From the Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103
Received for publication, June 26, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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The epidermal growth factor receptor (EGFR) is a
multisited and multifunctional transmembrane glycoprotein with
intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric
receptor undergoes dimerization resulting in kinase activation. The
consequences of kinase stimulation are the phosphorylation of its own
tyrosine residues (autophosphorylation) followed by association with
and activation of signal transducers. Deregulation of signaling
resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant
epidermal growth factor (EGF) receptor missing 267 amino acids from the
exoplasmic domain is common in human glioblastomas. The truncated
receptor (EGFRvIII/ The human epidermal growth factor receptor
(EGFR)1 is a transmembrane
glycoprotein with a cysteine-rich extracellular region and an
intracellular domain containing uninterrupted kinase site and multiple
autophosphorylation sites clustered at the C-terminal tail (see Ref. 1
and reviewed in Ref. 2) (Fig. 1). On the basis of internal sequence identity, the extracellular portion of the
EGFR has been subdivided into four domains. Domains I (amino acids
1-165) and III (aa 310-481) have 37% sequence identity, whereas
domains II (aa 166-309) and IV (aa 482-621) are rich in cysteines (3)
(see Fig. 1). These cysteines are linked by intra-chain disulfide
bonding (4). Domain III has been shown to bind directly with EGF, and
then two molecules of the monomeric receptor-ligand complex interact to
form a dimeric complex. Domain I is believed to be involved in the
second interaction (3, 4). The receptor dimerization results in kinase
activation. The earliest consequence of kinase activation is the
phosphorylation of its own tyrosine residues (autophosphorylation), and
this is followed by its association with and activation/phosphorylation
of signal transducers leading to mitogenesis. In addition, we have
demonstrated a phosphorylation-induced conformational alteration of the
EGFR (5). Such conformational change agrees well with the finding that
autophosphorylation also results in unmasking of cryptic cytoplasmic
domain(s) needed for receptor internalization (6).
EGFR) lacks EGF binding activity; however, the
kinase is constitutively active, and cells expressing the receptor are
tumorigenic. Our studies revealed that the high kinase activity of the
EGFR is due to self-dimerization, and contrary to earlier reports,
the kinase activity per molecule of the dimeric
EGFR is comparable
to that of the EGF-stimulated wild-type receptor. Furthermore, the
phosphorylation patterns of both receptors are similar as determined by
interaction with a conformation-specific antibody and by phosphopeptide
analysis. This eliminates the possibility that the defective
down-regulation of the
EGFR is due to its altered phosphorylation
pattern as has been suggested previously. Interestingly, the
receptor-receptor self-association is highly dependent on a
conformation induced by N-linked glycosylation. We have
identified four potential sites that might participate in
self-dimerization; these sites are located in a domain that plays an
important role in EGFR functioning.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural features of the WtEGFR and
EGFR. The 621 aa in the extracellular region
of the EGFR are divided into four domains; domains I (aa 1-165) and
III (aa 310-481) have 37% sequence identity. Domains II (aa 166-309)
and IV (aa 482-621) are rich in cysteine. Domain III is involved in
ligand binding, whereas domain I is important in ligand-induced
dimerization. There are 12 potential N-linked glycosylation
sites in the wild-type receptor; these sites are marked by the
letter N.
EGFR lacks aa 6-273 and the missing part is
denoted by dashes.
EGFR also lacks 4 of the 12 N-linked glycosylation sites. The scheme also shows the
locations of transmembrane domain (TM; aa 622-644),
tyrosine kinase domain (TK; aa 683-958), and the C-terminal
tail (C-ter, aa 959-1186) which contains all five
autophosphorylation sites.
Deregulation of signaling due to the aberrant expression of the EGFR
has been implicated in oncogenesis. Nearly 50% of grade IV gliomas
(glioblastoma multiforme) have amplified EGFR genes. In the
majority of such cases, the EGFR gene amplification is correlated with structural rearrangement of the gene, resulting in
in-frame deletions that preserve the reading frame of the receptor message. To date, three truncated forms of EGFR have been identified (7-9). The type III deletion mutant occurs in 17% of the
glioblastomas and is characterized by an 801-base pair in-frame
deletion resulting in the removal of N-terminal amino acid residues
6-273 from the extracellular domain of the intact 170-kDa EGFR (9)
(see Fig. 1). Although there is a consensus that this truncated
receptor (EGFRvIII/EGFR) lacks ligand binding activity and the
kinase is constitutively active (10-12), the subcellular localization of the receptor has not yet been conclusively established. In some
transfectants, a significant fraction of the receptor population was
reported to be intracellular (10, 11), whereas studies from another
laboratory appear to suggest that the receptors are predominantly on
the cell surface (12). In addition, the molecular mechanism by which
the transfectants acquire transforming activity is not clear. Studies
from Cavenee and co-workers (13) suggest that constitutive activation
of Ras-mitogen-activated protein kinase pathway contributes to the
transforming activity of the
EGFR, and the studies from Wong and
co-workers (14, 15) have demonstrated that the transformation is
mediated through constitutive activation of phosphatidylinositol
3-kinase and c-Jun N-terminal kinase and not through
Ras-mitogen-activated protein kinase pathway.
Although the EGFR undergoes ligand-independent kinase activation,
the extent of autophosphorylation of the
EGFR is significantly less
compared with that of the ligand-stimulated WtEGFR (11, 16). Since
phosphorylation-induced conformational change results in exposure of
sequence motifs involved in endocytic and lysosomal sorting and such
unmasking is thought to be obligatory for receptor down-regulation (6),
this may explain the persistent presence of the
EGFR on the cell
surface. It is possible that the endocytic codes are cryptic in the
receptors that are in partially active conformation. However, it is an
open question why the
EGFRs are not fully active although the
receptor is capable of self-dimerization. Is it due to the fact that
receptor dimerization is necessary but not sufficient for full kinase
activity? It is also not known why the
EGFR undergoes
self-association, whereas the WtEGFR does not. Such information will be
useful in developing strategies to control the activity of the aberrant receptor.
Contrary to earlier reports, our studies reported here suggest that
there is no difference between the dimeric EGFR and the ligand-stimulated WtEGFR with respect to kinase activity as determined by the extent of autophosphorylation. We also demonstrate by using a
conformation-specific antibody that detects an epitope exposed only
upon phosphorylation of tyrosines 992, 1068, and 1086 (17) that the
antibody recognizes the
EGFR as fully active receptor. This is
further confirmed by phosphopeptide analysis. This suggests that in
addition to its persistent presence on the cell surface, the high
kinase activity of the
EGFR also contributes to its tumorigenic
activity. Finally, we report that the ligand-independent dimerization
of the
EGFR is contingent upon core glycosylation. Out of 12 potential N-linked glycosylation sites in the receptor, the
four sites located in domain III are likely to be involved in inducing
a stable conformation needed for receptor-receptor association. Based
on these and related studies, we also propose models for self- and
ligand-induced receptor dimerization and how subdomains within the
extracellular region might influence such interactions.
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EXPERIMENTAL PROCEDURES |
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Materials--
The cross-linking reagent
3,3'-bis(sulfosuccinimido)suberate (BS3) was purchased from
Pierce, and tunicamycin and swainsonine were obtained from Sigma.
Endoglycosidase H was purchased from Glyco (Novato, CA). A substrate
for the EGFR (RRKGSTEEEAEYLRV) with an Km of 21 µM (18) containing the tyrosine 1173 autophosphorylation site was purchased from Infinity Biotech Research and Resourse (Upland,
PA). EGF was purified from mouse submaxillary glands and radiolabeled
with 125I by the chloramine-T procedure (17). Solid-phase
EGF was prepared by coupling EGF to Affi-Gel 15 (Bio-Rad) as described
(17). Labeled ATP was prepared with 32Pi and
-Prep A kit (Promega, Madison, WI) according to the manufacturer's directions. Specific radioactivity of [
-32P]ATP
was adjusted by adding unlabeled ATP (17). All radioisotopes were
purchased from ICN Biomedicals (Costa Mesa, CA).
Iodination of mAb 425-- The monoclonal antibody directed to an extracellular peptide epitope of the human EGFR (19) was iodinated by the chloramine-T method. 5 µg of protein A-purified mAb 425 in a total volume of 20 µl containing 0.15 M sodium phosphate, pH 7.5, and 0.25 mCi of Na125I was incubated at 20 °C with 30 µg of chloramine T. After 1 min, the iodination reaction was terminated by the addition of sodium metabisulfite followed by NaI and BSA. Under these conditions nearly 50% of 125I was incorporated into the protein as determined by trichloroacetic acid precipitation. The labeled antibody was separated from the free radioactivity by Sephadex G-10 chromatography.
EGFR Mutants and Cell Culture-- Human glioblastoma cell line, U87MG, as well as cells expressing the truncated receptor lacking the EGF binding activity and the wild-type receptor were obtained from Drs. W. K. Cavenee and H.-J. Su Huang. The generation and characterization of the mutant have previously been described (16). The transfected cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated cosmic bovine serum (HyClone, Logan, UT) and 0.4 mg/ml G418; parental U87MG cells were grown in the absence of G418. Plasma membranes from these cells were prepared as described (17).
Receptor sites/cell were determined by 125I-EGF binding assay as described previously (17). Briefly, 2.25 ng of 125I-EGF (5.6 × 105 cpm) in a total volume of 200 µl of Earle's balanced salt solution containing 20 mM HEPES, pH 7.5, 2.5 mg/ml BSA, and unlabeled EGF (25 ng/ml for cells expressing less than 1 × 105 receptor sites/cell and 100 ng/ml for cells expressing more than 1 × 105 receptor sites/cell) were incubated at 20 °C for 2 h with cells grown in 2-cm2 24-well plates. Nonspecific binding, which was 5-10% of the total binding, was measured by incubating the cells with labeled EGF in the presence of 200 nM unlabeled EGF. The EGF binding sites/cell in different mutants are shown in Table I.
For antibody binding, 13 ng of 125I-labeled mAb 425 (2 × 106 cpm) mixed with 30 ng of unlabeled antibody was incubated with cells under conditions described above. This represents the saturating concentration of the antibody for the receptor binding. Nonspecific binding that was 2-3% of the total binding was determined by incubating cells with labeled antibody in the presence of 50-fold excess of unlabeled antibody.
Biosynthetic Labeling of the EGFR-- This was done as described (17) except that incubation with Tran35S-label (100 µCi/ml; 1190 Ci/mmol) was carried out in methionine- and cysteine-free Dulbecco's modified Eagle's medium containing 2% cosmic bovine serum.
Antibodies--
Anti-peptide antibody Ab P2 was directed to
amino acid residues 964-979
(Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of
the cytoplasmic domain of the human -type platelet-derived growth
factor receptor. It was generated in rabbits using HPLC-purified peptide according to the method described previously (20). Monoclonal antibody, mAb 425, raised against human A431 carcinoma cells and polyclonal 170-kDa antibody to denatured EGFR were gifts from Dr. M. Das and were developed as described (19, 21). mAb 425 is directed to a
peptide epitope in the extracellular domain of the human EGFR, and it
recognizes only the native human receptor (19). The anti-EGFR
monoclonal antibody, Ab-15 (clone H9B4), directed to a cytoplasmic
domain, close to the C-terminal tail of the receptor was purchased from
NeoMarkers (Union City, CA). This antibody, like the polyclonal
anti-170-kDa antibody, is highly suitable for Western blot analysis. A
rabbit polyclonal anti-EGFR antibody coupled to agarose (catalog number
Sc-03AC) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA);
this antibody that is suitable for kinase assay is directed to the C
terminus of the receptor. The mouse monoclonal anti-phosphotyrosine
antibody, 1G2, used for purification of the tyrosine-phosphorylated
proteins, was generated as described and coupled to activated Sepharose (22).
Quantification of the 32P-Labeled EGFR--
This was
carried out as described (17) with some modifications. Briefly,
isolated membranes from cells expressing the wild-type or truncated
EGFRs were phosphorylated with [-32P]ATP either in the
presence (wild-type) or absence (truncated) of EGF under
autophosphorylation conditions (5). An aliquot of the labeled proteins
purified by anti-phosphotyrosine monoclonal antibody (1G2) was
subjected to immunoprecipitation with an antibody directed to a
cytoplasmic domain of the EGFR, analyzed by SDS-PAGE, and quantified as
described (17). The quantification of the receptor protein is based on
the assumption that there is no incorporation of 32P into
Ser/Thr residues of the EGFR. This assumption was validated in an
earlier report that no 32P-labeled human EGFR could be
detected in MI41, a murine NIH 3T3 cell line expressing a human EGFR
mutant in which all five acceptor tyrosine residues have been
substituted with phenylalanine (17).
Chemical Cross-linking of the EGFR-- The detergent-solubilized plasma membranes or 32P-labeled receptor preparation was incubated with BS3 at 4 °C for 10 min (23, 24). Excess cross-linker was inactivated by the addition of Tris, pH 7.5, and the samples were processed as described in the figure legends.
Immunoprecipitation Technique and Electrophoresis--
These
were carried out as described (17). Briefly, the labeled receptor
preparation was incubated with the indicated antibody at 4 °C
overnight in 15 µl (unless otherwise indicated) of 20 mM
HEPES, pH 7.4, 0.15 M NaCl, 0.2% Nonidet P-40, 2.5 mg/ml
BSA, 1 mM vanadate, and protease inhibitors. After
isolation of the immune complexes by protein A-Sepharose, the receptors
were analyzed by SDS-PAGE (7% gel unless otherwise indicated), and the
labeled bands were visualized either by autoradiography (for
32P-labeled receptors) or by fluorography (for
35S-labeled proteins) as described (17). The molecular
weight markers used were myosin (205,000), -galactosidase
(116,000), phosphorylase b (97,000), albumin (66,000), and
ovalbumin (45,000).
Western Blot Analysis--
This was carried out as described
(25). Briefly, the electrophoretically separated proteins were
transferred to poly(vinylidene difluoride) membranes (Millipore Corp.,
Bedford, MA) by electrophoresis at 4 °C overnight at 0.1 mA followed
by 1.5 h at 0.2 mA. After incubation with an antibody as specified
in figure legends, the antigen-antibody complex was visualized either
by ECL Plus reagents from Amersham Pharmacia Biotech or by
125I-protein A. The intensity of the band was quantified
using the ImageQuant program. The pre-stained molecular weight markers
used (with apparent molecular weight) were
2-macroglobulin (200,000),
-galactosidase (123,000),
fructose-6-phosphate kinase (84,000), and pyruvate kinase (63,000).
Phosphopeptide Analysis-- For phosphopeptide mapping, 32P-labeled wild-type EGFR was purified by EGF-Affi-Gel chromatography as described (17), whereas the truncated receptor was purified by an EGFR-specific antibody, mAb 425. Briefly, isolated membranes from cells expressing the intact EGFRs were solubilized with 0.5% Nonidet P-40 in 20 mM HEPES, pH 7.4, 0.15 M NaCl, 10% glycerol, and protease inhibitors (aprotinin, leupeptin, and phenylmethanesulfonyl fluoride). After centrifugation, the clarified supernatant was incubated at 4 °C for 2 h with EGF-Affi-Gel (1 mg/ml), and the gel beads were washed three times with the binding buffer to remove the unbound proteins. For the truncated receptor, the detergent-solubilized membrane extracts were incubated at 4 °C for 2 h with mAb 425 antibody, and the immune complex was isolated by protein A-Sepharose. The gel beads containing either the wild-type or truncated receptors were incubated with [32P]ATP under autophosphorylation conditions (5). After washing the beads to remove free ATP, the bound receptor was dissociated by heating with SDS-sample buffer and subjected to SDS-PAGE. After overnight in fixing solution (25% methanol, 10% acetic acid in water), the wet gels were processed as described (17) with some modifications. Briefly, the region of the dried gel corresponding to the receptor band was digested with 0.5 ml of sequencing grade trypsin (20 µg/ml; Roche Molecular Biochemicals) in 50 mM ammonium bicarbonate, pH 7.8, for 40 h at 37 °C with an addition of fresh trypsin after 20 h. After centrifugation, an aliquot of the clear supernatant was redigested with trypsin for 20 h at 37 °C, dried in vacuum, and dissolved in 0.1% trifluoroacetic acid in water. Equal counts of phosphopeptides derived from both receptor types were then subjected to reverse-phase HPLC analysis using a DeltaPak 6-µm C18 column (Waters; column size 3.9 × 150 mm) as described (17). Briefly, following injection, the column was washed with 10 ml of 0.1% trifluoroacetic acid in water, and then the phosphopeptides were eluted with a 0-60% acetonitrile gradient containing 0.1% trifluoroacetic acid with a flow rate of 1 ml/min. 0.5 ml fractions were collected, and the Cerenkov counts were determined.
Kinase Assay--
This was performed as described (18) with some
modifications. Briefly, the EGFR was immunoisolated using a polyclonal
antibody coupled to agarose; this antibody is directed to a cytoplasmic domain of the receptor. After washing three times, the gel beads were
incubated in a total volume of 50 µl containing 20 mM
HEPES, pH 7.5, 5% glycerol, 0.1% Nonidet P-40, 5 mM
MnCl2, 1 mM vanadate, 10 µM
[-32P]ATP (5 × 104 cpm/pmol), 200 µM peptide substrate, and protease inhibitors (leupeptin,
phenylmethanesulfonyl fluoride, and aprotinin). After 10 min at
20 °C, the reaction was terminated by the addition of EDTA (final
concentration 20 mM) and centrifuged. Trichloroacetic acid
was added to the supernatant to a final concentration of 5%. BSA was
added as carrier protein to facilitate the precipitation of the EGFR
that might have dissociated from the antibody during the incubation.
After 10 min at 4 °C, the reaction mixture was centrifuged at
4 °C and the supernatant was processed as described to
determine the substrate phosphorylation (18).
Sucrose Density Gradient Ultracentrifugation-- This was carried out as described (23) with some modifications. Briefly, the WGA-purified EGFRs were centrifuged at 50,000 rpm at 4 °C for 8.5 h in a SW Ti55 rotor through a linear gradient of 5-20% (w/v) sucrose in 20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.1% Nonidet P-40, 1 mM sodium vanadate, and 1 mM MnCl2. At the end of the run, fractions (150 µl) were collected and processed as described in legend for Fig. 3.
Isolation of Endoplasmic Reticulum from Tunicamycin-treated
Cells--
This was carried out as described (26). Briefly, confluent
cultures of cells grown in p150 plates were incubated at 37 °C for
20 h in growth medium containing 1 µg/ml tunicamycin. Under these conditions, the newly synthesized EGFRs lacking
N-linked oligosaccharides are associated with the
endoplasmic reticulum. The microsomal fraction that also contains
endoplasmic reticulum was isolated from these cells as described
(26).
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RESULTS |
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Mutation of the EGFR resulting in type III truncation occurs
spontaneously in vivo in glioma cells. This led us to use a
human glioma cell line, U87MG, as the host for the expression of the EGFR/EGFRvIII and, as a control, the wild-type receptor. The
EGFR
maintains its transforming activity in this host cell. Another advantage in selecting this cell line is that it is deficient in
endogenous EGFR (see below).
Receptor Numbers per Cell in U87MG.EGFR--
Earlier reports
suggested that the
EGFR lacks ligand binding activity (10, 11, 16).
Hence we used 125I-labeled mAb 425 antibody to determine
the number of receptor molecules per cell with an objective to
investigate whether both receptor types are expressed in comparable
numbers. Such studies should also allow us to determine the
endogenous receptor level in U87MG.
EGFR cells. This information is
also important for the studies described in this paper. mAb 425 is a
monoclonal antibody directed to a peptide epitope in the extracellular
domain of the EGFR, and it binds to the receptor with very high
affinity (19). In addition, earlier studies have demonstrated the
specific and saturable binding of this antibody to membranes from a
human carcinoma cell line, A431(19). Thus, mAb 425 is suitable for
determining the receptor numbers under circumstances where EGF cannot
be used. The concentration of the antibody used for receptor assay (215 ng/ml) leads to maximum binding to both U87MG.
EGFR and U87MG.WtEGFR cells since there was no further increase in the specific binding of
the antibody to either cell type when mAb 425 concentration was
increased by 3-fold (data not shown). Different laboratories including
ours have reported the use of saturating concentration of a ligand for
the estimation of receptor numbers (17, 27). Based on the antibody
binding, U87MG.
EGFR cells express 4.4 × 105
receptors per cell (see Table I). To
investigate whether the estimated numbers of antibody-binding sites
reflect the actual receptor sites, we also determined the receptor
numbers in U87MG.WtEGFR using 125I-EGF binding and compared
it with the antibody-binding sites. As shown in Table I, the receptor
sites calculated based on ligand binding (5.1 × 105)
were similar to the receptor numbers determined by antibody binding
(5.3 × 105) indicating the validity of this method.
This suggests that (i) the expression levels of both the receptors are
comparable (4.4 × 105 receptor sites/cell for
EGFR
versus ~5 × 105 receptor sites/cell for
WtEGFR) and (ii) the bulky size of the antibody of molecular mass
150-kDa relative to EGF of 6-kDa did not adversely influence its
binding to the receptor. We also determined the endogenous EGFRs in
U87MG.
EGFR by 125I-EGF binding (0.2 × 105 sites/cell), and it represents less than 5% of the
truncated receptor population. This is further confirmed by Western
blot analysis followed by densitometric scanning of the 170-kDa
wild-type receptor band and the 145-150-kDa truncated receptor band
(Fig. 2).
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Isolation of the Dimeric EGFR in Biologically Active State and
Its Kinase Activity--
Based on chemical cross-linking studies in
which intact cells were reacted with a cross-linker, it was concluded
that the autokinase activity of the
EGFR is 20% that of the
EGF-stimulated wild-type receptor (11, 16). Since the
EGFR is
capable of self-dimerization, it implies that receptor dimerization is
obligatory but not sufficient for full kinase activation. Hence to
examine the extent of dimerization and more importantly to investigate the kinase activity of the dimeric receptor, sedimentation studies were
performed to isolate the monomeric and dimeric forms of the
EGFR in
the biologically active state. For this purpose the
WGA-agarose-purified receptors from U87MG.
EGFR cell membranes were
subjected to sucrose sedimentation analysis, and the kinase activity of
the gradient fractions was assayed by autophosphorylation. As control,
the WtEGFR was also subjected to sedimentation analysis under similar conditions; however, the gradient fractions were assayed by
autophosphorylation in the presence of EGF. The identity of the EGFR in
the gradient fractions was confirmed by immunoprecipitation with mAb
425 antibody. As shown in Fig. 3, the
EGFR exists in two forms, a slow moving form and a fast moving form;
however, under similar conditions the WtEGFR moves as a single entity
with a mobility similar to that of the slow moving component of the
EGFR. To determine the molecular weights of the different
components, we analyzed the peak fractions by chemical cross-linking.
For this purpose, the 32P-labeled receptor preparations
were subjected to chemical cross-linking with 50 µM
BS3, immunoprecipitated with mAb 425, and then analyzed by
SDS-PAGE. As shown in Fig. 3 (insets), following
BS3 treatment of the faster moving peak from the
EGFR, a
cross-linked complex corresponding to a molecular weight of ~300,000
could be detected; the receptor population that escaped cross-linking is present as an 145-150-kDa band. The 145-150-kDa band and not the
300-kDa band was seen when cross-linking was carried out with the
slower moving peak. These suggest that the fast and the slow moving
components of the
EGFR represent the dimeric and the monomeric forms
of the receptor, respectively. No cross-linked complex could be seen
when the peak fraction from the WtEGFR was subjected to BS3 treatment and the 32P-labeled receptor
moved as a 170-kDa protein, a size consistent with the monomeric
form of the receptor. For the experiment described in Fig. 3, the
truncated receptor has undergone a series of treatments that not only
took more than 10 h to complete but also resulted in significant
receptor dilution. Despite these treatments, a significant portion of
the
EGFR exists in the dimeric state.
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We also determined the specific activity of monomeric EGFR kinase.
For this purpose we quantified both the wild-type and the truncated
receptors in the gradient fractions by Western blot analysis using a
polyclonal antibody directed to the denatured 170-kDa EGFR (see Fig.
4). Both the receptors were also
quantified by probing the blot with a monoclonal antibody directed to
an epitope located in the C-terminal tail of the EGFR. The relative intensities of the bands are similar to that of the blot probed with
the anti-170-kDa antibody. This strengthens the possibility that the
wild-type and truncated receptors react equally well in Western blot
with anti-170-kDa antibody as well as with the C-terminal antibody, and
hence the Western blot result reflects the actual amount of these two
proteins in the gradient fractions. As shown in Fig. 4, the kinase
activity per unit receptor protein for the dimeric receptor is
5-7-fold higher relative to the monomeric receptor. Furthermore, the
extent of autophosphorylation per molecule of the dimeric
EGFR
(fractions 15-18) is similar to that of the ligand-activated WtEGFR
(fractions 19-22) (see Fig. 4). This suggests that receptor
dimerization, at least with respect to the
EGFR, is sufficient for
full kinase activation.
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Interaction of the EGFR with an Antibody That Recognizes Only
the Activated Receptor--
The studies described above revealed that
there is no difference with respect to kinase activity between the
EGFR which undergoes dimerization in the absence of the ligand and
the EGF-stimulated wild-type receptor. This led us to investigate
whether the phosphorylation pattern and phosphorylation-mediated
conformational change of the receptors are also similar. Such
information is important since it has been proposed that the slow
endocytic rate of the
EGFR is due to its altered phosphorylation
(16). For this purpose we studied the interaction of a
conformation-specific antibody, Ab P2, with the
EGFR and compared it
with that of the WtEGFR. This anti-peptide antibody Ab P2 recognizes
the phosphorylated and hence activated EGFR and not the
unphosphorylated receptor; however, Ab P2 is not directed to
phosphotyrosine (5, 20). The receptor conformation recognized by the
antibody is positively regulated by phosphorylation of three tyrosine
residues at 992, 1068, and 1086. The phosphorylation of the other two
acceptor sites, namely tyrosines 1148 and 1173, does not play any role in antibody binding (17, 28). It should be mentioned in this context
that the interaction between Ab P2 and the activated receptor is
independent of EGF (5). To study the interaction between Ab P2 and the
EGFR, the detergent-solubilized membranes from the wild-type receptor
stimulated by EGF and the truncated receptor (both expressed in U87MG
cells) were phosphorylated by labeled ATP, and the
tyrosine-phosphorylated receptors were isolated by 1G2-Sepharose. Equal
amounts of the receptors were then subjected to immunoprecipitation by
Ab P2, and the receptor bands following SDS-PAGE were quantified by
densitometric scanning. The extent of precipitation of the
EGFR at
each of the antibody concentrations was similar to that of the WtEGFR
at the corresponding antibody concentration (Fig.
5). This indicates that the affinity of
the antibody for both receptor types is similar and the phosphorylation pattern, at least with respect to Tyr 992, 1068, and 1086, of the
EGFR is comparable to that of the WtEGFR. This is further confirmed
by phosphopeptide analysis of the receptors. The HPLC elution profiles
of the phosphopeptides from the
EGFR were similar to that of the
WtEGFR (Fig. 6). This suggests that the
inefficient internalization of the
EGFR is not due to its altered
phosphorylation.
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Glycosylation-induced Conformational Modification and Kinase
Activation of the EGFR--
The EGFR is a glycoprotein, and it has
12 potential N-linked glycosylation site (1) (see Fig. 1).
Earlier studies have demonstrated the importance of core glycosylation
in EGF binding and hence for kinase activation (29, 30). Since four of
the 12 sites are missing from the
EGFR and it lacks ligand binding activity, we investigated whether the conformation of the truncated receptor is influenced by glycosylation and if so whether such conformational modification plays any role in kinase activation. Like
the aglyco-WtEGFR which does not interact with mAb 425 (19), aglyco-
EGFR synthesized in the presence of tunicamycin is also not
capable of binding with the antibody (data not shown). As discussed
above (see Table I), this monoclonal antibody is directed to an
extracellular peptide epitope of the EGFR (19), and it also recognizes
the
EGFR. This suggests that the aglyco-
EGFR, like the
aglyco-WtEGFR, fails to attain certain conformation needed for mAb 425 binding. We also investigated if there is any relationship between the
conformational alteration induced by glycosylation and kinase
activation of the
EGFR. Cells expressing the
EGFR were
metabolically labeled with [35S]methionine in the absence
or presence of tunicamycin, and then the labeled receptors were
quantified by immunoprecipitation with a polyclonal anti-EGFR antibody
coupled to agarose. Based on this quantification, equal amounts of
the labeled receptors from each cell lysate were phosphorylated with
unlabeled ATP. The autophosphorylated receptors were immunoisolated by
1G2 and then analyzed by SDS-PAGE. Upon tunicamycin treatment, the
intensity of the truncated receptor band is drastically reduced to the
same extent as that of the wild-type receptor (Fig.
7). Similar results were also seen when 1G2-purified receptors were immunoprecipitated by a receptor-specific antibody (data not shown). It should be mentioned in this connection that although anti-phosphotyrosine antibody binding is independent of
the number of tyrosine residues in a receptor molecule being phosphorylated, nevertheless any phosphorylation (one or five per
molecule) is a reflection on its kinase activity. Thus the drastic
reduction in the intensity of the bands from Tu-treated cells suggests
the lower kinase activity of the aglyco-receptor compared with that of
the glycosylated receptor. We have also assayed the kinase activity of
the Tu-treated/untreated cells by exogenous substrate assay (Table
II). In this assay, equal amounts of the
glycosylated and aglyco-receptors (quantified by Western blot using an
C-terminal anti-EGFR antibody) were immunoisolated by a polyclonal
anti-EGFR antibody coupled to agarose, and then the kinase activity in
the gel beads was assayed by phosphorylation of an EGFR substrate (18).
As with the autokinase assay, the extent of phosphorylation of the
substrate by the aglyco-receptor was much less compared with that of
the control receptor (Table II). Thus, as with the EGFR, core
glycosylation of the
EGFR is needed to generate a conformation which
eventually leads to its kinase activation.
|
|
We also investigated the mechanism by which the carbohydrate chains are
involved in kinase activation. One possibility is that the
oligosaccharide chains participate directly in kinase stimulation, and
removal of the carbohydrate chains reverts the receptor back to its
kinase-inactive state. Alternatively, the function of the
N-linked glycosylation is to impart a stable kinase-active conformation to the receptor, and once the receptor attains such a
conformation, the carbohydrate chains are dispensable. To distinguish between these two possibilities, we studied the effect of enzymatic removal of the carbohydrate chains from the mature EGFR on its kinase activity. One such enzyme is endoglycosidase H that cleaves between the N-acetylglucosamine residues of the chitobiose
unit of N-glycans that are linked to asparagine. The high
mannose form of the truncated receptor which is a substrate for the
enzyme was synthesized by growing cells with
[35S]methionine in the presence of swainsonine, an
inhibitor of mannosidase II. The receptor synthesized in the presence
of swainsonine and not the control receptor is susceptible to
endoglycosidase H cleavage (data not shown). When equal numbers of
acid-insoluble counts from control- and swainsonine-treated cell
lysates were phosphorylated with unlabeled ATP and then the
tyrosine-phosphorylated receptors were purified by 1G2 antibody, the
intensity of the treated band was similar to that of the control band.
Thus, the modified receptor of Mr ~140,000 is
as active as the control receptor (Fig.
8A). Similar results were also
seen by exogenous substrate assay (data not shown). These suggest that
terminal processing blocked by swainsonine is not required for kinase
activation. Deglycosylation of the high mannose receptor by treatment
with endoglycosidase H results in a protein of
Mr ~115,000 (see Fig. 8B). The
intensities of both the 115-kDa and 140-kDa bands after phosphorylation
with unlabeled ATP and immunoisolation by 1G2 are similar suggesting that the autokinase activity of the deglycosylated receptor is similar
to that of the untreated receptor. Similar results have also been
reported earlier with the wild-type receptor in which complete removal
of the carbohydrate chains from the glycosylated receptor neither
abolished the ligand binding activity nor the kinase activation (29,
30). Our results suggest that (i) the initial N-linked
glycosylation but not its terminal processing is sufficient for kinase
activation; and (ii) the function of the glycosylation is to impart a
kinase-active conformation to the
EGFR, and it does not revert back
to its kinase-inactive conformation upon deglycosylation.
|
Aglyco-EGFR Synthesized in the Presence of Tunicamycin Lacks
Dimer Forming Ability--
Since self-dimerization/oligomerization is
obligatory for
EGFR kinase stimulation, we investigated whether the
reduced kinase activity of the aglyco-receptor is due to its loss of
dimer forming ability. It should be mentioned in this connection that
based on quantifying receptor protein by Western blot, the autokinase activity of the aglyco-
EGFR is 10-20% of the glycosylated receptor (data not shown). This agrees well with our finding that the kinase activity of the dimeric
EGFR is 5-7-fold higher relative to the monomeric receptor (see Fig. 4). To test whether the reduced kinase activity of the aglyco-receptor is indeed due to its loss of dimer forming ability, chemical cross-linking studies were carried out. The
microsomal fraction from Tu-treated cells contains the aglyco-receptors as well as the glycosylated receptors synthesized prior to tunicamycin treatment. The detergent-solubilized microsomal fraction was passed through WGA-agarose column, and the flow-through (source of
aglyco-receptor) was phosphorylated with [
-32P]ATP.
Following purification of the labeled receptor with 1G2, it was
subjected to cross-linking with BS3 and then
immunoprecipitated with a receptor-specific antibody. The glycosylated
receptor bound to WGA-agarose was eluted by
N-acetylglucosamine and incubated with labeled ATP. After
purification of the receptor with 1G2, it was incubated with
BS3 and then subjected to immunoprecipitation with an
anti-receptor antibody. As shown in Fig.
9A, lane 2 (left-hand
panel), following BS3 treatment, a cross-linked
complex corresponding to Mr ~300,000 could be
detected in the glycosylated receptor in addition to the
145-150-kDa band. The ~300-kDa band was absent when no
cross-linker was used (Fig. 9A, lane 1). However, under
similar conditions, no cross-linked complex could be seen when the
aglyco-receptor was treated with BS3 (see Fig. 9A,
lane 4), although the 115-kDa band corresponding to the monomeric
form of the receptor was present. Increasing the concentration of
BS3 to 250 µM had no effect on the mobility
of the aglyco-receptor (data not shown). This suggests that lower
kinase activity of the aglyco-receptor is due to its lack of dimer
forming ability. This was further confirmed by Western blot analysis
(Fig. 9B). In this experiment, detergent-solubilized plasma
membranes (source of the glycosylated receptor) and the WGA-agarose
flow-through from the Tu-treated microsomal fraction (source of the
aglyco-receptor) were subjected to cross-linking with 150 µM BS3, and following immunoprecipitation by
a receptor-specific antibody, the cross-linked receptors were subjected
to Western blot analysis. In glycosylated receptor, a 300-kDa band as
well as a band at ~145-150-kDa could be seen following cross-linking
(Fig. 9B, lane 2). The 300-kDa band could not be seen when
no cross-linker was used (Fig. 9B, lane 1). However, under
similar conditions, no cross-linked complex could be seen when the
aglyco-receptor was treated with BS3 (see Fig. 9B,
lane 4), although the 115-kDa band corresponding to the monomeric
form of the receptor was present. No cross-linked complex could be seen
when BS3 concentration was increased to 300 µM (data not shown).
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DISCUSSION |
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The major findings of our studies are as follows. (i) The
hyperactivation of the EGFR is due to its stable receptor-receptor self-association. (ii) The extent of kinase activation and the phosphorylation pattern of the
EGFR resulting from its
self-dimerization are the same as that of the ligand-induced
dimerization of the WtEGFR. (iii) Most interestingly, the
self-dimerization of the
EGFR leading to kinase activation is highly
dependent on a conformation induced by core glycosylation. These
studies were conducted by expressing both receptor types in U87MG, a
glioblastoma cell line. Our binding studies with
125I-labeled mAb 425 revealed that the expression levels of
both receptor types are comparable. In addition, the endogenous
receptor level in host cell is also low (~5% of the truncated
receptor) (see Table I). It should be mentioned in this connection that the
EGFR binding sites/cell determined in the present study differ significantly from the one reported by Huang et al. (16)
although both groups used the same cell line. By using
fluorescence-activated cell sorter analysis and comparing the intensity
of the stained cells with that of A431 cells, Huang et al.
(16) estimated that the expression level of
EGFR was 1-3 × 106 receptors/cell. Such drastic differences in receptor
number might be due to the differences in receptor assay
(125I-antibody binding versus flow cytometry
which is an indirect method) and/or difference in cell culture
including different passage number.
During the isolation of the dimeric form of the EGFR by sucrose
gradient centrifugation, the receptor preparation has undergone a
series of experimental manipulations that not only took considerable time to complete but also resulted in significant dilution of the
receptor. Despite these, nearly 40% of kinase activity could be
recovered in a dimeric state suggesting that the receptor-receptor interaction is highly stable. Thus, it is likely that under in vivo conditions in which the receptor concentration is very high (due to amplification), the
EGFR will be predominantly in dimeric form. Our studies also revealed that on a molar basis, the kinase activity of the dimeric receptor is 5-7-fold higher compared with that
of the monomeric receptor. We also showed that the kinase activity per
molecule of the dimeric
EGFR is similar to that of the
EGF-stimulated WtEGFR as determined by autophosphorylation assay. This
is in contrast to the earlier studies that reported that the kinase
activity as determined by phosphotyrosine content per molecule of the
EGFR is only 13-20% of the WtEGFR stimulated by EGF (13, 16).
Those studies were further supported by the finding that only 20% of
the receptor population could be chemically cross-linked. However, it
should be noted that chemical cross-linking is neither a quantitative
reaction nor does it provide any direct information as to the
activation state of the receptor. Another reason for differences
between our results and those of others (13, 16) might lie in the way
the studies were performed. Unlike the present study in which the
dimeric receptor complex was separated from the monomeric and hence
dormant receptor kinase to determine its activity, the earlier works
(13, 16) were conducted on a receptor preparation that contained a
mixture of monomeric and dimeric receptors. Our Western blot analysis
of the sucrose gradient fractions revealed that only 10-15% of the total receptor protein is present in the dimeric form. Thus the ~10-fold lower kinase activity per molecule of the
EGFR relative to the WtEGFR as reported by Huang et al. (16) could be
attributed to the presence of a significantly large population of the
monomeric receptor with very low kinase activity along with a small
population of the dimeric receptor and hence does not reflect the
actual kinase activity of the dimeric receptor.
The facts that the affinity and the extent of binding of the
conformation-specific antibody, Ab P2, with the EGFR are similar to
that of the EGF-activated wild-type receptor (Fig. 5) suggest a similar
phosphorylation-induced conformational change of both the receptors. It
should be mentioned in this context that Ab P2 recognizes an epitope in
the intracellular domain of the EGFR which is unmasked only upon
phosphorylation of tyrosines 992, 1068, and 1086, located in the
C-terminal tail of the receptor, as a unit. This suggests that the
EGFR is recognized by the antibody as an "active" molecule. This
is further supported by the phosphopeptide analysis which revealed that
there is no difference in the phosphorylation pattern of the
EGFR
with that of the wild-type receptor stimulated by EGF (Fig. 6). Thus
whether the receptor undergoes self-dimerization (as is the case with
the
EGFR) or ligand-induced dimerization, the outcome is the same.
Our results and hence the conclusion are in contrast to earlier reports
that correlated the inefficient down-regulation of the
EGFR with its
low autokinase activity and hence altered phosphorylation pattern.
Since phosphorylation-induced exposure of the otherwise cryptic
endocytic sequence motifs is needed for receptor internalization and
down-regulation (6), Huang et al. (16) suggested that the
cryptic internalization codes are not exposed in the
EGFR. Since we
could not detect any altered phosphorylation pattern of the
EGFR in
relation to the wild-type receptor, it suggests that the defective
endocytosis is not due to low or altered phosphorylation but due to a
mechanism that is not yet clear. Thus the high tumorigenic activity of
the
EGFR is probably not only due to the persistent presence of the receptor on the cell surface as has been suggested (16) but is also
possibly due to the higher kinase activity of the dimeric receptor
generated by self-association. It should be mentioned that due to high
stability of the dimeric receptor, most of the receptor population
under in vivo situation will be present in dimeric state.
The importance of the carbohydrate chains on the conformation of the
EGFR is underscored by our finding that like the aglyco-WtEGFR,
EGFR synthesized in the presence of tunicamycin fails to bind a
monoclonal antibody, mAb 425, directed to a peptide epitope in the
extracellular domain of the EGFR (data not shown). This antibody,
however, binds to the wild-type as well as truncated EGFR (see Table
I). These results suggest that glycosylation-mediated post-translational modification confers a conformation needed for mAb
425 binding to the wild-type receptor as well as to the truncated
receptor. The conformation induced by core glycosylation and recognized
by mAb 425 results in proper folding of the full-length receptor to
generate an EGF-binding active conformation, and once such a
conformation is attained, the carbohydrate chains are dispensable (29,
30). Although the
EGFR undergoes dimerization and kinase activation
in the absence of EGF binding, interestingly, our studies revealed that
core glycosylation is also needed for receptor-receptor self-association and hence for kinase activation. In addition, as with
the wild-type receptor, the glycosylation-induced conformation of the
truncated receptor is stable since removal of the carbohydrate chains
from the glycosylated receptor does not have any negative effect on
receptor dimerization and kinase activation (see Figs. 8 and 9).
There are 12 potential N-linked glycosylation sites in the
EGFR-2 in each of domains I and II and four in each of domains III and
IV ((2) see Fig. 1). Eight of the 12 chains are present in the EGFR.
It should be mentioned in this connection that the type II-truncated
EGFR expressed in certain glioblastomas lacks a major portion of domain
IV (amino acids 520-603) (9). Despite the fact that three of the four
glycosylation sites located in domain IV are also missing from the
receptor, type II receptor has EGF binding activity. Thus it is likely
that the oligosaccharide chains in domain IV have no influence on
ligand binding. This result together with the observations that mAb 425 (i) inhibits EGF binding to the wild-type receptor (19), (ii) mimics
EGF in its receptor binding profile (19), and (iii) interacts only with
the glycosylated
EGFR (lacking domain I and a major portion of
domain II and missing four carbohydrate chains) suggest that the four
N-linked oligosaccharide chains in domain III are probably sufficient to induce receptor-active conformation, i.e. a
conformation needed for EGF binding (for WtEGFR) or for
self-dimerization (for
EGFR).
The similar characteristics with respect to sugar requirements for
self-dimerization and for EGF binding appear to suggest that
glycosylation positively regulates receptor-receptor association, and
this is probably a prerequisite step for ligand binding. The fact that
unliganded intact receptor pre-exists in a monomeric form whereas the
truncated receptor undergoes self-dimerization suggests that there are
two opposing interactions in the EGFR. One interaction is involved in
receptor-receptor self-association and is dependent on glycosylation.
This site is probably located in domain III (see above). The other
exerts negative influence on protein-protein interaction, and this site
is located in domain I. In the full-length receptor, the protein is in
a monomeric state since the negative influence of domain I is much
stronger compared with the positive influence exerted by domain III.
However, EGF binding to domain III somehow overcomes the inhibitory
effect of domain I. Thus, although we think of activation by ligand as a positive step, it may, instead, simply reflect the ability of a
ligand to remove a negative constraint. Future studies with the
receptor in which different domains are swapped will allow us to
understand the role of individual domains in inter-receptor association
and dissociation. Furthermore, studies with the receptor in which
potential glycosylation sites are mutated will enable us to understand
the influence of individual oligosaccharide chains located in domain
III in receptor structure and function. More importantly, such
information will be useful in developing antibodies to block receptor
self-dimerization as a way to control aberrant EGFR.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Webster K. Cavenee and H.-J. Su
Huang of Ludwig Institute of Cancer Research, La Jolla, CA, for
providing us with U87MG.WtEGFR and U87MG.EGFR cells.
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FOOTNOTES |
---|
* This work was supported in part by a grant from the Foundation of the University of Medicine and Dentistry of New Jersey (to S. B.).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: Medical Science Bldg.,
Rm. C-567, Dept. of Pathology and Laboratory Medicine, UMDNJ-New Jersey
Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714. Tel.:
973-972-2623; Fax: 973-972-5909; E-mail: bishayee@umdnj.edu.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005599200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; Ab, antibody; BSA, bovine serum albumin; BS3, 3,3'-bis(sulfosuccinimido)suberate; EGF, epidermal growth factor; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin; WtEGFR, wild-type EGFR; aa, amino acid.
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