
Mineo
,
Gordon N.
Gill§, and
Richard G. W.
Anderson¶
From the Department of Cell Biology and Neuroscience, University
of Texas Southwestern Medical Center, Dallas, Texas 75235-9039 and the
§ Department of Medicine, University of California, San Diego,
La Jolla, California 92093-0650
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ABSTRACT |
In quiescent fibroblasts, epidermal growth factor
(EGF) receptors (EGFR) are initially concentrated in caveolae but
rapidly move out of this membrane domain in response to EGF. To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in
cells lacking endogenous EGFR. All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae. By
contrast, migration from caveolae required EGF binding, an active
receptor kinase domain, and at least one of the five tyrosine residues
present in the regulatory domain of the receptor. Movement appears to
be modulated by Src kinase, is blocked by activators of protein kinase
C, and occurs independently of internalization by clathrin-coated pits.
Two mutant receptors previously shown to induce an oncogenic phenotype
lack the ability to move from caveolae in response to EGF, suggesting
that a prolonged residence in this domain may contribute to abnormal
cell behavior.
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INTRODUCTION |
The most common mutant
EGF1 receptor (EGFR) found in
human tumor cells has a truncated extracellular domain, is
constitutively active at the cell surface, and is not down regulated by
either EGF or anti-EGFR IgG (1). Other oncogenic EGFR, those missing the cytoplasmic regulatory domain but having an active kinase domain,
also fail to down regulate (2). Down-regulation is the term used to
describe the attenuation of signal transduction by receptor-mediated
endocytosis. Detailed studies of the normal EGFR have found that
endocytosis under these conditions is a high affinity, saturable
process that involves the interaction of endocytic codes in the
receptor cytoplasmic tail with an unidentified set of molecules present
in clathrin-coated pits (3, 4). Receptors that can not down regulate,
therefore, are either missing the information required for capture or
are incapable of accessing coated pits. None of the studies carried out
so far have distinguished between these two mechanisms.
To determine which of these two mechanisms accounts for the behavior of
mutant, oncogenic EGFR that fail to down regulate, the location of the
unstimulated receptors must first be determined. EGFR might be randomly
distributed across the surface or confined to specialized membrane
domains. Membrane fractionation and immunocytochemistry has been used
to show that in quiescent fibroblasts wild-type EGFR (5, 6), as well as
other receptor (7) and non-receptor (8) tyrosine kinases, are highly
enriched in caveolae membrane fractions. Moreover, the first phases of
signal transduction initiated by EGF binding, such as activation of
tyrosine kinase activity (6, 7), phosphorylation of protein substrates
(7), recruitment of adaptors (6, 7, 9) and essential kinases (7), and activation of MAP kinase (7, 10), all appear to take place in caveolae
membranes. These and other studies (reviewed in Ref. 8) indicate that
entire signaling pathways are pre-organized in caveolae. Rapid signal
attenuation coincides with loss of receptors from caveolae (6) in
response to ligand binding. Depending on the cell type, it takes 3-30
min for EGFR to leave caveolae (6, 7). Obviously the exit of the
receptor from caveolae must be an important control point in EGFR
signal transduction. Spending too little or too much time at this
location after ligand binding may be deleterious to the cell.
Mutagenesis has been used to map the critical amino acids in EGFR
cytoplasmic tail responsible for both receptor down-regulation and
signal transduction. This region of the receptor contains both a kinase
and a regulatory domain (11). A mutation in the kinase domain that
inactivates kinase activity (M721K) completely abolishes high affinity
receptor internalization (12). At least one of the tyrosine residues in
the regulatory domain that normally is a substrate for the kinase
appears to be required for down-regulation by EGF (13). On the other
hand, a constitutively active and tyrosine-phosphorylated receptor
that is unable to bind EGF appears not to be spontaneously internalized
(14), suggesting separate roles for EGF binding and tyrosine
phosphorylation during internalization. Through an analysis of the
behavior of truncated receptors, an 18-amino acid sequence between
amino acids 973 and 991 was found to be necessary for ligand-stimulated
receptor down-regulation and cytosolic calcium increase (3). This
region, which consists of a YXX
motif flanked by numerous
negatively charged amino acids, contains the binding site for clathrin
AP2. Unexpectedly, excision of this region from the tail does not
affect ligand-induced down-regulation (15). There are two tyrosine
residues in the kinase domain (Tyr-891 and Tyr-920) that are not
autophosphorylated but are consensus sequences for pp60Src
kinase phosphorylation (16). Activation of G-protein-coupled receptors
may stimulate pp60Src kinase phosphorylation of these sites
(17-21). It is not known if the phosphorylation of these residues
exerts any control over receptor internalization. Finally,
PKC-dependent phosphorylation of Thr-654 blocks
ligand-induced down-regulation (4).
Little is known about the mechanism responsible for targeting EGFR to
caveolae membrane or what releases the receptor in response to ligand
binding. It is possible that those mutations that disrupt EGF-stimulated internalization prevent migration of the receptor out of
caveolae. Mutations that had this effect would provide valuable insight
into the mechanism of receptor sequestration by caveolae. For these
reasons, we have used previously characterized cell lines expressing
wild-type and mutant EGFR to study the plasma membrane distribution of
these receptors before and after EGF stimulation. We find that all of
the mutant receptors that are internalization impaired also do not exit
from caveolae in response to EGF. This suggests that internalization of
those growth factor receptors that are concentrated in caveolae is a
three-step process that involves exit from caveolae, migration in the
bulk plasma membrane, and capture by coated pits.
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EXPERIMENTAL PROCEDURES |
Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum,
iodixanol, penicillin, G418, and streptomycin were from Life Technologies, Inc. EGF, tyrphostin AG1478, tyrphostin A9, and PP2 were
from Calbiochem (San Diego, CA). Puromycin, tetracycline, endothelin-1,
and lysophosphatidic acid (LPA) were from Sigma. Anti-caveolin pAb IgG,
anti-Raf-1 mAb IgG, and anti-PKC
mAb IgG were from Transduction
Laboratory (Lexington, KY). Anti-phosphorylated Erk pAb IgG was from
Promega (Madison, WI), and anti-phosphotyrosine mAb IgG was from
Upstate Biotechnology (Lake Placid, NY). Anti-EGFR pAb,
agarose-conjugated mAb anti-phosphotyrosine IgG, and anti-erbB2 mAb IgG
were from Santa Cruz Biotechnology (Santa Cruz, CA). Carboxyl truncated
EGFR were detected using a pAb (designated N13) raised against the 13 amino-terminal acids of EGFR. Horseradish peroxidase-conjugated goat
anti-mouse and anti-rabbit IgG were from Cappel (Durham, NC).
125I-Protein A, prestained molecular weight markers, and
ECL reagents were from Amersham Pharmacia Biotech. All chemicals for
SDS-PAGE electrophoresis were from Bio-Rad. Polyvinylidene difluoride
membranes were from Millipore (Bedford, MA).
Methods
Cell Culture--
Rat1B cells and normal human fibroblasts were
cultured in DMEM supplemented with 10% v/v fetal bovine serum, 1 mM glutamate, 100 µg/ml penicillin, and 100 µg/ml
streptomycin. Cells were grown to near confluence (3-4 days) before
removing serum and incubating an additional 24-48 h in the presence of
DMEM alone.
The stable HeLa cell lines (HtTA) expressing either wild-type or K44A
mutant dynamin were generously provided by Dr. Sandra L. Schmid
(Department of Cell Biology, Scripps Research Institute, La Jolla, CA).
The cells were maintained in DMEM with 10% fetal calf serum in the
presence of 200 ng/ml puromycin, 400 µg/ml G418, 2 µg/ml
tetracycline, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Sub
confluent cultures were harvested and plated at a density of 1.2 × 106 cells/100-mm culture dishes and grown in the
presence (uninduced) or absence (induced) of 2 µg/ml tetracycline in
DMEM plus 10% fetal calf serum for 48 h before experiments. For
the last 24 h, the cells were cultured in the absence of serum.
Stable transfected B82 mouse L cells, which lack endogenous EGFR,
expressing either wild-type or mutant human EGFR were grown in DMEM
plus 10% fetal calf serum, 80 nM methotrexate, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Cells were grown to near confluence (2-4 days) and incubated overnight in the presence of DMEM
without serum before each experiment.
NR6 cells that lack endogenous EGFR were infected with a retrovirus
expressing de 2-7 EGFR, selected in G418 and a clonal line prepared.
These cells were maintained in DMEM plus 10% fetal calf serum
containing high glucose (4.5 g/liter) in the presence of 400 µg/ml
G418, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Cells were
grown to near confluence (2-4 days) and incubated overnight in the
presence of DMEM without serum before each experiment.
Cytosol Acidification and Potassium Depletion--
Human
fibroblasts were incubated in DMEM without serum for 24 h before
the experiments. To acidify the cytosol, cells were washed twice with
medium A (DMEM plus 20 mM Hepes, pH 7.4) and then incubated
in medium A for 30 min at 37 °C. Cells were rinsed twice with medium
B (medium A plus 10 mM acetic acid, pH 5.0) and incubated
in media B for 10 min at 37 °C. Acidified cells were washed twice in
phosphate-buffered saline and incubated in the absence or presence of
EGF (50 ng/ml) for the indicated times in DMEM. Intracellular potassium
depletion (22) was carried out by rinsing cells twice with medium A and
incubating them in the same medium for 30 min at 37 °C. Cells were
rinsed with buffer A (50 mM Hepes, 100 mM NaCl,
pH 7.4) and incubated in hypotonic buffer A (buffer A diluted 1:1 with
water) for 5 min at 37 °C. The cells were rinsed three times in
buffer A and incubated in the same buffer for 30 min at 37 °C,
washed twice with phosphate-buffered saline before adding EGF (50 ng/ml) and incubating for the indicated times in DMEM.
Caveolae Isolation--
Caveolae were isolated by the
detergent-free method of Smart et al. (5). The cells were
washed in ice-cold buffer B (20 mM Tricine pH 7.8, 1 mM EDTA, and 250 mM sucrose) and collected by
scraping in the same buffer. Cells were lysed by Dounce homogenization in buffer B containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml soybean trypsin inhibitor, and 1 µg/ml benzamidine. A postnuclear supernatant fraction was prepared by spinning the lysate at
90 × g for 10 min. The postnuclear supernatant was
layered over 23 ml of ice-cold 30% Percoll in buffer B. After
centrifugation at 85,000 × g for 30 min, the cytosol
and plasma membrane fractions were collected. The plasma membrane
fraction was briefly sonicated (six 50-Joule bursts at 10 watts each),
mixed with buffer C (50% w/v iodixanol in buffer B plus 40 mM sucrose) to a final iodixanol concentration of 23% and
overlaid with 6 ml of linear (10-20%) gradient of iodixanol in buffer
B. Samples were centrifuged at 53,000 × g for 90 min.
The bottom 4 ml of the gradient was pooled and designated non-caveolae
membrane. The top 5 ml of the gradient was mixed with 4 ml of buffer C,
overlaid with 1 ml of 15% w/v iodixanol in buffer B, followed by 0.5 ml of 5% w/v iodixanol in buffer B. The gradients were centrifuged at
53,000 × g for 90 min, and caveolae were collected
from the 5%/15% interface (0.5 ml).
SDS-PAGE and Western Blotting--
Protein concentrations were
determined by the method of Bradford (Bio-Rad) using bovine serum
albumin as a standard. Samples were dissolved in SDS-PAGE sample buffer
(62 mM Tris, pH 8, 0.5% w/v SDS, 10% glycerol, 0.5% w/v
bromphenol blue), loaded on a 4% stacking gel, and separated using a
5-15% linear gradient gel. Proteins were transferred to
polyvinylidene difluoride membrane by electrophoretic transfer at 50 V
for 2 h on ice. After membranes were incubated with buffer D (20 mM Tris, pH 7.6, 137 mM NaCl) plus 5% nonfat
dry milk and 0.5% Tween 20 for 1 h at room temperature. The
membranes were then incubated with primary antibodies diluted in buffer
D plus 1% nonfat dry milk and 0.2% Tween 20 for 2 h at room
temperature. The membranes were washed with buffer D plus 0.2% milk
and 0.2% Tween 20 for 15 min and twice for 5 min at room temperature,
and then incubated with the appropriate horseradish peroxidase-conjugated goat anti-IgG antibody (0.1 µg/ml) in buffer D
plus 1% milk and 0.2% Tween 20 for 1 h. The membranes were then washed once for 15 min and four times for 5 min each with buffer D plus
0.2% milk and 0.2% Tween 20. Staining was detected using enhanced
chemiluminescence (ECL). Apparent molecular masses were estimated using
prestained molecular weight markers (broad range). To measure the
relative amounts of EGFR in each fraction, the horseradish
peroxidase-conjugated goat anti-IgG antibody was replaced with
125I-protein A. Gels were incubated in the presence of 1 µCi/ml 125I-protein A for 1 h. The membranes were
then washed once for 15 min and four times for 5 min each with buffer D
plus 0.2% milk and 0.2% Tween 20. The radioactive intensity of the
EGF receptor band (180 kDa) was measured using a Molecular Dynamics
PhosphorImager (Sunnyvale, CA).
Immunoprecipitation--
The cells were washed in ice-cold
buffer B and collected by scraping in the same buffer. Cells were lysed
by Dounce homogenization in buffer B containing protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml benzamidine, 1 µg/ml soybean trypsin inhibitor) and phosphatase inhibitors (0.5 mM sodium vanadate, 10 mM sodium pyrophosphate,
and 10 mM sodium fluoride). A postnuclear supernatant
fraction was prepared by spinning the lysate at 90 × g
for 10 min. The samples were diluted with 2× buffer E (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 60 mM
octylglucoside) to make samples at the protein concentration (300 µg/ml). Ten µl (2 µg) of agarose-conjugated monoclonal
anti-phosphotyrosine IgG was added to the 1-ml sample and incubated at
4 °C overnight. The samples were washed twice with buffer F (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 500 mM NaCl, 1% Triton X-100), twice with buffer G (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 250 mM NaCl, 1% Triton X-100), and twice with buffer H (10 mM Tris-HCl, pH 7.5, 5 mM EDTA). The beads were
mixed with SDS sample buffer, separated by gel electrophoresis using a
5-15% gradient gel, and immunoblotted with the indicated antibody.
Other Methods--
Each experimental results presented is a
representative example from three to five trials using identical conditions.
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RESULTS |
Regulation of EGFR Loss from Caveolae--
Previously, we reported
that EGF receptors are concentrated in caveolae membrane fractions
isolated from human fibroblasts (5). We used quantitative
immunoblotting to estimate the percentage of total surface EGF
receptors that were in the caveolae fraction of these cells before and
after incubation in the presence of EGF (Table
I, HF). Plasma membranes from quiescent
fibroblasts were used to prepare caveolae and non-caveolae fractions.
Immunoblots of each fraction showed that we recovered most of the
plasma membrane EGF receptors in the two fractions and that 60.5% of
these receptors were in the caveolae fraction. EGFR was enriched
11.6-fold in the caveolae fraction relative to the whole membrane.
After exposure to EGF, there was a marked decline in the total number
of receptors in the plasma membrane and a decrease in the percentage of
these receptors in the caveolae fraction.
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Table I
Relative amount of EGFR in membrane fractions prepared from either
normal human fibroblasts (HF), Rat-1 cells (Rat-1), or B82 cells (B82)
transfected with wild-type receptor
Human fibroblasts and Rat-1 cells were incubated in the presence (+) or
absence ( ) of EGF for 20 min at 37 °C while the B82 cells were
analyzed without any treatments. The plasma membrane (PM), non-caveolae
membrane (NCM), and caveolae membrane (CM) were isolated, and equal
amounts of protein from each sample were separated by gel
electrophoresis. Samples were immunoblotted with a pAb EGFR using
125I-protein A. The radioactive intensity of the EGF receptor
band (180 kDa) was measured as described and expressed as a percentage
of the total plasma membrane radioactivity. The results are the average
of three trials (HF and Rat-1 (EGF)), two trials (HF (+EGF) and B82
cells), or one trial (Rat-1 (+EGF)).
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Previously, we have used Rat-1 cells to study the dynamics of EGFR
behavior (6). Like the human fibroblast, the caveolae fraction of these
cells was enriched in EGFR (Table I, Rat-1). In quiescent cells, 48.6%
of the receptors were in the caveolae fraction (Rat-1 - EGF) and they
were enriched 18-fold. After 20 min in the presence of EGF, the
caveolae fraction contained only 14% of the remaining EGFR. To
determine if receptor loss was coupled to internalization by
clathrin-coated pits, we used immunoblotting to look at the effects of
cytosol acidification and potassium depletion, two treatments that
inhibit receptor-mediated endocytosis (22, 23), on EGFR migration from
caveolae (Fig. 1). In untreated human
fibroblasts, EGFR was concentrated in the caveolae fraction (A, lane 1). The receptor in this
fraction markedly declined after a 20-min incubation in the presence of
EGF at 37 °C (compare lanes 1 and
2). Caveolin-1, by contrast, did not change during the
incubation. Neither cytosol acidification (lanes
3 and 4) nor potassium depletion (lanes 5 and 6) prevented loss of EGFR
from the caveolae fraction. Moreover, incubation of cells in the
presence of EGF at 4 °C (lanes 7 and
8), conditions that block uptake by coated pits (24), had
little effect on receptor loss from caveolae.
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Fig. 1.
Effects of inhibiting receptor-mediated
endocytosis (A and B) and tyrosine
kinase activity (C) on migration of EGFR from
caveolae. A, serum-starved, normal human fibroblasts
were either not treated (lanes 1 and
2) or subjected to cytosol acidification (lanes 3 and 4), potassium depletion (lanes 5 and 6), or 4 °C (lanes 7, 8). Each set of
cells was then washed and incubated for 0 or 20 min in the presence of
50 ng/ml EGF at either 37 °C (lanes 1-6) or
4 °C (lanes 7 and 8). Caveolae
fractions were prepared and separated by gel electrophoresis (10 µg/lane) using 5-15% gradient gels and immunoblotted either with
anti-EGFR IgG (EGFR) or anti-caveolin-1 IgG
(Caveolin). B, stable HeLa cell lines expressing
either wild-type (WT) or K44A (K44A) mutant
dynamin were incubated in the absence of 2 µg/ml tetracycline for
48 h to stimulate expression of the respective protein. The cells
were cultured in the absence of serum for 24 h before being
incubated in the presence of 100 ng/ml EGF for 0, 2, 20, or 60 min at
37 °C. Caveolae fractions (CM) were isolated, separated
by gel electrophoresis (10 µg/lane) and immunoblotted with either
anti-EGFR IgG (EGFR) or anti-caveolin-1 IgG
(Caveolin). C, serum-starved, normal human
fibroblasts were either not treated (lanes 7 and
8) or incubated in the presence of 10 µM
tyrphostin AG1478 (lanes 1 and 2), 10 µM PP2 (lanes 3 and 4),
or 10 µM tyrphostin A9 (lanes 5 and
6) for 10 min before EGF was added to the dish and the cells
incubated further for the indicated time. Caveolae fractions were
isolated and immunoblotted.
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Similar results were obtained with HeLa cells expressing K44A dynamin
(Fig. 1B), a dominant-negative acting mutant dynamin I that
blocks internalization of EGFR by coated pits (25). Caveolae fractions
were prepared from cells expressing either wild-type (WT) or
K44A (K44A) dynamin at various times after they were
incubated in the presence of EGF. Immunoblots showed that the
concentration of EGFR (EGFR) declined in these fractions
with the same kinetics regardless of the dynamin that was being
expressed (compare lanes 1-4 with
lanes 5-8). Caveolin-1, by contrast, did not
change (Caveolin). Therefore, EGFR loss from the caveolae
fraction is not linked to receptor internalization by coated pits.
Mutations that block the intrinsic tyrosine kinase activity of EGFR
prevent receptor internalization by coated pits (12). We used various
tyrosine kinase inhibitors to determine if kinase activity was required
for receptor migration out of caveolae (Fig. 1C). Tyrphostin
AG1478 preferentially inhibits EGFR kinase (26). The presence of this
inhibitor markedly blocked the ability of EGF to stimulate receptor
migration out of the caveolae fraction (compare lanes
1 and 2 with lanes 7 and
8). We next looked at inhibitors that are more specific for
Src kinases (PP2; Ref. 27) and the platelet-derived growth factor
receptor kinase (tyrphostin A9; Ref. 28). The Src kinase inhibitor PP2
partially blocked EGFR migration compared with AG1478 (compare
lanes 3 and 4 with lanes
7 and 8). By contrast, tyrphostin A9 had no
affect on migration (compare lanes 5 and
6 with lanes 7 and 8).
Phorbol 12-myristate 13-acetate (PMA) stimulates PKC phosphorylation of
EGFR at threonine 654 and blocks EGF-induced, high affinity
internalization of the receptor (4). To determine if PMA has any affect
on EGFR loss from caveolae, we preincubated normal human fibroblasts in
the presence or absence of 100 nM PMA for 10 min and then
added EGF for various times (Fig. 2). Caveolae were isolated and immunoblotted with antibodies to either EGFR
or caveolin-1 (A). In the absence of PMA, receptor loss
occurred with normal kinetics (EGFR, lanes
1-3). By contrast, the presence of PMA blocked receptor
loss from the caveolae fraction (lanes 6 and
7). PMA alone had no effect on the concentration of either EGFR (lanes 4 and 5) or caveolin-1
(Caveolin, lanes 1-7) in the caveolae
fraction.
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Fig. 2.
Effect of PMA on the migration of EGFR from
caveolae. A, serum-starved, normal human fibroblasts
were either not treated (lanes 1-3) or incubated
in the presence of 100 nM PMA (lanes 4-7) for the indicated time at 37 °C. EGF (50 ng/ml) was
either added (lanes 2, 3,
6, and 7) or not added (lanes 1, 4, and 5) to the dish and the cells
further incubated for the indicated time. Caveolae fractions were
prepared and separated by gel electrophoresis (10 µg/lane) using
5-15% gradient gels and immunoblotted either with anti-EGFR IgG
(EGFR) or anti-caveolin-1 IgG (Caveolin).
B, B82 cells transfected with either T654A mutant
(T654A) or wild-type (WT) EGFR were incubated in
the presence (lanes 7-12) or absence
(lanes 1-6) of 100 nM PMA for 10 min
before adding 100 ng/ml EGF to the dish (lanes 2,
3, 5, 6, 8, 9,
11, and 12) and incubated further for the
indicated time. Caveolae fractions were prepared and separated by gel
electrophoresis (10 µg/lane) using 5-15% gradient gels and
immunoblotted either with anti-EGFR IgG (EGFR) or
anti-caveolin-1 IgG (Caveolin).
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To determine if threonine phosphorylation was required for the effect
of PMA, matched sets of quiescent B82 mouse L cells expressing either
wild-type (Fig. 2B, lanes 4-6 and
10-12) or T654A mutant (lanes 1-3
and 7-9) EGFR were incubated in the presence (lanes 7-12) or absence (lanes
1-6) of PMA for 10 min before adding EGF for various times
(Fig. 2B). Using caveolin-1 as the gel load control
(Caveolin), we saw that EGF alone clearly stimulated a rapid
decline in the concentration of both wild-type and mutant receptors in
the caveolae fraction (compare lanes 1-3 with
lanes 4-6). By contrast, PMA blocked the loss of
wild-type receptor (lanes 10-12) but did not
prevent the loss of T654A EGFR (lanes 7-9). PMA
had no effect on the localization of either wild-type or mutant
unoccupied receptors to caveolae (compare lane 7 with lane 10). These results suggest that
PKC-dependent phosphorylation of EGFR at Thr-654 prevents
exit of the receptor from caveolae.
EGFR can be activated by G-protein-coupled receptors (GPCR) such as
endothelin ET-1 (21, 29) and LPA (30), which leads to the
phosphorylation of Erk. In some cells, Src kinases may be intermediates
in this reaction and act by phosphorylating two tyrosine residues in
the kinase domain of the receptor (Tyr-891, Tyr-920) that are not
substrates for autophosphorylation (16, 19, 20). Other studies suggest,
in addition, that GPCRs stimulate autophosphorylation of EGFR (29). To
see if caveolae EGFR activated by GPCR behave differently than when
stimulated by EGF, we looked at the effects of endothelin ET-1 and LPA
(Fig. 3). We first determined if these
ligands stimulated EGFR phosphorylation (A). A postnuclear supernatant fraction was prepared from Rat-1 cells after no incubation (lane 1) or following incubation in the presence
of EGF (lanes 2-4), endothelin ET-1
(lanes 5-7), or LPA (lanes
8-10). Tyrosine-phosphorylated proteins were
immunoprecipitated from this fraction and immunoblotted with anti-EGFR
IgG. Low amounts of EGFR were present in immunoprecipitates from
untreated cells (lane 1) but markedly increased
within 3 min after exposure to EGF (lane 2). The
concentration of phosphorylated receptors increased further after 20 min but declined by 60 min. Both endothelin ET-1 (Et-1) and
LPA (LPA) also stimulated the appearance of EGFR in the
anti-PY IgG immunoprecipitates (compare lanes
5-7 and lanes 8-10). We reproducibly
found that the kinetics of receptor phosphorylation were different for
LPA than for endothelin ET-1 (compare lanes 5-7
with lanes 8-10).
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Fig. 3.
Effects of endothelin ET-1 and LPA on the
migration of EGFR from caveolae. A, serum-starved Rat
1B cells were incubated in the absence (lane 1)
or presence of 50 ng/ml EGF (lanes 2-4), 100 nM endothelin ET-1 (lanes 5-7), or
100 µM lysophosphatidic acid (lanes 8-10) for the indicated times. The postnuclear supernatant
was prepared and immunoprecipitated with anti-phosphotyrosine IgG.
Immunoprecipitated proteins were separated by gel electrophoresis (300 µg/lane) using 5-15% gradient gels and immunoblotted with anti-EGFR
IgG (pEGFR). B, Rat-1B cells were treated
according to the protocol described in A before the caveolae
fractions were isolated, separated by gel electrophoresis (10 µg/lane) and immunoblotted with anti-EGFR IgG (EGFR),
anti-phosphorylated MAP kinase IgG (pMAPK), or
anti-caveolin-1 IgG (Caveolin).
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Cells incubated in the presence of EGF rapidly lost EGFR from the
caveolae membrane fraction (Fig. 3B; EGFR,
lanes 1-4), which was accompanied by a transient
increase in the concentration of phosphorylated Erk1 and Erk2
(pMAPK, lanes 1-4) in this fraction. By contrast, neither endothelin ET-1 (EGFR, lanes
5-7) nor LPA (LPA, lanes
8-10) induced EGFR loss from the caveolae fraction. Each of
these ligands did, however, stimulate activation of caveolae MAP kinase
(pMAPK, lanes 5-10), an indication
that EGFR in this fraction was activated. MAP kinase activation
occurred rapidly and was sustained when endothelin ET-1 was added to
the media (lanes 5-7) while there was a
significant lag before LPA activated MAP kinase. LPA also reproducibly
induced the appearance of higher amounts of activated MAP kinase than
did the other ligands. Interestingly, whereas LPA and EGF stimulated
Raf-1 recruitment to caveolae, endothelin ET-1 did not (data not
shown). These results indicate that all three ligands stimulate EGFR
phosphorylation (A) but only EGF causes receptor migration
out of caveolae (B).
Mutant EGFR Impaired in Migration from Caveolae--
The presence
of EGFR in caveolae must be regulated through interactions between the
receptor and specific molecular elements within the caveolae membrane.
So far the results suggest that two sets of interactions are necessary.
One set specifies the localization of receptors to caveolae fraction.
The other controls receptor migration to a different membrane domain in
response to specific stimuli. We examined the behavior of mutant
receptors to try and identify the critical parts of the EGFR necessary
for both localization and migration.
We used either B82 mouse fibroblasts or NR6 cells transfected with
cDNAs coding for various mutant EGFR. Neither of these cells
express endogenous EGFR, but after transfection each cell expressed
comparable numbers of the indicated receptor. B82 cells expressing
wild-type cDNA have 10-fold more receptors than Rat-1 cells (31).
Quantitative immunoblotting showed that the caveolae fraction contained
42% of these receptors and they were enriched 6.7-fold (Table I, B82).
Cells were incubated in the presence of EGF for various times before
isolating caveolae and assaying for the presence of EGFR by
immunoblotting (Fig. 4). All the
receptors we tested were concentrated in the caveolae membrane fraction
before EGF was added to the media (lane 1). After
the addition of EGF, however, we identified two classes of receptors:
those that immediately disappeared from the caveolae fraction and those
retained for the time course of the experiment (lanes
1-3). The migratory receptors included the wild-type,
T654A, c'958f993-1186, c'958f1022-1186,
c'958f991-1022, and c'1022. By contrast, M721K, c'991, c'958,
c'688, c'647, and de 2-7 were all retained in caveolae after addition
of EGF. The comparative behavior of these mutant receptors indicates
that migration of receptors out of caveolae membrane requires both the
autophosphorylation of at least one of the five tyrosine residues in
the receptor regulatory domain and the binding of EGF.
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Fig. 4.
Ability of normal and mutant EGFR to migrate
from caveolae, activate caveolae MAP kinase, and recruit Raf-1 to the
caveolae. The domain structure of EGFR for wild-type and 11 mutant
EGFR is illustrated. B82 cells and NR6 (only the de 2-7 receptors)
cells transfected with the indicated EGFR were grown for 24 h in
the absence of serum before they were incubated in the presence of 100 ng/ml EGF for 0, 20, or 60 min (lanes 1-3); 0 or
20 min (lanes 4 and 5); or 0 or 3 min
(lanes 6 and 7) at 37 °C. The
caveolae fractions were isolated, separated by gel electrophoresis (10 µg/lane), and immunoblotted with anti-EGFR IgG (lanes 1-3), anti-phosphorylated MAP kinase IgG (lanes 4 and 5), or anti-Raf-1 IgG (lanes 6 and 7).
|
|
As is the case for normal human fibroblasts (7) and Rat-1 cells (9),
the highest concentration of plasma membrane-associated Erk1 and Erk2
in B82 and NR6 cells was in the caveolae fraction (data not shown). All
of the mutant receptors that contained functional kinase domains
(wild-type, T654A, c'958f993-1186, c'958f1022-1186, c'958f991-1022, c'1022, c'991, and c'958) were able to
stimulate the activation of these MAP kinases in response to EGF
(lanes 4 and 5). In cells expressing
de 2-7, however, the same amount of activated MAP kinase was present
in the caveolae fractions regardless of whether the cells had been
exposed to EGF (de 2-7, lanes 4 and
5). This is consistent with reports that this mutant receptor has a basal tyrosine kinase activity and does not bind EGF
(32, 33).
We also monitored Raf-1 recruitment to the caveolae fraction in cells
expressing wild-type and mutant receptors. In cells expressing
receptors that contained a portion of the regulatory domain, EGF
stimulated the recruitment of Raf-1 to the caveolae fraction (Fig. 4,
lanes 6 and 7). Receptors containing
active kinase domains but lacking the regulatory domain also stimulate Raf-1 recruitment (c'991 and c'958,
lanes 6 and 7). Interestingly, even
though the concentration of c'958 and c'991 (lanes
1-3) did not decline, recruitment of Raf-1 to caveolae
membrane was transient (data not shown). Receptors lacking kinase
activity (M721K and c'647, lanes
6 and 7) did not stimulate Raf-1 recruitment.
Normal and Mutant EGFR Activate ErbB2--
EGFR lacking the
phosphotyrosine sites for recruiting SHC and Grb2 stimulate downstream
signaling as effectively as wild-type receptors (34), which is
consistent with our observation that the binding of EGF to c'958 very
effectively stimulated MAP kinase activation in caveolae (Fig. 4). One
possibility is that the truncated receptor forms a heterodimer with
ErbB2 (35, 36) and activates MAP kinase through this intermediate. We
used immunoblotting to study the dynamics of ErbB2 localization to the
caveolae membrane fraction (Fig. 5).
Caveolae (A, lanes 1-4) and
non-caveolae (A, lanes 5-8) membrane
fractions isolated from quiescent normal human fibroblasts were
separated by gel electrophoresis (equal protein loads) and
immunoblotted with anti-EGFR IgG (A, EGFR),
anti-ErbB2 IgG (A, ErbB2), or anti-caveolin-1 IgG
(A, Caveolin). Both EGFR and ErbB2 were highly
enriched in caveolae fractions (compare caveolae to non-caveolae). The
addition of EGF to media stimulated the rapid loss of EGFR from the
caveolae fraction (caveolae) but had only a modest effect on
the level of ErbB2 in this fraction. Even after a 60-min incubation in
the presence of EGF, the concentration of ErbB2 in the caveolae
fraction remained high. Therefore, even though ErbB2 and EGFR appear to
be in close physical proximity in caveolae, EGFR preferentially
migrates to non-caveolae membrane in response to EGF.
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|
Fig. 5.
ErbB2 is concentrated in caveolae
membrane. A, normal human fibroblasts were
serum-starved for 24 h and incubated in the presence of 50 ng/ml
EGF for 0, 2, 20, and 60 min at 37 °C. Caveolae fractions
(lanes 1-4) and non-caveolae fractions
(lanes 5-8) were isolated, separated by gel
electrophoresis (5 µg/lane), and immunoblotted with anti-EGFR IgG
(EGFR), anti-ErbB2 IgG (ErbB2), or
anti-caveolin-1 IgG (Caveolin). B, B82 cells
transfected with the wild-type (lanes 1-4),
kinase-negative M721K (lanes 5-8), c'958
truncated (lanes 9-12), or c'991 truncated
(lanes 13-16) EGFR were serum-starved for
24 h before incubating in the presence of 100 ng/ml EGF for 0, 2, 20, or 60 min at 37 °C. Caveolae fractions were isolated, separated
by gel electrophoresis (10 µg/lane), and immunoblotted with anti-EGFR
IgG (EGFR), anti-ErbB2 IgG (ErbB2), or
anti-caveolin-1 IgG (Caveolin). C, B82 cells
transfected with the wild-type (lanes 1-4),
kinase-negative M721K (lanes 5-8), c'958
truncated (lanes 9-12), or c'991 truncated
(lanes 13-16) EGFR were serum-starved for
24 h before incubating in the presence of 100 ng/ml EGF for 0, 2, 20, or 60 min at 37 °C. At the end of the incubations, postnuclear
supernatant fractions were prepared and phosphotyrosine-containing
proteins were immunoprecipitated. Each precipitate was separated by gel
electrophoresis and immunoblotted with anti-ErbB2 IgG.
(pErbB2).
|
|
We next looked at the effects of EGF on the distribution of ErbB2 in
cells expressing truncated receptors (Fig. 5B). EGF
stimulated the loss of the wild-type receptor (EGFR) from
caveolae fraction but the concentration of ErbB2 remained relatively
unchanged (WT, compare EGFR with
ErbB2). EGF did not stimulate the loss of kinase-negative receptors (M721K) from the caveolae fraction, and the amount
of ErbB2 in this fraction did not decline. Likewise, even though EGF
did not stimulate the loss of the kinase-active, truncated receptors
c'958 and c'991 from the caveolae fraction, the concentration of ErbB2
remained unchanged (B, compare EGFR with
ErbB2 in c'958 and c'991). These
results suggest that truncated receptors may remain in close proximity
to ErbB2 for extended periods of time after cells are exposed to
EGF.
A potential measure of how long EGFR interacts with ErbB2 is the length
of time ErbB2 remains phosphorylated after EGF binds. Cells transfected
with wild-type, M721K, c'958, or c'991 receptors were starved of serum
for 24 h before they were incubated in the presence of EGF for
various times (Fig. 5C). At the end of each incubation,
tyrosine-phosphorylated proteins were immunoprecipitated from whole
cell lysates, separated by gel electrophoresis, and immunoblotted with
anti-ErbB2 IgG mAb. The amount of ErbB2 immunoprecipitated from
wild-type EGFR cells increased between 0 and 20 min of EGF stimulation
but declined by 60 min (WT), which paralleled the loss of
EGFR from the caveolae fraction (B, WT). EGF did
not stimulate phosphorylation of ErbB2 in cells expressing
kinase-negative receptors (M721K). By contrast, the amount
of phosphorylated ErbB2 in cells expressing either c'958 or c'991
receptors (c'958, c'991) was as high at 60 min as
at 20 min (compare lanes 11, 12,
15, and 16 with lanes 3 and
4).
| |
DISCUSSION |
Regulation of Receptor Migration--
All of the normal and mutant
EGFR we tested were found to be concentrated in the caveolae membrane
fraction of quiescent cells. This included c'647, which only has a
11-amino acid cytoplasmic tail, and de 2-7, which is missing amino
acids 6-274 of the extracellular domain. Therefore, the information
required for targeting the receptor to caveolae membrane is contained
within amino acid 275-647 of the receptor. This rules out a role for
the putative caveolin-1 binding motif (37) in receptor clustering and
focuses attention on the possibility that the transmembrane domain
targets growth factor receptors to caveolae membrane.
The rapid exit of EGFR appears to require autophosphorylation of at
least one of the five tyrosine residues in the regulatory domain of the
receptor. Kinase-negative receptors (M721K) did not migrate (Fig. 4)
while attaching as few as 31 amino acids of the regulatory domain
(amino acids 991-1022) to the carboxyl end of an active kinase domain
(at amino acid 958) was sufficient. This construct (c'958
f991-1022) lacks most of the actin binding region in the
receptor (amino acids 984-996; Ref. 38), so actin does not play a
direct role in receptor exit from caveolae. Even though endothelin ET-1
and LPA stimulated EGFR phosphorylation, this population of
phosphorylated receptors did not migrate out of caveolae (Fig. 3).
Previous studies indicate that both cytokines can stimulate the
Src-dependent phosphorylation of EGFR (19, 20) and that
Tyr-891 and Tyr-920 in the kinase domain of the receptor are consensus
sites for Src phosphorylation (16). Therefore, these two tyrosine
residues, which are in the kinase domain, may play an additional role
in controlling receptor traffic out of caveolae. The ability of the
inhibitor PP2 to partially block receptor exit from caveolae indicates
a role for Src family kinases, too. This is in agreement with the
finding that overexpression of Src kinase stimulates an increase in the
rate of EGFR endocytosis (39). Finally, most likely EGF binding is also
required to get receptors to move out of caveolae because de 2-7,
which does not bind EGF (32, 33), was concentrated in caveolae even
though it has a constitutively active kinase domain (see Fig. 4).
With the exception of c'991, all of the mutant receptors that are
internalization-impaired also do not disappear from the caveolae
membrane fraction in response to EGF binding. Moreover, both PMA and
tyrosine kinase inhibitors, which are known to prevent coated
pit-mediated internalization of EGF (31), blocked exit. In the case of
c'991, we incubated cells in the presence of EGF for up to 60 min (Fig.
4) but still saw little loss of receptor from the caveolae fraction.
Previous studies have shown that internalization of c'991 receptors is
markedly slower than wild-type receptors (13), with only ~50%
internalization by 60 min. One explanation for our findings is that
only those c'991 receptors present in non-caveolae membranes are
initially internalized whereas those in caveolae exit very slowly.
PMA blocked the loss of EGFR from the caveolae fraction, possibly by
inhibiting receptor tyrosine kinase activity (4). By contrast, the
migration of full-length receptors bearing a T654A substitution was not
affected by PMA. PMA causes T654A receptors to have a lower affinity
for EGF than wild-type receptors do in the absence of PMA (4).
Likewise, EGFR in cells expressing K44A dynamin have a low affinity for
EGF (40). The fact that both populations of receptors migrated out of
caveolae membrane with normal kinetics suggests that the affinity of
EGF for EGFR has little influence on the exit of the receptor from
caveolae. Finally, migration from caveolae and internalization by
coated pits appears not to be linked because four independent methods of inhibiting receptor-mediated endocytosis (4 °C, K+
depletion, cytosol acidification, and K44A dynamin) had no effect on
EGF-stimulated receptor loss from caveolae fractions.
EGFR Molecular Interactions in Caveolae--
An EGFR truncated at
the COOH terminus of the kinase domain (c'958) was just as effective as
wild-type receptor at stimulating the phosphorylation of the Erk1 and
Erk2 in caveolae fraction (Fig. 4). EGFR form heterodimers with other
members of the ErbB family (35, 36, 41) and previous studies have shown
that truncated EGFR (c'973) can signal through ErbB2 (34). Since truncated EGFR appear not to migrate out of caveolae in response to
EGF, we were curious how they would be able to interact with ErbB2 if
the two receptors were located in different regions of the plasma
membrane. Our results suggest, however, that both truncated EGFR and
ErbB2 are concentrated together in caveolae membranes. We cannot be
sure that both types of receptors are in the same caveola, but these
results raise the possibility that a common targeting motif causes them
both to accumulate in the same membrane domain. The close proximity of
the two receptors in caveolae might facilitate heterodimer formation,
which would facilitate the transfer of information laterally through
complimentary signaling pathways (35).
In contrast to EGFR, the concentration of ErbB2 in caveolae only
declined slightly in response to EGF, which suggests that interactions
between these two signaling pathways may be restricted to caveolae
membranes. The inefficient movement of ErbB2 out of caveolae may be
related to its impaired internalization by clathrin-coated pits (42).
Activation of two different types of truncated receptors (c'958 and
c'991) did not cause any loss of ErbB2. The prolonged presence of both
truncated EGFR and ErbB2 in caveolae after EGF binding would result in
persistent signal transduction through the ErbB2 pathway. Indeed, the
EGF-stimulated tyrosine phosphorylation pattern of ErbB2 was markedly
prolonged for truncated receptors (Fig. 5C, c'958
and c'991) compared with wild-type receptors (Fig. 5C, WT).
EGF signal transmission is initiated by the combinatorial interactions
between receptor and multiple molecular intermediates. Each set of
interactions is dictated by: (a) the local concentration of
the collaborating molecules and (b) the molecular ecology of the surrounding membrane. The current study, together with previous work, indicates that EGFR can be located in at least three different membrane compartments after EGF binds; caveolae, bulk plasma membrane, and clathrin-coated pits. It is possible, therefore, that interactions occurring in caveolae may induce one set of signaling events while another set of signals is broadcast once the receptor reaches clathrin-coated pits. Through this type of receptor behavior, signal
transduction becomes compartmentalized at the cell surface, thereby
allowing a single class of receptors to transmit different signals from
distinct locations in the cell. For example, if EGFR can only interact
with ErbB2 when the two are in caveolae, then a critical determinant in
EGF signaling through ErbB2 is whether or not EGFR is in caveolae at a
time when EGF is bound.
It should be possible to identify the signaling pathways that emanate
from each of the various compartments visited by a migratory receptor
by pinpointing where EGF stimulates the combinatorial interactions
required for the initiation of a specific signaling event. For example,
we have shown previously that EGF stimulates the recruitment and
activation of Raf-1 in caveolae (6), which is linked to the activation
of a resident population of MAP kinase (7). Therefore, activation of
the entire MAP kinase pathway can occur in caveolae, even in
vitro (10). In the current study, we show that both LPA and
endothelin ET-1 stimulate the phosphorylation of those EGFR that appear
to be concentrated in caveolae (Fig. 3), suggesting that lateral
signaling between these two pathways can occur in this compartment. We
also found that kinase-active de 2-7 was constitutively located in
caveolae, and recent evidence indicates that this mutant EGFR causes
the continuous activation of both c-Jun N-terminal kinase (43) and
phosphatidylinositol 3-kinase (1). Caveolae may be the compartment
where pathways dependent on the activation of these molecules
originate. On the other hand, kinase-active receptors that are unable
to move out of caveolae (c'991) cannot stimulate cell motility (44).
The COOH-terminal addition of regulatory domain tyrosines to these receptors renders them competent to stimulate cell movement and to exit
from caveolae. The signaling pathways in control of cell migration,
therefore, may originate from receptors that are not in caveolae. We
also showed in the current study that PMA prevents movement of
wild-type receptor from caveolae but not T654A mutant receptors. The
ability of PMA to inhibit EGF-stimulated mitogenesis is lost in cells
expressing T654Y mutant receptors (45). This raises the possibility
that EGFR control mitogenesis from a non-caveolae compartment. We must
stress that these examples merely indicate that caveolae have the
capacity to initiate certain signaling pathways. More work is needed to
determine if they are an exclusive site.
Mislocalized Receptors Are Oncogenic--
An important conclusion
of this study is that abolishing high affinity internalization of EGFR
also impairs the ability of the receptor to migrate out of caveolae.
Numerous studies have documented that multiple EGFR signaling
intermediates are concentrated in the caveolae fraction (8). The
prolonged residence of kinase-active receptors in caveolae, therefore,
offers the potential for an unregulated stimulation of those signaling
pathways that use these intermediates. Indeed, two (c'958 and de 2-7)
of the three mutant kinase-active receptors that were impaired in
leaving caveolae have previously been found to be oncogenic (2, 46,
47). Our study suggests that the oncogenic phenotype of cells
expressing these receptors may be caused by the inability of these
receptors to migrate out of caveolae membrane. If this is true, moving
these mutant receptors out of caveolae should allow the cells to revert to a normal phenotype.
| |
ACKNOWLEDGEMENTS |
We thank William Donzell and Ann-Sofi Horton
for their valuable technical assistance and Sue Knight for
administrative assistance.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL 20948, GM 43169, and CA58689 and by a grant from the Perot Family Foundation.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 correspondence should be addressed: Dept. of Cell
Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039.
| |
ABBREVIATIONS |
The abbreviations used are:
EGF, epidermal
growth factor;
EGFR, epidermal growth factor receptor;
DMEM, Dulbecco's modified Eagle's medium;
PMA, phorbol 12-myristate
13-acetate;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
LPA, lysophosphatidic acid;
MAP, mitogen-activated protein;
GPCR, G-protein-coupled receptor;
PKC, protein kinase C.
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