1 Institute of Pathology, University of Oslo, Rikshospitalet University
Hospital, N-0027 Oslo, Norway
2 MSD Cardiovascular Research Center, Rikshospitalet University Hospital and
Department of Pharmacology, University of Oslo, N-0316 Oslo, Norway
* Author for correspondence (e-mail: espenst{at}ulrik.uio.no )
Accepted 19 December 2001
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Summary |
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Key words: Caveolae, Caveolin, Cholesterol, EGFR, Rafts
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Introduction |
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It should be noted that caveolin-enriched membrane domains isolated by
fractionation contain both caveolae and lipid rafts in general. Lipid rafts
are membrane domains enriched in cholesterol and sphingolipids. Although
caveolae also have such characteristics, lipid rafts also exist in the plasma
membrane outside morphologically defined caveolae, as well as in cells not
expressing caveolin (for reviews, see Brown
and London, 1998; Kurzchalia
and Parton, 1999
; Simons and
Ikonen, 2000
; Simons and
Toomre, 2000
). Electron microscopy (EM) is required to discover
whether the EGFR is localized within morphologically identifiable caveolae. In
the classical studies by the groups of Cohen
(Haigler et al., 1979
) and
Hopkins (Hopkins et al., 1985
;
Miller et al., 1986
), the EGFR
was localized either directly using anti-EGFR-coated colloidal gold
(Hopkins et al., 1985
;
Miller et al., 1986
) or
indirectly by the localization of bound EGF-ferritin complexes
(Haigler et al., 1979
). These
studies showed that in nonstimulated cells the EGFR is more or less randomly
distributed at the plasma membrane and not concentrated within any specific
morphologically identifiable microdomain. Incubation with EGF at 4°C did
not seem to change the random distribution of EGFR
(Hopkins et al., 1985
;
Miller et al., 1986
;
Torrisi et al., 1999
).
However, upon chase at 37°C, Hopkins et al., reported that the EGFR
relocated into noncoated plasma membrane invaginations resembling caveolae
(Hopkins et al., 1985
). Again,
other studies showed that the EGFR relocates into clathrin-coated pits
(Ringerike et al., 1998
;
Stang et al., 2000
;
Torrisi et al., 1999
).
The importance of rafts in signal transduction has been demonstrated
indirectly in several studies where the composition of rafts has been
modulated by changes in the plasma membrane cholesterol content (reviewed by
Incardona and Eaton, 2000;
Kurzchalia and Parton, 1999
;
Simons and Toomre, 2000
).
Cholesterol depletion has previously been shown to cause activation of Erk,
and further incubation with EGF enhanced the effect, causing hyperactivation
of Erk (Furuchi and Anderson,
1998
). This could suggest that cholesterol depletion directly
affects EGF-induced activation of the EGFR; however, this was not addressed in
the published study (Furuchi and Anderson,
1998
). Cholesterol depletion will change the biophysical
properties of the plasma membrane, making it more fluid (reviewed by
Burger et al., 2000
;
Yeagle, 1985
). Studies of EGFR
activation upon reconstitution of EGFR into liposomes have shown that the
lipid composition influences EGFR kinase activity. Introduction of cholesterol
into the liposome membrane resulted in decreased kinase activity, thus
indicating that membrane fluidity affects the tyrosine kinase activity of
reconstituted EGFR (Ge et al.,
2001
).
In the present work we have studied the plasma membrane localization of EGFR with respect to caveolae and lipid rafts and characterized the role of cholesterol in control of EGF binding, EGFR dimerization and phosphorylation of the EGFR. To avoid complicating effects of endocytosis of the EGFR, all experiments have been performed at 4°C. Our results show that although only a small percentage of EGFR is localized within caveolae, the cholesterol content of the plasma membrane is critical for control of EGFR activity. This suggests that localization to lipid rafts might control the EGFR and, as support for this, we find that a significant number of EGFR colocalize with the raft-localized GPI-anchored protein, placental alkaline phosphatase (PLAP), upon antibody-induced patching.
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Materials and Methods |
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Antibodies
Antibodies used were rabbit anti-caveolin 1 (Transduction Laboratories,
Lexington, KT), mouse anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA;
Neomarkers, Lab Vision, Fremont, CA), sheep anti-EGFR (Gibco BRL Life
Technologies, Gaithersburg, MD), mouse anti-phosphorylated EGFR (PY1173,
Upstate Biotechnology, Lake Placid, NY), mouse anti-human PLAP (DAKO,
Carpinteria, CA), rabbit anti-human transferrin receptor (HybriDomus, Nota
Bene Scientific, Hellebaek, Denmark), rabbit anti-mouse IgG (Cappel, ICN
Biomedicals, Costa Mesa, CA). Rabbit anti-sheep IgG, alkaline
phosphatase-conjugated donkey anti-sheep IgG, peroxidase-conjugated donkey
anti-mouse IgG and peroxidase-conjugated donkey anti-sheep IgG were all from
Jackson ImmunoResearch Laboratories, West Grove, PA.
Cell culture and treatment
The human laryngeal carcinoma cell line HEp-2 was grown in Dulbecco's
modified Eagle's medium (3.7 g/l sodium bicarbonate) (BioWhittaker,
Walkersville, MD) containing 2 mM L-glutamine (BioWhittaker) and 1x
penicillin-streptomycin-fungizone mixture (17-745, BioWhittaker) supplemented
with 5% (v/v) fetal bovine serum (FBS) (BioWhittaker). The human epidermal
carcinoma cell line A431 was grown in the same medium, but with 10% (v/v) FBS.
HEp-2 cells were plated at a density of 15,000 cells/cm2 and A431
cells at a density of 25,000 cells/cm2 48 hours prior to
experiments. EGF (0.1, 1.0 or 10 nM) was added to cells in minimal essential
medium (MEM) (Gibco BRL) without HCO3- and with 0.1%
(w/v) bovine serum albumin (BSA) for 15 minutes on ice. The cells were then
washed three times with ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 1 mM
Na2HPO4 and 2 mM NaH2PO4) to
remove unbound ligand. To deplete cholesterol from the plasma membrane, cells
were incubated with 10 mM methyl-ß-cyclodextrin (MßCD) in MEM, for
15 minutes at 37°C, or with 1 µg/ml U18666A
(3-ß-(2-diethylaminoethoxy)-androstenone-HCl) (BioMol Research
Laboratories, Plymouth Meeting, PA) for 48 hours in DMEM. To add cholesterol
to the plasma membrane, the cells were incubated with MEM containing
MßCD-cholesterol (water-soluble cholesterol, 0.4 mg cholesterol/ml)
for 30 minutes at 37°C.
Western blotting
Cells were lysed in lysis buffer (10 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50 mM
NaF, 30 mM sodium pyrophosphate, 1% Triton X-100, 1 mM
Na3VO4 and 1 mM phenyl methyl sulphonyl fluoride (PMSF))
on ice for 10 minutes and subjected to western blotting, as described
(Ringerike et al., 1998). The
reactive proteins were detected using an enhanced chemiluminescence method
(ECL, Amersham Pharmacia Biotech, Piscataway, NJ). For quantitation of band
intensity, proteins were electrotransferred to polyvinylidene difluoride
membranes (Hybond-P, Amersham Pharmacia Biotech) upon SDS-PAGE. The membranes
were incubated with sheep anti-EGFR antibodies before incubation with alkaline
phosphatase-conjugated anti-sheep IgG. Immunobinding was detected by the
enhanced chemifluorescence method (ECF, Amersham Pharmacia Biotech), and the
chemifluorescence was measured by a phosphorofluoroImager (Molecular Imager
FX, Bio-Rad, Hercules, CA).
Analysis of 125I-EGF binding
HEp-2 cells plated in 24- or 48-well cell culture plates at half the normal
density were preincubated with or without 10 mM MßCD, 1 µg/ml U18666A
or 0.4 mg/ml water-soluble cholesterol, as described above. Binding of
125I-EGF was performed and data analysed essentially as previously
described (Ringerike et al.,
1998). To measure the total number of EGF binding sites, binding
was performed for 15 minutes on ice with a saturating concentration (8 nM) of
125I-EGF (Amersham Pharmacia Biotech) in the absence (total
binding) or presence (nonspecific binding) of 150 nM unlabeled EGF. The cells
were then washed in ice-cold PBS, lysed in 1 M NaOH, and bound radioactivity
was measured.
Crosslinking of the EGFR
Cells were washed with ice-cold PBS before incubation on ice for 30 minutes
with the nonpermeable crosslinking reagent bis(sulfosuccinimidyl) suberate
(BS3) (3 mM in PBS) (Pierce, Rockford, IL). In all experiments, a
freshly prepared solution of BS3 was used. The crosslinking
reaction was terminated by adding glycine (1 M) to a final concentration of
250 mM and further incubation on ice for 5 minutes. The cells were washed in
PBS, scraped loose and transferred to test tubes followed by centrifugation at
420 g for 5 minutes at 4°C. The cell pellets were lysed on
ice in lysis buffer containing 250 mM glycine. The lysates were homogenized
using QIAshredderTM (Qiagen, Valencia, CA) before being subjected to
SDS-PAGE using 4-15% gradient gels (Bio-Rad) and western blotting with
quantitation of band intensity as described above.
Biotinylation of the EGFR
HEp-2 cells, nontreated or preincubated with U18666A, MßCD, or
water-soluble cholesterol, as described above, were washed three times in PBS
containing 1 mM CaCl2 and 0.5 mM MgCl2 (CaMg-PBS) before
incubation with 1.8 mM EZ-LinkTMSulfo-NHS-LC-Biotin (Pierce, Rockford,
IL) for 1 hour on ice. To stop the reaction, cells were washed three times
with CaMg-PBS and incubated with 20 mM glycine in CaMg-PBS for 10 minutes on
ice. Cells were washed with CaMg-PBS and lysed in immunoprecipitation buffer
(PBS containing 10 mM EDTA, 1% (v/v) Triton X-100, 10 mM NaF, 200 U/ml
aprotinin (Fluka Chemie AG, Buchs, Switzerland), 1 mM PMSF, 1 mM
N-ethylmaleimide and 1 mM Na3VO4) on ice 20 minutes
before the lysates were centrifuged at 20,000 g for 15 minutes
at 4°C. Anti-EGFR antibodies bound to protein G-coupled Sepharose beads
(Amersham Pharmacia Biotech) were added to the supernatant fraction, and
immunoprecipitation was performed at 4°C for 1 hour. The immunoprecipitate
was washed and subjected to SDS-PAGE and western blotting. Biotinylated EGFR
was detected by use of alkaline phosphatase conjugated streptavidine (DAKO
Corporation).
Fluorescence microscopy
Cells were plated on 12 mm coverslips (MENZEL-GLÄSER®, Germany) in
24-well microtiter plates, two to three days prior to experiments. Cells were
fixed in 4% (w/v) paraformaldehyde in Soerensen's buffer (0.162 M
Na2HPO4, 0.038 M NaH2PO4) for 20
minutes at room temperature and washed three times in cytoskeleton buffer (137
mM NaCl, 5 mM KCl, 1.1 mM Na2HPO4-H2O, 0.4 mM
KH2PO4, 5.5 mM glucose, 4 mM NaHCO3, 10 mM
MES, 2 mM EGTA, 2 mM MgCl2). To localize cholesterol, coverslips
were incubated with 50 µg/ml filipin in PBS containing 0.2% BSA and 0.2%
gelatin (Bio-Rad) for 30 minutes at room temperature, washed and mounted using
DAKO fluorescent mounting medium with 15 mM NaN3 (DAKO). To
visualize the filipin labeling, cells were viewed using a Leitz DM RXE
microscope equipped with a UV filter and an F-view digital camera.
Immunoelectron microscopy (immuno-EM)
To patch PLAP, HEp-2 cells were incubated with mouse anti-PLAP antibodies,
followed by rabbit anti-mouse IgG and, finally, 5 nm protein A-gold (purchased
from G. Posthuma, Utrecht, The Netherlands), each for 30 minutes on ice. Cells
with patched PLAP, or otherwise preincubated as described in legends to
figures, were fixed with paraformaldehyde (4% w/v) and glutaraldehyde (0.1%
w/v) in Soerensen's phosphate buffer and processed for cryosectioning and
immunolabeling (Griffiths et al.,
1984). Bound antibodies were visualized using protein A-gold. When
the primary antibody was mouse IgG or sheep IgG, incubation with rabbit
anti-mouse IgG or rabbit anti-sheep IgG, respectively, was used as an
intermediate reagent between the primary antibody and protein A-gold. The
sections were examined using a Philips CM 120 electron microscope. To estimate
the number of EGFR at the plasma membrane, EGFR labeling density was
quantitated. The number of gold particles representing EGFR labeling at the
plasma membrane of randomly chosen cells was counted, the length of the plasma
membrane was measured and the number of gold particles per unit membrane
length was calculated.
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Results |
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|
We also localized the EGFR in cells incubated with EGF at 4°C. Double labeling of cells incubated with 10 nM EGF on ice for 15 minutes showed no change in the number of EGFR localized to caveolae compared with the number in nonstimulated cells (data not shown). In the same cells, we also studied the plasma membrane distribution of bound EGF to investigate whether EGFR localized to caveolae bound EGF with higher efficiency than did EGFR outside caveolae. These experiments demonstrated the same distribution of EGF labeling as for EGFR with 6% localizing to caveolae in A431 cells and 5% in HEp-2 cells (Fig. 2).
|
Most previous reports on caveolar localization of EGFR have been based on
fractionation experiments. Since fractionation techniques will not separate
rafts in general from caveolae, we also studied the localization of EGFR to
lipid rafts in general. As rafts, with the exception of caveolae, cannot be
identified by morphological criteria, we identified rafts indirectly after
patching of raft-associated molecules. Rafts in HEp-2 cells were visualized by
patching the GPI-anchored protein PLAP
(Harder et al., 1998).
Following incubation with mouse anti-PLAP, the bound antibodies were
crosslinked using rabbit anti-mouse antibodies followed by 5 nm protein
A-gold. To see whether binding of EGF influences the possible
raft-localization of the EGFR, in some experiments EGF was included during the
protein A-gold incubation. The antibody-induced crosslinking of PLAP resulted
in clearly identifiable patched areas at the plasma membrane. To determine
EGFR localization with regard to rafts, sections were labeled using antibodies
to the EGFR or, as a control, anti-transferrin receptor (TfR) antibodies
(Fig. 3). The labeling
demonstrated that the EGFR localized both within and outside rafts.
Quantitation of EGFR within anti-PLAP patched rafts showed that about 40% of
EGFR localizing to the plasma membrane is within rafts in nonstimulated cells.
Similar quantitation of TfR labeling showed that only about 6% of TfR at the
plasma membrane colocalized with patched PLAP, confirming that
anti-PLAP-induced patches represent rafts and not just unspecific trapping of
plasma membrane components. Labeling of cells incubated with EGF during the
final incubation with protein A-gold showed that ligand binding on ice did not
affect the EGFR localization. Both EGFR and bound EGF showed the same
quantitative distribution as the EGFR in nonstimulated cells
(Fig. 4).
|
|
Cholesterol depletion increases the number of EGFR at the plasma
membrane and causes EGFR hyperactivation
Our present immuno-EM results, as well as several previous studies,
indicate that a substantial amount of the EGFR is localized within rafts;
rafts are cholesterol-rich membrane domains. To study whether cholesterol
directly affects EGFR activation, we used two established methods to deplete
the plasma membrane of cholesterol. MßCD extracts cholesterol
(Kilsdonk et al., 1995;
Klein et al., 1995
), whereas
U18666A causes a redistribution of cholesterol from the plasma membrane to
late endosomes (Liscum and Klansek,
1998
). The effect of U18666A was initially investigated by
fluorescence microscopy. Filipin staining demonstrated that cholesterol was
redistributed from the plasma membrane to intracellular vesicles
(Fig. 5). To make sure that the
different drugs had the expected effect on clathrin-coated pits and caveolae,
drug-treated cells were prepared for immuno-EM. Both cholesterol-depleting
drugs caused flattening of clathrin-coated pits, and invaginated caveolae were
no longer observed (data not shown).
|
The effect of cholesterol depletion on expression and activation of EGFR
was examined by western blotting, by using antibodies recognizing either all
EGFR or the EGFR phosphorylated on tyrosine 1173 (PY1173). Both
cholesterol-depleting drugs caused an increase in EGFR tyrosine
phosphorylation (Fig. 6). In
A431 cells increased phosphorylation of the EGFR could be seen even in the
absence of added EGF, while the effect of cholesterol depletion was observed
only upon incubation with EGF in HEp-2 cells. The different EGFR expression
levels in the two cell lines most likely explain this difference.
Additionally, A431 cells are known to produce growth factors that activate the
EGFR in an autocrine fashion (Van de
Vijver et al., 1991). To examine whether the increased activation
of EGFR induced by treatment with MßCD or U18666A was a direct effect of
changes in the plasma membrane cholesterol content, we incubated cells with
water-soluble cholesterol to increase the cholesterol content of the plasma
membrane. Incubation with water-soluble cholesterol was observed to result in
diminished EGF-induced phosphorylation of the EGFR
(Fig. 7).
|
|
The EGFR usually exists in two affinity states. To examine whether cholesterol depletion increased EGFR phosphorylation by increasing the number of high affinity EGFR, we measured binding of iodinated EGF to cells and performed Scatchard analysis. When cholesterol-depleted cells and cells enriched in cholesterol were compared with untreated cells, we found no difference in the fraction of high-affinity binding sites (data not shown). However, saturation binding experiments demonstrated that specific binding of EGF was increased upon cholesterol depletion and decreased in cells enriched in cholesterol (Fig. 8).
|
Binding of EGF involves dimerization of the EGFR, and it was recently
suggested that the predominant mechanism for dimerization is the formation of
a complex of one EGF molecule and an EGFR dimer, followed by the direct arrest
of a second EGF molecule (Sako et al.,
2000). By this mechanism, increases in EGF-induced EGFR
dimerization can cause increased EGF binding without necessarily affecting
binding affinity. To examine whether changes in plasma membrane cholesterol
content caused changes in EGFR dimerization, we took advantage of a chemical
crosslinking reagent previously used for this purpose
(Johannessen et al., 2001
;
Sorkin and Carpenter, 1991
).
Control cells, MßCD-treated cells and cells enriched in cholesterol were
upon incubation with or without EGF for 15 minutes on ice incubated with the
membrane-impermeable crosslinking reagent BS3, and the cell lysates
were subsequently subjected to western blotting with antibodies to EGFR. As
demonstrated in Fig. 9,
cholesterol depletion caused increased EGF-induced EGFR dimerization, whereas
addition of water-soluble cholesterol inhibited EGF-induced EGFR dimerization.
The increased dimerization in cholesterol-depleted cells was 21%.
|
Neither cholesterol depletion, nor incubation with water soluble cholesterol, affected the total level of EGFR (Figs 6, 7). However, a possible explanation for the observed changes in EGF binding, EGFR activation and EGFR dimerization could be altered EGFR distribution with more EGFR localizing to the plasma membrane. To investigate this possibility, the amount of EGFR localized at the plasma membrane was measured by biotinylation of the plasma membrane. By calculating the fraction of biotinylated EGFR, we found that cholesterol depletion indeed caused a change in EGFR distribution. Even though the total level of EGFR expression was unaltered, more EGFR became biotinylated, demonstrating that more EGFR localized to the plasma membrane (Fig. 10). The increased number of EGFR at the plasma membrane upon cholesterol depletion was confirmed by immuno-EM. Although labeling density varied from cell to cell within the same specimen, quantitation showed that labeling density for EGFR at the plasma membrane on average increased by approximately 25% upon both MßCD and U18666A treatment of HEp-2 cells.
|
![]() |
Discussion |
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In the present study we have used immuno-EM to characterize the plasma
membrane distribution of the EGFR. Based on the colocalization between EGFR
and caveolin 1 we show that only a limited number of EGFR resides within
caveolae. As noncoated invaginations, most likely representing caveolae,
previously have been shown to occupy approximately 7% of the plasma membrane
in A431 cells (Parton, 1994),
our results could indicate that the EGFR is randomly distributed at the plasma
membrane. Our results are in conflict with those of several fractionation
studies, where the authors have concluded that the EGFR is enriched in
caveolae, but in agreement with prior EM studies
(Haigler et al., 1979
;
Hopkins et al., 1985
;
Miller et al., 1986
;
Torrisi et al., 1999
). Based
on fractionation experiments, as much as 40-60% of EGFR has been reported to
localize to caveolae (Mineo et al.,
1999
). These numbers correspond well with the number of EGFR we
find colocalized with patched PLAP, indicating that this represents the number
of EGFR localizing to rafts in general and not only to caveolae.
Different results with respect to the impact of ligand binding on
localization of EGFR have been reported. Whereas some authors conclude that
activated EGFR remains in caveolin-positive membrane fractions
(Couet et al., 1997;
Waugh et al., 1999
), Mineo et
al. found that activated EGFR relocates to caveolin-negative membranes even at
4°C (Mineo et al., 1999
).
Our results showed no change in EGFR distribution upon incubation with EGF at
4°C. Previously we have shown that as much as 50% of EGFR activated at
4°C relocated to coated pits upon chase at 37°C
(Stang et al., 2000
), but
whether this represents a relocation with respect to lipid rafts is unclear.
In fact, the flattening of clathrin-coated pits upon cholesterol depletion
illustrates the importance of cholesterol for the integrity and function of
clathrin-coated pits (Rodal et al.,
1999
).
Labeling for EGF showed the same distribution as found for EGFR. This indicates that, at high ligand concentrations, ligand binds to EGFR with the same efficiency within and outside rafts. Attempts to localize high-affinity EGFR by incubation with limiting concentrations of EGF failed because of inefficient labeling intensity for EGF. The mechanisms determining high versus low affinity binding of ligand are still unclear. The finding that the percentage of EGFR localizing to caveolae corresponds to the percentage of high-affinity EGFR in the cells examined, could suggest that caveolar localization is important for high-affinity binding. However, considering the finding that conditions disrupting the organization of caveolae had no effect on the fraction of high-affinity EGFR, this is very unlikely.
Changes in the cholesterol content of the plasma membrane had significant
effects on the amount of EGF bound to cells as well as on dimerization and
activation of the EGFR. However, whether these effects are direct or indirect
consequences of changes in cholesterol content is unclear. A possible
explanation for the increase in binding of EGF and for the increase in
dimerization and activation of the EGFR could be the altered sub-cellular
distribution of the EGFR and increased number of EGFR at the plasma membrane
upon cholesterol depletion. The increase in plasma membrane EGFR is most
likely due to inhibited endocytosis of the EGFR. Although the major
endocytosis of EGFR is ligand induced, EGFR is also constitutively endocytosed
and recycled in a ligand-independent manner
(Herbst et al., 1994). As
cholesterol depletion inhibits endocytosis but, as shown for the transferrin
receptor, not recycling (Subtil et al.,
1999
), the number of EGFR available for binding of EGF will
increase as an indirect consequence of cholesterol depletion. However, the
effect on clathrin-dependent endocytosis does not explain the inhibitory
effects found after incubation with water-soluble cholesterol. This suggests
that cholesterol also controls the EGFR in a direct fashion. Upon cholesterol
depletion, the fluidity of the plasma membrane increases with increased
possibility of lateral movement of the EGFR. Increased lateral mobility will
probably increase the possibility of EGF-induced EGFR dimerization and thereby
EGFR activation.
How EGF binds to monomeric versus dimeric EGFR is an unresolved issue. Our
results showed that dimeric EGFR could be detected only upon incubation with
EGF. This could support the theory that dimerization is induced by the initial
binding of EGF to monomeric EGFR and that eventually dimers are formed.
However, whether dimerization involves binding of one or more EGF molecules is
unclear. Sako et al. recently claimed that single molecule tracking revealed
that the predominant mechanism of dimerization involves the formation of a
complex of one EGF molecule and one EGFR dimer, followed by the direct arrest
of a second EGF molecule (Sako et al.,
2000). This would suggest that dimerization occurs before binding
of the second EGF molecule. This sequence of events could explain how changes
in cholesterol content and ensuing changes in membrane fluidity might affect
EGF binding. Increased EGF-induced EGFR dimerization due to increased lateral
mobility will also increase the chance for binding of a second EGF molecule.
Increased cholesterol content of the plasma membrane will have the opposite
effect on fluidity and thereby, in theory, the opposite effect on EGFR
dimerization and total EGF binding. As activation of the EGFR depends on
dimerization, such a model for EGF binding will explain not only the observed
changes in EGF binding and EGFR dimerization, but also the changes in EGFR
activation.
It is important to realize that cholesterol depletion could inhibit
interaction between the EGFR and other molecules involved in control of EGFR
activation. The ganglioside GM3 is known to modulate EGFR activity
(Bremer et al., 1986;
Meuillet et al., 1999
;
Meuillet et al., 2000
) either
directly (Rebbaa et al., 1996
)
or via a phosphatase (Suarez Pestana et
al., 1997
). Changes in GM3 content do not affect EGF binding but,
whereas GM3 depletion increases EGFR autophosphorylation, addition of GM3
decreases EGFR autophosphorylation
(Meuillet et al., 1999
;
Meuillet et al., 2000
).
Membranes enriched in GM3, also known as the `glycosphingolipid signaling
domain' or the `glycosignaling domain' can, like lipid rafts, be isolated as a
low density detergent insoluble membrane fraction. However, as reported for
the EGFR, GM3-containing membranes can be separated from caveolin-containing
membranes (Iwabuchi et al.,
1998
). The GM3-positive membranes contain less cholesterol than
caveolin-positive membranes and rafts in general
(Iwabuchi et al., 1998
).
However, as incubation with MßCD, and most likely also incubation with
U18666A, depletes cholesterol not only from rafts
(Ilangumaran and Hoessli,
1998
), we cannot exclude the possibility that disorganization of
GM3-positive glycosignaling domains can cause at least some of the observed
effects on EGFR activation.
In conclusion, contrary to results based on subcellular fractionation, immuno-EM studies show that only small amounts of EGFR localize to caveolae. However, a significant amount of EGFR was found to colocalize with patched PLAP. This demonstrates that a significant amount of EGFR localizes to rafts. Cholesterol was found to be important in control of EGFR activation and its depletion probably has multiple effects on EGFR localization and activation. Whether cholesterol controls the EGFR directly or only indirectly, by regulating endocytosis through clathrin-coated pits and thereby affecting the level of EGFR at the plasma membrane, is still unclear.
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Acknowledgments |
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References |
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