(Received for publication, September 11, 1996, and in revised form, November 15, 1996)
From the Ludwig Institute for Cancer Research,
§ Department of Medicine,
Department of Chemisty and
Biochemistry and §§ Center for Molecular
Genetics, University of California at San Diego,
La Jolla, California 92093-0660,
Pharmingen, Inc.,
San Diego, California 92121, and the ¶¶ Department of
Pathology, University of Utah School of Medicine,
Salt Lake City, Utah 84132
Deregulation of signaling by the epidermal growth
factor receptor (EGFR) is common in human malignancy progression. One
mutant EGFR (variously named EGFR, de2-7 EGFR, or EGFRvIII), which
occurs frequently in human cancers, lacks a portion of the
extracellular ligand-binding domain due to genomic deletions that
eliminate exons 2 to 7 and confers a dramatic enhancement of brain
tumor cell tumorigenicity in vivo. In order to dissect the
molecular mechanisms of this activity, we analyzed location,
autophosphorylation, and attenuation of the mutant receptors. The
mutant receptors were expressed on the cell surface and constitutively
autophosphorylated at a significantly decreased level compared with
wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR,
the constitutively active
EGFR were not down-regulated, suggesting
that the altered conformation of the mutant did not result in exposure
of receptor sequence motifs required for endocytosis and lysosomal
sorting. Mutational analysis showed that the enhanced tumorigenicity
was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these
activities suggesting that the biological functions of
EGFR are due
to low constitutive activation with mitogenic effects amplified by
failure to attenuate signaling by receptor down-regulation.
Ligand binding to wild-type epidermal growth factor receptor (wt EGFR)1 results in receptor dimerization, kinase activation, and autophosphorylation that provides both docking sites for proteins involved in signal transduction and exposure of endocytic and lysosomal targeting sequence codes required for receptor internalization and down-regulation (1). The biochemical and biological roles of each autophosphorylation site in wt EGFR have been explored by mutational analysis, and mutation of any single autophosphorylation site does not significantly abrogate binding of the activated receptor to specific SH2-containing proteins associated with distinct signaling pathways (2). Likewise, such single mutations are generally incapable of reducing the biological functions of the receptor in in vitro models (3, 4). Correspondingly, the mitogenic and transforming activities of wild-type receptor were diminished only when combinations of favorable autophosphorylation sites (i.e. Tyr-1068, Tyr-1148, and Tyr-1173) were mutated (3), suggesting that the autophosphorylation sites of wt EGFR may have less specificity for signaling proteins and can compensate for each other. Sites of tyrosine phosphorylation may be provided via heterodimerization with other members of the erb B family of receptors (5-7). Point mutations that inactivated the tyrosine kinase activity of wt EGFR eliminated occupancy-induced receptor internalization (8, 9, 40, 41), whereas mutant receptors lacking multiple autophosphorylation sites also lacked the ability to undergo ligand-induced endocytosis, suggesting that kinase-regulated receptor internalization is mediated by the phosphotyrosine residues (1, 10). The role of the kinase and phosphotyrosine residues in EGFR down-regulation may be more indirect. Evidence suggests that autophosphorylation of EGFR results in a more open conformation (11) to expose the otherwise cryptic internalization/lysosomal targeting motifs to the receptor trafficking machinery, thereby triggering receptor down-regulation processes. Receptors lacking internalization sequences can lead to cell transformation (12). Thus, the normally strict controls on cell proliferation may loosen when regulatory constraints intrinsic to the EGFR structure are relaxed by these mutations.
Mutations of the EGFR gene occur in many types of human tumors where
growth deregulation is pervasive (13-15). In glial tumors of the
central nervous system, abnormalities of the EGFR are restricted to
grade III (anaplastic astrocytoma) and especially grade IV (glioblastoma multiforme) disease (16-18). Several clinical and histopathological studies have shown that such abnormalities are related to a shorter interval to relapse and decreased survival rates,
suggesting that they play an important role in glioma malignancy progression (18, 19). Nearly 50% of grade IV tumors have significantly amplified EGFR genes, and of these, the majority also show
rearrangements of the gene so that these gliomas express both wt EGFR
and the mutant forms (16, 20, 21). The most common of the
rearrangements are genomic alterations that eliminate a DNA fragment
containing exons 2-7 of the gene and generate a mutant receptor
(variously called EGFR, de2-7 EGFR, or EGFRvIII) with an in-frame
deletion of 267 amino acids from the extracellular domain (21-24).
This specific genetic alteration is also found frequently in lung and breast cancers (15, 25). In previous studies we found that expression
of
EGFR in glioblastoma cells markedly enhanced tumor cell growth in
nude mice (26), but the molecular mechanisms of the enhanced
tumorigenicity have not been elucidated. Two defects have been
described for the
EGFR, lack of EGF binding and constitutive autophosphorylation that was unaffected by ligand but whose extent was
low compared with that of ligand-activated wt EGFR in the same cell.
The importance of autophosphorylation as a marker of constitutive
kinase activation of
EGFR to tumor growth was thus unclear.
Here we report that EGFR are expressed on the cell surface but are
defective in down-regulation due to low rates of receptor endocytosis.
Both the intrinsic tyrosine kinase activity and each of the major sites
of tyrosine autophosphorylation located in the regulatory carboxyl
terminus are essential for the enhanced tumorigenesis characteristic of
EGFR. Unlike wt EGFR, a single point mutation in any of the major
autophosphorylation sites is sufficient to abrogate the in
vivo growth advantage conferred by
EGFR. Thus deletion of exons
2-7 abolished ligand binding but created a constitutively active EGFR
that was strictly dependent for biological activity on intact
autophosphorylation. Defective attenuation due to failure to
down-regulate activated receptors appears to be the major mechanism
that enhances tumorigenicity of these weakly phosphorylated EGFR
mutants. Thus deletion of exons 2-7 results in a partially active EGFR
that couples to mitogenic signal transduction but not to vesicular
trafficking pathways.
Anti-EGFR antibodies included mouse monoclonals
528 and 225 (27) and 13A9 (28) directed against the ectodomain, rabbit polyclonal sera, Ksm, directed against residues 642-663 in the submembrane domain (provided by J. Kyte, University of California at
San Diego, La Jolla, Ca), and mouse monoclonal C13 directed against
residues 991-1018 in the carboxyl terminus. The monoclonal antibodies
specific for EGFR, D806, D1133, and D124, were generated against the
fusion junction in the ectodomain of the mutant
receptor.2 The monoclonal
antiphosphotyrosine antibody PY20 was from Transduction Laboratories
(Lexington, KY), and the rabbit polyclonal antiphosphotyrosine sera
were prepared as described (29).
All
mutant EGFR cDNAs were first constructed in pBluescript SK()
vector (Stratagene) and then transferred to a retrovirus-based vector,
pLRNL (30). The DK cDNA was generated by replacing the 769-base
pair EcoRI fragment (nucleotides 2317-3085) (31) of the
EGFR cDNA (26) with the corresponding fragment from the kinase-inactive EGF receptor cDNA, K721M (8). DY1, DY2, and DY3
represent the
EGFR with point mutation(s) which replace tyrosine with phenylalanine at residue 1173, residues 1068 and 1173, and residues 1068, 1148, and 1173, respectively. DY1, DY2, and DY3 were
constructed by replacing the 1.2-kilobase pair
BglII/HindIII fragment (nucleotides 2951 to the
carboxyl-terminal end) of pSK
EGFR with the corresponding DNA
fragments from pX-TF1, pX-TF2, and pX-TF3 (32, 33), respectively. DY5
is the
EGFR with five tyrosine to phenylalanine mutations at codons
992, 1068, 1086, 1148, and 1173. DY5 was created by a double
recombinant polymerase chain reaction (34) which generated an
additional two point mutations in the DY3 cDNA template. Primers
used in this construction were as follows: (a)
5
-TGTACCATCGATGTCTA-3
(sense, nucleotides 3001-3017); (b) 5
-TGTGGGATGAGGAACTCGTCGGCAT-3
(antisense, nucleotides 3221-3245); (c)
5
-ATGCCGACGAGTTCCTCATCCCACA-3, (sense, nucleotides 3221-3245);
(d) 5
-TCGTCTATGCTGTCCTC-3
(antisense, nucleotides 3421-3437); (e) 5
-GAGGACAGCATAGACGA-3
(sense, nucleotides
3421-3437); (f) 5
-GGCTGATTGTGAAAGACAGGATTCT-3
(antisense,
nucleotides 3503-3527); (g) 5
-AGAATCCTGTCTTTCACAATCAGCC-3
(sense, nucleotides 3503-3527); and (h)
5
-TTGCCCACTGCAGTGCT-3
(antisense, nucleotides 3574-3590). Briefly, two DNA fragments, A and B, were produced by using two pairs
of primers, a + b and c + d, respectively in polymerase chain reaction mixtures
containing DY3 cDNA. The other two DNA fragments, C and D, were
generated by the same method using the other two sets of primers,
e + f and g + h. Equimolar amounts of
fragments A and B were used with primers a and d
to generate recombinant fragment E. Fragments C and D were mixed to
produce recombinant fragment F by the same method using primers
e and h. Finally, fragments E and F were used to
generate recombinant fragment G by using primers a and
h. Fragment G, after digestion with ClaI and
PstI, was used to replace the corresponding region (nucleotides 3007-3582) in the DY3 cDNA. DY4 is the
EGFR with four point mutations which changed codons 992, 1068, 1086, and 1148 from tyrosine to phenylalanine. DY4 was constructed by replacing the
0.25-kilobase pair PvuII/XhoI fragment
(nucleotides 3762 to the carboxyl-terminal end) in the DY5 cDNA
with the equivalent region of
EGFR. Amphotropic viruses which
carried the mutant EGFR were generated as described (26).
The human glioblastoma cell line U87MG (ATCC) was
infected with mutant EGFR viruses and selected in a medium containing
400 µg of G418 per ml (26). 5 × 105 cells in 100 µl of staining buffer (phosphate-buffer saline (PBS), 1% calf serum,
0.1% sodium azide) were exposed (30-60 min) to anti-EGFR monoclonal
antibody 528 which recognized both wild-type and mutant EGFR, or cells
were exposed to monoclonal antibody D806 specific for mutant receptors,
and then exposed to fluorescein isothiocyanate-conjugated goat
anti-mouse immunoglobulin antibody (PharMingen, Inc.) for 30-60 min.
Stained cells were analyzed with a FACSort (Becton Dickinson). For cell
sorting, sodium azide was omitted from the staining buffer, and the
brightest 5% of the stained cells were selected with a FACStar (Becton
Dickinson). The sorted cells were cultured and reanalyzed. The number
of DK and DY mutant receptors on the cell surface was estimated by
comparing the fluorescence intensity of these cells with that of
U87MG.EGFR cells which expressed 2 × 106
EGFR
per cell (26).
Cells (4 × 105 cells per well in a 12-well plate or 2.5 × 106 cells per 100-mm dish) were cultured overnight in
medium containing 10% dialyzed fetal bovine serum. The cells were
exposed to 100 ng/ml EGF (Collaborative Biomedical Products) for 5 min
at 37 °C. For Western blotting, cells were washed with PBS, lysed in
a PBS buffer containing 0.06 M Tris-Cl, pH 6.8, 1.3% SDS,
12.8% (v/v) glycerol, 1.3% 2-mercaptoethanol, 2 mM
Na3VO4, 5 µg/ml leupeptin, 5 µg/ml
antipain, 5 µg/ml pepstatin, and 0.25% bromophenol blue, and then
boiled. The lysates were electrophoresed through SDS, 7.5%
polyacrylamide gels and the proteins transferred to nitrocellulose membranes (Bio-Rad). For immunoprecipitation/Western blotting, cells
were lysed in a PBS buffer containing 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 0.2% sodium azide, 0.04% sodium fluoride,
and 5 µg/ml each of the protease inhibitors. Cellular debris was
removed by centrifugation at 12,000 × g for 5 min at 4 °C. EGFR were immunoisolated using a EGFR-specific monoclonal antibody, D1133, or anti-EGFR monoclonal antibody, 528, electrophoresed, and transferred to nitrocellulose membranes. Blots
were probed with horseradish peroxidase-conjugated anti-phosphotyrosine
monoclonal antibody PY20, and the proteins were visualized using the
enhanced chemiluminescence detection system (Amersham Corp.). The blots were stripped and reprobed with the C13 anti-EGFR monoclonal
antibody in the enhanced chemiluminescence system. EGFR concentration
and phosphorylation were determined by scanning the autoradiograms with
a densitometer. Multiple exposures were obtained to ensure linearity of
response, and relative phosphorylation was calculated under these
conditions. The level of EGFR phosphorylation was normalized to the
protein concentration. The enhanced chemiluminescence was employed to
minimize the possibilities of residual signal when stripping blots
probed with 125I-protein A.
For additional quantitation of phosphotyrosine content, confluent
monolayers of U87MG or U87MG.EGFR cells on 100-mm dishes were
untreated or treated with EGF or 225 IgG (27), rinsed with PBS, and
extracted with 500 µl of boiling SDS sample buffer (5% SDS, 50 mM Tris-Cl, pH 6.8, 5% 2-mercaptoethanol, 1 mM
Na3VO4). Equal amounts of protein were loaded
on 5-15% gradient gels, transferred to nitrocellulose in the presence
of 0.1 mM Na3VO4, and blocked with
2.5% BSA, 0.1 mM Na3VO4, and
0.005% Tween 20. Tyrosine phosphate was detected using 5 µg/ml
affinity purified antiphosphotyrosine polyclonal antibodies in
conjunction with 50 ng/ml 125I-Protein A for 30 min. Dried
blots were exposed to storage phosphor plates followed by imaging using
a Bio-Rad G250 Molecular Imager. Profiles from the appropriate lanes
were used to determine the difference between the phosphotyrosine
pattern of either control or U87MG cells expressing
EGFR using the
Molecular Analyst software package.
Cells were plated on
fibronectin-coated coverslips 48 h prior to the experiment and
were either untreated or treated with 100 ng/ml EGF for 2 h and
then fixed for 10 min at room temperature with freshly prepared 3.6%
paraformaldehyde, 0.024% saponin, 1 mM
Na3VO4 in PBS. Free aldehyde groups were
quenched with 0.1% NaBH4 for 10 min, and nonspecific sites
were blocked with 1% BSA. To detect total EGFR, a mixture of anti-EGFR
monoclonal antibodies was used (528, 13A9, 225; 10 µg/ml each). To
detect EGFR, D1133 monoclonal antibody was used at a concentration
of 10 µg/ml. All antibody and rinse solutions contained 1% BSA,
0.012% saponin, and 1 mM Na3VO4.
Coverslips were incubated with antireceptor antibodies together with 10 µg/ml affinity purified rabbit antiphosphotyrosine antibodies for
1 h followed by staining with fluorescein isothiocyanate-labeled goat anti-mouse and Texas Red-labeled goat anti-rabbit antibodies (1:100; Cappel Laboratories) for 45 min. The coverslips were mounted in
ProLong antifade medium (Molecular Probes, Inc.) and viewed with a
Nikon inverted fluorescence microscope with a 40 × oil immersion
objective. A multi-dye filter set was used in which excitation filters
of 485 nm (fluorescein isothiocyanate) and 575 nm (Texas Red) were
selected from a computer-controlled filter wheel in conjunction with a
multi-wavelength emitter and dichroic filter set (460, 525, and 640 nm;
XF56 set from Omega Optical). Fluorescein isothiocyanate and Texas Red
images were acquired separately using a Photometrics cooled CCD camera
and a Macintosh workstation running OncorImage software. Images were
scaled to 256 gray levels using Adobe Photoshop 3.0 on the Macintosh
and transferred to TMAX-100 film using an AFGA film recorder.
The specific internalization rates of
wild-type EGFR (wt EGFR) and EGFR were determined by measuring
endocytosis of 125I-labeled 13A9 monoclonal antibody. The
antibody binds equally well to both the wt EGFR and
EGFR and does
not affect the binding of EGF to the wt EGFR. This allowed evaluation
of receptor endocytosis in both the empty and occupied state. 13A9
antibody was iodinated with 125I (Amersham Corp.) to a
specific activity of 1500 cpm/fmol as described previously (35). Cells
grown to confluency on fibronectin-coated plates were incubated at
0 °C for 90 min with 4 nM 125I-labeled 13A9
in the presence or absence of 1 µg/ml EGF. The cells were rapidly
warmed to 37 °C by the addition of prewarmed medium. The relative
amount of internalized versus surface-associated antibody
was determined at 2-min intervals by acid stripping as described (9).
The stripping efficiency was approximately 93%. The data were
converted to internalization plots as described previously (9) and
normalized to total surface binding of 13A9 (36).
Cells expressing wild-type and
mutant EGFR were washed in PBS containing 2 mM EDTA and 2 mM EGTA before being solubilized and homogenized at 4 °C
in buffer containing 10 mM Hepes, pH 7.9, 10 mM
NaCl, 1% glycerol, 1% Triton X-100, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, 4 mM benzamidine, and 1 mM
phenylmethylsulfonyl fluoride. Cell lysates were centrifuged at
12,000 × g for 5 min at 4 °C. Mutant receptor
supernatants were incubated with a 1:50 dilution of monoclonal antibody
D124, and wt EGFR supernatant was incubated with 1:100 dilution of
monoclonal antibody, 13A9, at 4 °C for 1 h. Protein A-Sepharose
was then added for 30 min at 4 °C. Immunoprecipitates were washed
four times in buffer containing 10 mM Hepes, pH 7.9, 300 mM NaCl, 1% glycerol, 2 µg/ml leupeptin, 4 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride. Immunoisolated wt EGFR was treated with 200 ng/ml EGF for 10 min at 22 °C prior to start of the kinase assay. Kinase reaction
mixtures contained 12.5 mM Hepes, pH 7.4, 10 mM
MnCl2, 50 µM Na3VO4,
10 µM [-32P]ATP (1.0-2.8 × 104 cpm/pmol), immunoprecipitated EGFR, and 100 µM 1173 peptide (RRKGSTAENAEYLRV). Phosphorylation
reactions proceeded for 10 min at 22 °C and were terminated by the
addition of trichloroacetic acid (final concentration, 6.1%). BSA (100 µg) was added to facilitate the precipitation of EGFR, and the
reactions were incubated at 4 °C for 10 min. Precipitated receptors
were centrifuged for 5 min at 12,000 × g, and
phosphate incorporation was determined by spotting the supernatant on
phosphocellulose paper (Whatman P-81, 2.4-cm circle). The
phosphocellulose was washed four times in 500 ml of 75 mM phosphoric acid for 15 min at 4 °C, rinsed in acetone, and dried. The amount of retained radioactivity on the phosphocellulose was then
measured. Protein concentrations were determined by separating receptors and protein standards on 7.5% SDS-polyacrylamide gels, transferring to Immobilon, probing with the anti-EGFR polyclonal antibody, Ksm, followed by an alkaline phosphatase conjugated anti-rabbit antibody, and scanning the blots with a densitometer.
For subcutaneous inoculation, U87MG cells
(1 × 106) and U87MG cells expressing mutant EGFR
(1 × 106 or 2 × 105) were suspended
in 0.1 ml of PBS and injected simultaneously into the left or right
flanks, respectively, of 4- to 5-wk-old female nude mice of BALB/c
background. The growing tumors were measured twice a week with a
caliper, and tumor volumes were calculated using width (a)
and length (b) measurements
(a2b/2, where a < b) (37). For intracerebral stereotactic implantation, 1 × 105-5 × 105 cells in 5 µl of PBS
were inoculated into the corpus striatum in the right hemisphere of the
nude mouse brain (38). Brains were removed 12 days postimplantation,
embedded in OCT compound (Miles), frozen in liquid nitrogen, and stored
at 80 °C. Thin cryostat sections (5-6 µm) were stained with 528 antibody and counter-stained with hematoxylin. Tumors were
microscopically measured, and the volumes of tumors were calculated as
described above.
The autophosphorylation
of wt EGFR induced by ligand binding results in a conformational change
proposed to expose cryptic endocytic and lysosomal targeting codes
required for induced internalization and down-regulation (11, 39).
Kinase-inactive EGFR are defective in ligand-induced down-regulation
(40, 41), whereas deletion of the trafficking sequence codes in the
carboxyl terminus of kinase-active EGFR results in
ligand-dependent transformation and tumorigenesis by these
attenuation-defective EGFR (12, 42). The extent of autophosphorylation
of mutant EGFR was much less than that of ligand-activated wt EGFR
(26), raising the question of whether these mutant receptors can
undergo the endocytosis and down-regulation characteristic of activated
EGFR. Receptor distribution was first analyzed by immunofluorescence
microscopy using monoclonal antibodies directed to the fusion junction
in the ectodomain of
EGFR, to wt EGFR, and to phosphotyrosine. As shown in Fig. 1, both wild-type and
EGFR were
predominantly located at the cell surface of U87MG cells (Fig. 1,
a and e). Staining of permeabilized cells
localized both wt EGFR and
EGFR diffusely on the cell surface with
enhancements at cell margins in agreement with previous reports (43,
44). After addition of EGF, wt EGFR were lost from the cell surface and
appeared in perinuclear vesicles corresponding to endosomes and
lysosomes (Fig. 1c). In contrast, the distribution of
EGFR was not changed upon addition of EGF, and receptors remained at
the cell surface (Fig. 1g). In cells expressing wt EGFR,
phosphotyrosine staining was most marked in focal adhesions (Fig.
1b) and, after addition of EGF, increased phosphotyrosine
co-localized with internalized EGFR in perinuclear vesicles (Fig.
1d). In cells expressing
EGFR, phosphotyrosine was
detected primarily with a punctate distribution at the cell surface
which corresponded to the distribution of
EGFR (Fig. 1f);
after addition of EGF this staining at the cell surface did not change
(Fig. 1h).
To determine whether the phosphorylation detected in these assays could
be primarily attributed to EGFR (Fig. 1, e-h), extracts from U87MG and U87MG.
EGFR cells were analyzed by Western blotting with anti-phosphotyrosine antibodies. Phosphorimage quantitation indicated that a single peak corresponding to
EGFR accounted for the
majority of the measurable increase in phosphotyrosine (Fig.
2A). Identical results were obtained when
these cells were chronically treated with EGF or with the antagonist
monoclonal anti-EGFR antibody 225 (data not shown), confirming lack of
effect of both ligand agonist and an antagonist monoclonal antibody on tyrosine kinase activity of
EGFR. To confirm that the single peak
corresponding to 140-155 kDa was indeed
EGFR, the mutant receptors were repeatedly immunodepleted from U87MG.
EGFR cell lysates (Fig. 2B, lanes 3-4) using a monoclonal antibody
specific to
EGFR. In the
EGFR-depleted lysate the overwhelming
majority of phosphotyrosine immunoreactivity in the 140-155-kDa region on Western blots had disappeared (Fig. 2B, lane 5),
indicating that phosphotyrosine immunoreactivity detected at this
region (Fig. 2B, lane 2) is
EGFR.
Because the rate-limiting step in down-regulation is internalization
from the cell surface (9, 45), the rates of endocytosis of wt EGFR and
EGFR were directly measured using the 13A9 monoclonal antibody that
recognizes both. Previous studies have indicated that the rate of
endocytosis of monoclonal antibodies bound to the ectodomain of wt EGFR
accurately reflects basal and ligand-stimulated internalization
kinetics (9). The rate of endocytosis of
EGFR corresponded to the
basal rate of endocytosis of non-activated and kinase-inactive EGFR
(Fig. 3) (9) and was consistent with random entrapment
in coated pits (9). In sharp contrast, ligand-activated wt EGFR
exhibited a significantly higher rate of endocytosis.
EGFR were thus
defective in down-regulation at least in part due to a low rate of
constitutive internalization equivalent to that of non-activated wt
EGFR.
Essential Sites of Autophosphorylation in
The major
phosphotyrosine containing protein in U87MG.EGFR cells was the
EGFR itself (Figs. 1 and 2) suggesting that autophosphorylation was
critical to biological signaling by this mutant receptor. To determine
the contribution of the intrinsic tyrosine kinase activity and
autophosphorylation of
EGFR to tumorigenicity, several derivative
EGFR mutants were constructed (Fig. 4A).
The kinase activity of
EGFR was inactivated by introducing a point
mutation in the ATP-binding site which changed lysine 721 to methionine (8) (DK, Fig. 4A). Other
EGFR cDNAs were
constructed with point mutations that altered the tyrosine
phosphorylation sites identified in wt EGFR (Tyr-992, Tyr-1068,
Tyr-1086, Tyr-1148, and Tyr-1173) (46-48), either singly (DY1, Y1173F)
or in combinations (DY2-5). The constructs were introduced into U87MG
cells individually by retroviral-mediated gene transfer and selected,
and populations expressing levels of mutant receptors similar to that
of U87MG.
EGFR were sorted and expanded for further analysis (Fig.
4A). As a control, U87MG cells infected with viruses
carrying the wt EGFR gene and expressing a similar level of wt EGFR
were also selected by the same procedure. The presence of equivalent
amounts of mutant (140-155 kDa) and wt receptors (170 kDa) in the
cells was confirmed by probing Western blots using anti-EGFR antibodies
(Fig. 4B, lower panel).
While EGFR was constitutively autophosphorylated (Fig. 4B,
upper panel, lanes 3 and 4), DK EGFR was devoid of any
significant tyrosine phosphorylation (Fig. 4B, upper panel, lane
5). Tyrosine phosphorylation of DK EGFR could not be restored by
activation of the endogenous wt EGFR in the same cells; only a minor
amount of DK EGFR phosphorylation was observed (Fig. 4B, upper
panel, lane 6), indicating a lack of substantial
cross-phosphorylation between ligand-activated wild-type and mutant
receptors. This conclusion was supported by the lack of phosphorylation
of wt EGFR which co-existed with the constitutively active
EGFR in the same cells (Fig. 4B, upper panel, lane 3).
Autophosphorylation of mutant receptors was principally at tyrosine
residues 1068, 1148, and 1173, because receptors mutated at these sites
(DY3) showed almost complete loss of phosphorylation (Fig. 4B,
upper panel, lanes 11 and 12). Mutants which retained
one (DY2 and DY4) or two (DY1) of these residues were still
phosphorylated (Fig. 4B, upper panel, lanes 7-10, 13, and
14). Receptors with mutations of all five tyrosine residues
(DY5) were somewhat phosphorylated, albeit at significantly reduced
levels (Fig. 4B, upper panel, lanes 15 and
16).
In order to quantify autophosphorylation of the various mutant EGFRs,
receptors were immunoisolated before probing with anti-phosphotyrosine antibody. Fig. 5A shows that the extent of
phosphorylation per molecule of EGFR was approximately 12% that of
wt EGFR activated by EGF treatment. Analysis of the DY series of Tyr to
Phe mutations indicated that Tyr-1173 appeared to be the major
autophosphorylation site since a single mutation at this residue
decreased phosphorylation to 35% that of
EGFR. Furthermore, mutant
receptor DY4, which retained only the Tyr-1173 site, was phosphorylated
to 42% that of
EGFR (Fig. 5B). Phosphorylation of mutant
receptors with alterations in more than one autophosphorylation site
was even lower, with decreases to 25 and 1% for DY2 and DY3,
respectively (Fig. 5B). The phosphorylation level of DY5 was
3% that observed for
EGFR, suggesting that mutation of all five
tyrosines resulted in accessibility of the intrinsic kinase to some
minor phosphotyrosine site. In contrast, mutation of the kinase domain
completely abolished autophosphorylation of
EGFR (Fig.
5B).
Autophosphorylation of EGFR tyrosine residues by ligand binding not
only activates binding sites for signaling molecules but may also
regulate the catalytic activity of the receptor by altering the
conformation of the kinase domain and/or by changing the access of
cellular substrates to the kinase active site (49, 50). This raised the
question of whether mutations of the tyrosine residues in EGFR
affected the kinase activity. To evaluate the contribution of the
kinase domain and the phosphorylation sites of
EGFR in the enhanced
tumorigenic effect, the kinase activities of
EGFR containing
mutations in tyrosine residues were determined. Cells overexpressing
wild-type or mutant receptors were solubilized, and the receptors were
immunoisolated using anti-wt EGFR or anti-
EGFR specific antibodies.
Receptor kinase activity was assayed in vitro using the
synthetic substrate, 1173 peptide (51). While point mutation of Lys-721
inactivated the kinase activity of the DK receptor, mutations of
tyrosine residues did not have a measurable effect on the in
vitro kinase activity of the various DY receptors (Table
I).
|
In order to confirm the tumorigenic importance of the
intrinsic tyrosine kinase activity of EGFR, the ability of U87MG.DK cells to form tumors in nude mice was measured. U87MG.
EGFR cells grew markedly faster than U87MG cells when the same number of cells
(1 × 106) were implanted subcutaneously into the
flanks of nude mice (Fig. 6A), in agreement
with a previous report (26). Reducing the number of implanted
U87MG.
EGFR cells 5-fold still resulted in a profound growth
advantage of the mutant receptor containing cells when compared with
parental U87MG cells. U87MG.DK cells which expressed the same level of
DK receptors as that of
EGFR in U87MG.
EGFR cells showed a
similarly low tumorigenic potential as parental U87MG cells (Fig.
6A), indicating that inactivation of the intrinsic tyrosine
kinase activity abolished the enhancement of tumorigenicity mediated by
EGFR.
To determine whether the loss of growth advantage of DK receptor
containing U87MG cells was specific to the subcutaneous site, these
glioblastoma cells were stereotactically implanted into the brains of
nude mice. Animals which received 1 × 105
U87MG.EGFR cells had tumor masses of approximately 60 mm3 12 days postimplantation which were more than 100-fold
larger than tumors from the inoculation of 5 × 105
U87MG.DK or parental U87MG cells (Fig. 7,
a-c). Western blotting analysis showed that tumors obtained
from U87MG.DK implantations expressed endogenous EGFR of 170 kDa as
well as smaller species of 140-155 kDa corresponding to the DK
receptor. These DK receptors were expressed at a level comparable with
that of U87MG.DK cells grown in vitro and were not
tyrosine-phosphorylated (data not shown), demonstrating lack of
activity and stable expression of kinase-inactive DK EGFR in
vivo. Taken together, these data indicate that the intrinsic
kinase activity is essential for the enhanced tumorigenicity mediated
by
EGFR.
Mutations of the autophosphorylation sites significantly affected the
ability of EGFR to enhance tumor formation in nude mice. The
kinetics of subcutaneous tumor growth shown in Fig. 6B
indicate that mutation of any of the autophosphorylation sites, singly
or in combination, dramatically reduced the enhanced tumorigenic activity conferred by the
EGFR in U87MG cells. Cells bearing these
phosphorylation site mutants, with the exception of DY3, grew only
slightly faster than the same number (1 × 106) of
implanted parental U87MG cells, and much more slowly than the tumors
resulting from 5-fold fewer inoculated U87MG.
EGFR cells. U87MG.DY3
cells repeatedly formed tumors more slowly than the parental cells, and
the expression of the DY3 mutant receptor was not stable either in
tissue culture or in tumors. Similar results were obtained when these
cells were injected into the brains of nude mice (Fig. 7,
d-h). The average volume of tumors derived from cells
expressing
EGFR with tyrosine autophosphorylation site mutations was
30-600-fold less than that of tumors formed from 5-fold fewer
U87MG.
EGFR cells. These data strongly suggest that the tumorigenic
effect of
EGFR was mediated through phosphorylation of its
carboxyl-terminal tyrosine residues. Overexpression of wt EGFR did not
confer significantly enhanced tumorigenic activity in U87MG cells in
either subcutaneous or brain implantations (Fig. 6B and Fig.
7i), indicating that ligand-dependent wt EGFR
functioned less effectively than constitutively active
EGFR in the
animal model.
Deletion of amino acid residues 6-274 located in the ectodomain
of EGFR creates a highly tumorigenic receptor tyrosine kinase as
measured by growth of subcutaneously and intracerebrally implanted glioma expressor cells (26). Because this mutant receptor occurs in a
variety of human tumors (13-15), the basis for its enhanced proliferative effects are of considerable interest. The EGFR has
several features in common with v-erbB, the principal
oncogene of avian erythroblastosis virus (52), including failure to
bind ligand and constitutive activation of intrinsic protein tyrosine kinase activity as assessed by autophosphorylation (53, 54). v-erbB resulted from retroviral insertion that reproducibly
deleted most of the ectodomain of the avian EGFR (55), whereas
EGFR results from a common gene deletion that occurs naturally during malignant progression (18, 21, 24, 56). However, the transforming activity and tissue specificity of v-erbB is dependent on
additional truncation and/or mutation of the carboxyl terminus (57,
58), raising the question of whether a small deletion of the
ligand-binding domain in
EGFR alone is capable of eliciting any
biological effect. The present studies confirm the hypothesis that the
constitutive tyrosine kinase activity resulting from ectodomain
deletion is essential to tumorigenic effects in human tumor cells,
where this mutant receptor naturally occurs. Since the extent of
autophosphorylation of
EGFR was small compared with ligand-activated
wild-type EGFR, the basis for strong mitogenic signaling was uncertain.
The present studies indicate that enhanced tumorigenesis of
EGFR is
strictly dependent on intact autophosphorylation sites located in the
regulatory carboxyl terminus. Our observation that
EGFR are
defective in endocytosis and down-regulation suggests that the effects
of the small extent of constitutive activation are greatly amplified due to failure of normal attenuation mechanisms.
Individual autophosphorylation sites in several tyrosine kinase
receptors, an event particularly well studied in PDGF receptors, exhibit high specificity for binding SH2 and PTB domains (59); mutation
of individual sites abrogates signaling via particular proteins (60).
Autophosphorylation sites of EGFR appear to exhibit less specificity
(2), although heterodimerization with and transphosphorylation of
molecules such as erbB2 and erbB3 may provide coupling to specific
molecules such as phosphatidylinositol 3-kinase (61). In EGFR,
Tyr-1173, the favored autophosphorylation site in ligand-activated wt
EGFR (46), appears to be the major site of autophosphorylation.
Mutation of Tyr-1173 reduced its autophosphorylation by ~65%, and
mutation of all known autophosphorylation sites except Tyr-1173 yielded
a receptor with ~45% the extent of autophosphorylation. Mutation of
the three major autophosphorylation sites at Tyr-1068, Tyr-1148, and
Tyr-1173 abolished detectable autophosphorylation.
Mutation of any of these major autophosphorylation sites alone (Y1173F
[DY1] Figs. 6 and 7, Y1068F [DY6], and Y1148F [DY8], data not
shown) or in combination (DY3, DY4, DY5) abolished the enhanced
tumorigenesis characteristic of
EGFR. Since these measurements
reflect the average extent of phosphorylation per receptor, these
values may reflect heterogeneity in the phosphorylation of any one
receptor. The requirement that all major sites of tyrosine autophosphorylation be intact for enhanced tumorigenesis could reflect
a combinatorial role for interaction of multiple SH2 and phosphotyrosine binding domain proteins with constitutively activated
EGFR. Alternatively, given the finding that
EGFR are
autophosphorylated to only ~10% the level of ligand-activated
wild-type receptors, all autophosphorylation sites may be
required to reach a critical threshold. These characteristics
distinguish
EGFR from wt EGFR where it is necessary to mutate
multiple tyrosine autophosphorylation sites to block
ligand-dependent transformation (3).
There is evidence that EGFR couple to the mitogen-activated protein
kinase signaling pathway utilized by wt EGFR (62) but more weakly (63).
Montgomery et al. (63) observed activation of
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase and mitogen-activated protein kinases in NIH 3T3 cells
expressing
EGFR. The extent of activation was modest presumably due
to enhanced tyrosine phosphatase activity. Further studies confirmed
minimal activation of mitogen-activated protein kinases but could not
detect increases in GTP-bound Ras (64). However, Prigent et
al. (65) detected a 2-fold increase in GTP-bound Ras in U87MG
cells expressing
EGFR and observed that anti-Ras antibodies
inhibited DNA synthesis in these cells. Both Moscatello et
al. (64) and Prigent et al. (65) observed constitutive association of Grb2 with
EGFR. Interestingly while binding to Grb2
has been proposed to function in endocytosis of wt EGFR (66),
EGFR
are defective in endocytosis despite binding Grb2. While
EGFR may
couple to additional signaling pathways, it appears to use the same
mitogenic signal transduction pathways as wt EGFR consistent with the
low level constitutive activation of the mutant receptor. Moreover, it
has been reported (64) that
EGFR is constitutively dimerized. We
noted a small (<5%) extent of dimerization of
EGFR that was
unaffected by addition of ligand.3 The
extent of dimerization observed was roughly proportional to the extent
of activation of
EGFR (~10%) and efficacy of cross-linking.
EGFR thus exhibits neither strong homo- nor hetero-dimerization.
Autophosphorylation of EGFR provides sites both for assembly of
components of the signal transduction cascade and for exposure of
cryptic sequences in the carboxyl terminus necessary for ligand-induced endocytosis and down-regulation. In the absence of the carboxyl terminus, ligand-activated EGFR exhibit enhanced tumorigenesis due to
failure to increase both ligand and receptor internalization (12, 42,
67-69). Ectodomain-deleted EGFR are also defective in endocytosis
and down-regulation. A previous report that
EGFR localized within
cells rather than on the cell surface (70) was not confirmed in the
present studies nor in similar studies using several other glioblastoma
cell lines (71).4 Instead
EGFR expressed in
brain tumor cells are mainly localized to the cell surface reflecting a
low constitutive rate of endocytosis relative to the normal rate of
receptor recycling (35). These constitutively active receptors thus
escape the major mechanism used to attenuate receptor signaling.
Although Moscatello et al. (64) recently reported that 53%
of transfected
EGFR expressed in NIH3T3 cells were located on the
cell surface, they also showed 59% of the wt EGFR expressed in the
same cell type was on the cell surface, indicating that the
distribution of the EGFR may be cell type-specific. Indeed, we have
examined the relative distribution of wt EGFR and
EGFR in both the
NIH3T3 variant NR6, which lacks endogenous EGFR, and the human mammary
epithelial cell line 184A1, which expresses high levels of endogenous
EGFR. The distribution of wt EGFR was different between NR6 and 184A1
cells, being almost exclusively cell-surface localized in NR6 cells and
distributed between surface and intracellular compartments in 184A1
cells. The addition of EGF shifted the wt EGFR to an intracellular
compartment in both cell types.5
Significantly, the distribution of the
EGFR was the same as unstimulated wt EGFR in both cell types and was not altered by addition
of EGF. These results suggest that although the
EGFR displays
tyrosine kinase activity, it may interact with the cellular trafficking
machinery in the same way as empty wt EGFR.
The constitutive kinase activation, autophosphorylation levels set near
the threshold for enhanced mitogenesis, and the defect in endocytosis
may all reflect partial activation of EGFR. Conformational changes
sufficient to allow autophosphorylation to ~10% is sufficient for
receptor mitogenic signaling but insufficient for exposure of endocytic
sequence codes necessary for down-regulation and attenuation of signal
transduction. The recent observation that the kinase activities of wt
EGFR and
EGFR can be differentially blocked by various tyrosine
kinase inhibitors is consistent with the idea that the conformational
changes induced by ectodomain deletion in
EGFR differs from
ligand-activated wt EGFR (72). We hypothesize that deletion of exons
2-7 removes inhibitory constraints sufficient to allow low level
constitutive activation of the intrinsic protein tyrosine kinase
activity of EGFR. Although specific autophosphorylation sites may
engage specific targets, removal of any one site abolishes the
tumorigenic effect of the mutant receptor. Since the level of
constitutive activation appears near the threshold for mitogenic responses, we propose that failure to internalize
EGFR is a critical defect allowing inappropriate growth signals. Persistence of
EGFR at
the cell surface, the apparent site of assembly of many signaling complexes, would prolong and enhance its low level of activity.
EGFR
is thus seen as active for mitogenic signaling but escapes detection by
the endocytic trafficking system so that signaling is unattenuated.