(Received for publication, February 1, 1995; and in revised form, May 8, 1995)
From the
A single point mutation, Glu
Val,
equivalent to the activating mutation in the Neu oncogene, was inserted
in the transmembrane domain of the human epidermal growth factor (EGF)
receptor. Unlike the wild type, Glu
-EGF receptor,
transfected in NIH3T3 cells, gave rise to focal transformation and
growth in agar even in the absence of EGF. Constitutive activity of
mutant EGF receptor amounted to 20% of that of wild type receptor
stimulated by EGF. In addition, the mutant receptor was more sensitive
to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3
cells expressing Glu
-EGF receptor showed a transformed
phenotype and were not arrested in G
upon serum
deprivation. The mutant receptor was constitutively autophosphorylated,
and several other cellular proteins were phosphorylated on tyrosine in
absence of the ligand. Among these, the SHC adaptor protein was
phosphorylated in absence of EGF, the other adaptor, GRB-2, was
constitutively associated with the Glu
EGF receptor in vivo and in vitro, and mitogen-activated protein
kinase was constitutively phosphorylated. In contrast, other EGF
receptor substrates, like phospholipase C
, were not phosphorylated
in absence of EGF. The mutant receptor showed a higher sensitivity to
cleavage by calpain both in absence and presence of EGF, appeared as a
170- and 150-kDa doublet in cell extracts, and a specific calpain
inhibitor blocked the appearance of the 150-kDa form. Since the calpain
cleavage site is located in the receptor cytoplasmic tail, this finding
suggests that the Glu
mutation induces a slightly
different conformation in the EGF receptor intracellular domain. In
conclusion, our data show that a point mutation in the EGF receptor
transmembrane domain was able to constitutively activate the receptor
and to induce transformation via constitutive activation of the Ras
pathway.
Oncogenes have been proposed to derive from surface receptors by
three types of mutations, resulting in a constitutive activation of
tyrosine kinase activity and cell transformation. In the first type,
v-ErbB oncogene arose from the avian EGF ()receptor by
deletion of the extracellular ligand binding domain(1) ,
suggesting that the extracellular domain imposes a negative constraint
that is relieved by EGF binding. In the second type, a point mutation
in the colony-stimulating factor receptor, which corresponds to the
major activating mutation in v-Fms is again located in the
extracellular, ligand binding domain and induces a constitutive
activation of its intrinsic kinase activity without affecting
colony-stimulating factor binding(2, 24) . The third
example is the single substitution of a hydrophobic residue in the
transmembrane domain of Neu, LH, FGF3, and insulin
receptors(3, 4, 5, 18) . The diverse
ways through which constitutive activation of growth factor and hormone
receptors occurs indicate that ligand-induced conformational changes
can be mimicked by structural changes in several positions of the
receptor.
The receptor for EGF, a 170-kDa glycoprotein, is a member of the protein tyrosine kinase family(6, 7) . Like other growth factor receptors, the EGF receptor is composed of three domains: the extracellular, ligand binding domain, a single transmembrane domain, and a cytoplasmic portion containing the kinase domain with a C-terminal tail including the autophosphorylation sites. EGF binding to the receptor triggers its tyrosine kinase activity, which is essential to induce all responses to EGF(8, 9) , leading to autophosphorylation of the receptor and tyrosine phosphorylation of specific cellular substrates (for review, see (10) and (11) ). Tyrosine autophosphorylation regulates the biological activity of the EGF-R by influencing its own kinase activity (12, 13) and by creating binding sites for physiologically important substrates containing sequence motifs called Src homology (SH2) domains.
The
role of the transmembrane domain in various growth factor and hormone
receptors has been studied for the past several years. The activating
mutation in Neu/c-ErbB2, a substitution of a hydrophobic with a charged
residue (Val to Glu
), is located in the
transmembrane domain(14, 15) . This mutant has a
constitutively activated tyrosine kinase, increased level of
autophosphorylation, and constitutive coupling and activation of
phospholipase C
(PLC
)(16, 17) . Modulation
of receptor activity through the transmembrane domain region has been
described also in other cases. A similar mutation has been shown to
constitutively activate the insulin receptor kinase activity and its
metabolic effects (3, 4) , the luteinizing receptor
(LH), which gives rise to a familiar precocious male
puberty(5) , and the FGF3 receptor causing the most common
genetic form of dwarfism(18) . In contrast, the identical point
mutation in the EGF receptor has been previously reported not to affect
at all its biological and kinase activity(19, 20) .
To further address the role of the transmembrane domain in
regulating EGF receptor function, we have created the same point
mutation present in the transmembrane domain of Neu/ErbB2 by exchanging
Val for Glu. In this report, we show that this mutation
constitutively activates EGF receptor biological and transforming
ability and constitutively activates the Ras kinase cascade without
affecting ligand-dependent activation.
NIH3T3 cells, which contain
about 5,000 receptor/cell, were used for transfections. Transfections,
G418 selection, foci formations, growth in low serum, and growth in
agar were performed as previously described (27) . As
determined by I-EGF binding and Scatchard analysis, the
mutant receptors were expressed at 3.7
10
receptor/cell. As control, a NIH3T3 line (Cl 17) expressing 4
10
human wild type EGF-R/cell was
used(27) . Cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% newborn calf serum (NCS)
with penicillin, streptomycin, and glutamine.
For
immunoprecipitation experiments, total cellular lysates (3 mg of
protein) were incubated with appropriate antibodies for 4 h at 4
°C. For non-conjugated antibodies (SHC and PLC antibodies),
immunocomplexes were collected by binding to GammaBind G-Sepharose 4B.
Immunocomplexes were washed five times with an ice-cold buffer (HNTG)
containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 10%
glycerol, 0.1% Triton X-100; immunoprecipitates solubilized in 1
Laemmli buffer (38) were boiled and run on
SDS-polyacrylamide gels.
For immunoblots, analysis proteins were
transferred to nitrocellulose filters. Membranes were then incubated at
room temperature for 2 h in 5% bovine serum albumin-TBS buffer (20
mM Tris-HCl, pH 7.5, 150 mM NaCl). Blots were
incubated with primary antibodies for 2 h, washed in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), and
incubated with I-protein A for 1 h. For mGRB-2 Ab, blots
were incubated with
I-goat anti-mouse for 2 h.
Blots were exposed to Kodak X-OMAT x-ray film at -80 °C and developed.
Cell lysates (2 mg of proteins) were incubated with 5 µg of immobilized GST-GRB-2 protein at 4 °C for 90 min. The protein complexes were washed three times with HNTG buffer and boiled in Laemmli buffer. Protein complexes were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were washed and incubated with polyclonal anti-EGF-R Ab, as described above.
To test whether the point mutation in the transmembrane
domain of the EGF receptor was able to induce transformation, as in the
case of the the Neu oncogene, Val (corresponding to
Val
in the Neu/c-ErbB2 proto-oncogene) was converted into
a glutamic acid, and the mutant receptor transfected into NIH3T3 cells.
As shown in Table 1, the transforming activity of
Glu-EGF receptor was evident both in the presence and in
the absence of EGF, while transformation by the normal receptor is
strictly dependent on the presence of the ligand. Mutant
receptor-induced transformation in the absence of the ligand was lower,
only 20% of that of wild type in presence of EGF, but was clearly
evident and reproducible in foci formation, growth in agar, and growth
in low serum. In the presence of EGF, transformation was similar in
mutant and wild type receptor. In addition, the mutant receptor showed
a higher sensitivity to EGF for transformation. Indeed at 5 ng/ml EGF,
maximum transforming activity was achieved in the mutant while only 25%
of maximum with wild type receptor (Table 1, lowerpanel). The higher sensitivity to EGF is an intrinsic
property of the mutant receptor not due to a difference in affinity for
EGF. Low affinity mutant receptor had a K
of 3.6 nM for EGF, very similar to that of wild
type receptor (3.2 nM) ( (12) and data not shown). In
addition, NIH3T3 cells transfected with the mutant receptor clearly
showed a morphological transformed phenotype, could not be arrested in
G
by serum deprivation, but were always actively
proliferating, as indicated in Table 2. These findings
demonstrate that the single point mutation in the transmembrane domain
of the EGF receptor is sufficient to stimulate its oncogenic potential,
rendering it, at least partially, ligand independent. This result is
important because in the case of Neu/ErbB2, there have been contrasting
reports on whether the same point mutation is really essential for
transformation (14) or whether the proto-oncogene itself is
able to transform, depending on the level of expression(26) .
In the case of wild type EGF receptor, transformation is only and
strictly ligand-dependent and not dependent on the level of
expression(25, 27) . In addition, in our experiments,
both mutant and wild type receptors are expressed at similar levels, at
about 4
10
receptors/cells as determined by
Scatchard analysis (data not shown and Fig. 1). Our findings are
different from those by Kasles et al.(19) and
Carpenter et al.(20) , which have previously reported
that the same point mutation in the EGF receptor was not transforming.
However, Carpenter (20) has used B82 fibroblasts instead of
NIH3T3 cells, and in their successive experiments with NR6 cells even
wild type receptor was not transforming in the presence of
physiological concentrations of EGF (22) . It is therefore
possible that the difference is dependent on the higher sensitivity and
reproducibility of the NIH3T3 transformation
assays(25, 26, 27) .
Figure 1:
Autophosphorylation and tyrosine
phosphorylation of cellular proteins by Glu-EGF receptor
in the absence of EGF. Panel A, NIH3T3 expressing wild type (lanes3, 4) and mutant EGF-R (lanes1, 2) were incubated for 30 min with and without
100 ng/ml EGF and lysates prepared. Total cellular extracts (150 µg
of proteins) were run on a 7.5% SDS-PAGE gel, transferred on a
nitrocellulose filter, and reacted with anti-phosphotyrosine Ab. Lanes1 and 3, extracts from untreated
cells; lanes2 and 4, extracts from cells
preincubated with EGF. Arrow indicates EGF-R. Exposure of the
autoradiogram was for 18 h. Panel B, total cellular extracts
prepared from untreated (lanes1, 3) or
EGF-treated cells (lanes2, 4) were run on a
10% SDS-PAGE gel, transferred on a nitrocellulose filter, and reacted
with anti-phosphotyrosine Ab. Lanes1 and 2,
extracts from cells expressing mutant EGF-R; lanes3 and 4, from wild type (W.T.) expressing cells.
Exposure of the autoradiogram was for 48 h.
To elucidate the
mechanism by which the point mutation in the transmembrane domain of
the receptor is able to induce transformation, we performed a
biochemical characterization of the Glu-EGF receptor. As
shown in Fig. 1(panelA), the mutant receptor
was autophosphorylated in the absence of EGF, albeit at a lower level
than in the presence of EGF. Quantitation of the band intensity
indicated that, in the absence of EGF, the mutant receptor was
phosphorylated 4-fold less than in the presence of EGF. This finding
correlates well with the 5-fold biological activity observed in the
absence of EGF (Table 1). In addition, tyrosine-kinase activity
of the receptor toward cellular substrates, assayed with anti-pY
antibodies, was higher than that of wild type receptor stimulated with
EGF and was again evident in the absence of the ligand (Fig. 1, panelB).
A band of approximately 52 kDa appeared
to be phosphorylated specifically in the absence of EGF in Glu cell extracts (Fig. 1, panelB). To test
whether part of this 52-kDa band corresponded to the SHC adaptor
protein, extracts were immunoprecipitated with anti-pY antibody and
then blotted with anti-SCH antibody. As shown in Fig. 2(panelA), SHC was indeed phosphorylated
by the mutant receptor in the absence of EGF, while phosphorylation by
the wild type receptor occurred only in the presence of EGF. In
agreement with the finding reported by Pelicci et
al.(28) , low tyrosine phosphorylation of the 52-kDa form
was evident in NIH3T3 cells expressing wild type receptors even without
EGF (13% of EGF-dependent phosphorylation). However, basal
phosphorylation was much more intense in the mutant-expressing cells
(85% of EGF-induced). In addition, in mutant-expressing cells also the
46-kDa form of SHC was phosphorylated in the absence of EGF at levels
comparable with those observed in the presence of EGF. In contrast,
another SH2-containing substrate, PLC
, was not constitutively
phosphorylated in cells expressing the mutant receptor, while in the
presence of EGF, PLC
was 3-fold more phosphorylated by mutant than
wild type receptor (Fig. 2, panelB). On the
contrary, the point-mutated Neu induces a constitutive PLC
phosphorylation, while SHC and GRB-2 were not analyzed(17) .
Interestingly, the only other EGF receptor mutant that possesses
transforming activity in absence of EGF, Dc 214, was obtained by
deletion of the whole receptor cytoplasmic tail. Dc 214 is able to
phosphorylate SHC and GAP and to stimulate Ras in the GTP-bound state
activating the MAP kinase pathway, while it does not phosphorylate
PLC
(29, 30) . These findings indicate that
phosphorylation and activation of SH2-containing substrates is specific
and may suggest a priority in the pathway of signal transduction by the
EGF receptor. Indeed, SHC is the only EGF-R substrate found associated
in high amounts with EGF receptor; 10% of the total SHC is associated
with the activated EGF receptor, compared with only 1% of total
PLC
(30) . In addition, SHC is also phosphorylated by
physiological levels of receptor, as in parental NIH3T3
cells(31) . As shown in Fig. 2, panelC, in mutant-expressing cells GRB-2 is associated with
SHC in the absence of EGF (55% of EGF-induced), whereas in wild type
receptor-expressing cells GRB-2 is associated with SHC only in the
presence of EGF.
Figure 2:
Phosphorylation of SHC, PLC, and
GRB-2 association with SHC by wild type (W.T.) and mutant
EGF-R. Panel A, top total cellular lysates (3 mg of proteins)
from cells expressing wild type (lanes1, 2)
and mutant EGF-R (lanes3, 4) were
immunoprecipitated by anti-phosphotyrosine Ab, blotted on
nitrocellulose filters, and reacted with anti-SHC-SH2 Ab. Lanes1 and 3, untreated cells; lanes2 and 4, cells pretreated with EGF. The arrows indicate SHC proteins. Exposure of the autoradiogram was for 20 h. Bottom, 150 µg of total lysates were run on a 10% SDS-PAGE
gel, transferred on a nitrocellulose filter, and reacted with
anti-SHC-SH2 Ab. Arrow indicates SHC bands. Exposure of the
autoradiogram was for 16 h. In Panel B, top, lysates
(3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with anti-PLC
Ab, transferred to nitrocellulose filters, and reacted with
antiphosphotyrosine Ab. Lanes1 and 3,
untreated cells; lanes2 and 4, cells
pretreated with EGF. The arrow indicates PLC
. Exposure of
the autoradiogram was for 20 h. Bottom, 150 µg of lysates
were run on a 7.5% SDS-PAGE gel, transferred on a nitrocellulose
filter, and reacted with anti-PLC
Ab. Arrow indicates
PLC
. Exposure of the autoradiogram was for 78 h. In panel
C, top, lysates (3 mg of proteins) from cells expressing
wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with
anti-SHC-CH Ab, transferred to nitrocellulose filters, and reacted with
monoclonal anti-GRB-2 Ab. Lanes1 and 3,
untreated cells; lanes2 and 4, treated with
EGF. The arrow indicates GRB-2. Exposure of the autoradiogram
was for 72 h. Bottom, 100 µg of total cell lysates were
run on a 12.5% SDS-PAGE gel, transferred on a nitrocellulose filter,
and reacted with polyclonal anti-GRB-2 Ab. Arrow indicates
GRB-2. Exposure of the autoradiogram was for 16
h.
We then tested whether GRB-2 was also constitutively associated with the mutant EGF receptor. In Fig. 3, panelA, GRB-2 was coimmunoprecipitated with the mutant receptor both in absence and presence of EGF while only in the presence of the ligand with wild type receptor. Quantitation indicated that constitutive association amounted to 60% of that in the presence of EGF. Furthermore, in vitro the GST-GRB-2 fusion protein was able to interact with the mutant receptor both when stimulated or not with EGF (Fig. 3, panelB). These data indicate that GRB-2 is able to associate constitutively with mutant receptor.
Figure 3: GRB-2 association with wild type (W.T.) and mutant EGF receptors in vivo and in vitro. Panel A, top, lysates (3 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were immunoprecipitated with polyclonal anti-EGF-R Ab, transferred to nitrocellulose filters, and reacted with polyclonal anti-GRB-2 Ab. Lanes1 and 3, untreated cells; lanes2 and 4, treated with EGF. The arrow indicates GRB-2. Exposure of the autoradiogram was for 72 h. Bottom, 100 µg of total cell lysates were run on a 12.5% SDS-PAGE gel, transferred on to a nitrocellulose filter, and reacted with polyclonal anti-GRB-2 Ab. Arrow indicates GRB-2. Exposure of the autoradiogram was for 72 h. Panel B, lysates (2 mg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were incubated with 5 µg of GST-GRB-2 fusion protein run on 7.5% gels, transferred to nitrocellulose filters, and reacted with anti-EGF-R Ab. Lanes1 and 3, extracts from untreated cells; lanes2 and 4, treated with EGF. EGF-R is indicated. Exposure of the autoradiogram was for 16 h.
Since binding of the
transmembrane mutant receptor to GRB-2 should bring Sos in contact with
Ras and stimulate Ras in the GTP-bound state, we tested whether the
downstream Ras-activated kinase pathway was constitutively activated in
mutant-expressing cells. As shown in Fig. 4, MAP kinase was
constitutively phosphorylated in mutant-expressing cells (50% of
EGF-induced), indicating that the mitogenic pathway of Ras was
activated and hence might be responsible for the transformed phenotype
induced by Glu-EGF receptor. Constitutive MAP kinase
activation has been found so far only in cell lines expressing the
oncogenic form of Ras and Raf(32, 33) . Interestingly,
MAP kinase activation has been implicated in the G
-G
transition phase(34, 35) , and a constitutive
MAP kinase activity could explain why EGF receptor mutant-expressing
cells could not be arrested in G
(Table 1).
Figure 4: Phosphorylation of MAP kinase in cells expressing wild type (W.T.) and mutant EGF receptors. Lysates (50 µg of proteins) from cells expressing wild type (lanes1, 2) and mutant EGF-R (lanes3, 4) were run on a 10% gel, transferred to a nitrocellulose filter, and reacted with anti-MAP kinase Ab. Lanes1 and 3, extracts from untreated cells; lanes2 and 4, treated with EGF. Phosphorylated MAP kinase (pp42) and MAP kinase (p42) are indicated. Exposure of the autoradiogram was for 16 h.
As
evident from Fig. 1, the mutant EGF receptor was present in two
forms of 170 and 150 kDa, both in the absence and presence of EGF, and
both forms were tyrosine phosphorylated. The 150-kDa form was
previously described to be a proteolytic digest of the intact receptor
due to a calcium-dependent protease, calpain, which cleaves the
cytoplasmic tail of the EGF receptor(36) . This finding,
consistently observed with mutant receptor in immunoprecipitates by
anti-pY and anti-EGF-R antibodies, suggests that Glu receptor is more susceptible to cleavage by calpain. We tested
six different clones and two pools of transfectants derived from
independent transfections with the mutant receptor, and we always
observed the 170- and 150-kDa forms (data not shown), indicating that
the presence of two EGF-R bands is not a clonal artifact. To confirm
that the 150-kDa form was due to the proteolytic activity of calpain,
we prepared extracts using a specific calpain inhibitor in addition to
the other protease inhibitors always present in the lysis buffer. As
shown in Fig. 5, in the presence of the calpain inhibitor, the
mutant receptor appeared as a 170-kDa form, indicating that the smaller
band is really due to calpain cleavage. Indeed, with the wild type
EGF-R, we never observed the 150-kDa form in extracts untreated with
EGF. In some cases, the 150-kDa form was evident upon EGF treatment (Fig. 5, lane4) but never as intense as with
the mutant receptor. Thus, possibly the cleavage site is exposed upon
EGF treatment in the wild type receptor and by the Glu
mutation in the mutant receptor. This suggests that the mutation
may induce a conformational change in the cytoplasmic domain of the
receptor, which mimicks ligand activation. Quantitation of the 170- and
150-kDa forms indicated that they were present almost at a 1:1 ratio
(60 and 40%, respectively). The cleavage site of calpain is
Lys
, which is equivalent to removal of the last 150
amino acids of the receptor cytoplasmic tail. The cleavage into the
150-kDa form cannot be responsible for the transforming phenotype of
the mutant receptor. We have previously shown that removal of the last
123 or 165 amino acids of the EGF receptor dramatically decreases its
biological and transforming ability to less than 10% of the wild type
and abolishes substrate phosphorylation(37, 38) .
Therefore, the transmembrane point mutation and not the deletion of
part of the C-terminal receptor tail is responsible for constitutive
receptor activity.
Figure 5:
Calpain cleaves Glu-EGF
receptor in cells untreated and treated with EGF. Lysates from mutant (lanes3, 4) and wild type (W.T.)
EGF-R-expressing cells (lanes1-4) were
prepared in the absence (left panel) or presence (right
panel) of a specific calpain inhibitor I, run on a 10% SDS-PAGE
gel, transferred on to a nitrocellulose filter, and reacted with
anti-phosphotyrosine Ab. Lanes1 and 2,
extracts from untreated cells; lanes3 and 4, extracts from cells treated with EGF. Arrow indicates the EGF-R. Exposure of the autoradiogram was for 18
h.
Dimerization is considered the first event
occurring after ligand binding able to bring together the two kinase
domains that phosphorylate each other and start the chain of tyrosine
phosphorylation(39) . We therefore tested whether the point
mutation in the transmembrane domain of the EGF-R was able to induce
dimerization and whether dimer formation in the absence of EGF could
account for the increased kinase activity and the transforming ability.
However, in agreement with other findings(19, 20) ,
cross-linking experiments showed that Glu mutant receptor
was able to dimerize in the presence of EGF but not in its absence (Fig. 6). Therefore, formation of dimer receptors is not an
essential step for the constitutive functional activity of the
transmembrane mutant receptor, while it may contribute to its full
activity in the presence of EGF.
Figure 6: In vivo cross-linking of wild type and mutant EGF receptors. NIH3T3 cells expressing wild type (lanes1, 2) and mutant EGF receptors (lanes3, 4) were incubated in the presence (lanes2, 4) or absence of EGF (lanes1, 3). Lysates were prepared with 3 mM bis-sulfosuccimidyl suberate as cross-linking reagent and immunoprecipitated with anti-EGF-R Ab, run on a 6% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and reacted with anti-EGF-R Ab. Arrows indicate the monomer and dimer forms of EGF-R. Exposure of the gel was for 18 h.
In conclusion, substitution of a hydrophobic with a negatively charged residue constitutively activates the EGF receptor as well as Neu, LH, FGF3, and insulin receptors. The mechanism by which this single substitution is able to activate different receptors is still not resolved. Three-dimensional structure studies performed on peptides corresponding to the transmembrane domain of Neu indicate that there is no significant difference in the conformation of wild type and mutated sequences, but the results are consistent with alternative models involving receptor-packing interactions(40) . Further studies are necessary to address this point.