From the Division of Cytokine Biology, Center for
Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, Maryland 20892, the § Department of Protein
Chemistry and Biophysics, Berlex Biosciences,
Richmond, California 94804, and the ¶ Department of
Pathology, University of Alabama at Birmingham and Veterans
Administration Medical Center, Birmingham, Alabama 35294
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ABSTRACT |
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The epidermal growth factor receptor (EGFR)
mediates the actions of a family of bioactive peptides that include
epidermal growth factor (EGF) and amphiregulin (AR). Here we have
studied AR and EGF mitogenic signaling in EGFR-devoid NR6 fibroblasts that ectopically express either wild type EGFR (WT) or a truncated EGFR
that lacks the three major sites of autophosphorylation (c'1000). COOH-terminal truncation of the EGFR significantly impairs the ability
of AR to (i) stimulate DNA synthesis, (ii) elicit Elk-1 transactivation, and (iii) generate sustained enzymatic activation of
mitogen-activated protein kinase. EGFR truncation had no significant effect on AR binding to receptor but did result in defective GRB2 adaptor function. In contrast, EGFR truncation did not impair EGF
mitogenic signaling, and in c'1000 cells EGF was able to stimulate the
association of ErbB2 with GRB2 and SHC. Elk-1 transactivation was
monitored when either ErbB2 or a truncated dominant-negative ErbB2
mutant (ErbB2-(1-813)) was overexpressed in cells. Overexpression of
full-length ErbB2 resulted in a strong constitutive transactivation of
Elk-1 in c'1000 but only slightly stimulated Elk-1 in WT or parental
NR6 cells. Conversely, overexpression of ErbB2-(1-813) inhibited
EGF-stimulated Elk-1 transactivation in c'1000 but not in WT cells.
Thus, the cytoplasmic tail of the EGFR plays a critical role in AR
mitogenic signaling but is dispensable for EGF, since EGF-activated
truncated EGFRs can signal through ErbB2.
The ErbB family of receptors that include the epidermal growth
factor receptor (EGFR,1
ErbB1, HER1) (1), ErbB2 (HER2, neu) (2), ErbB3 (HER3) (3), and ErbB4
(HER4) (4) mediate the biological actions of a family of growth factors
that are structurally related to EGF. Signaling from ErbBs involves a
process of receptor homo- and heterodimerization that is initiated by
specific engagement of ligand by the extracellular domain of one or
more of the ErbB receptors (reviewed in Ref. 5). Within the family of
EGF-like growth factors are a subset of bioactive peptides that have
all been shown to bind to and activate the EGFR and include EGF (6, 7),
transforming growth factor Like EGF (reviewed in Ref. 7), AR is a potent mitogen for fibroblasts
(19), keratinocytes (19-21), and both normal and malignant epithelial
cells (11, 16, 19, 22-27). AR is commonly overexpressed in human
malignancies of the colon (22, 28), stomach (29-31), breast (32), and
in the pancreas, overexpression correlates with reduced patient
survival (26, 33). In vitro, AR has been shown to function
in an autocrine manner to drive the proliferation of malignant cells of
the colon (22), breast (24), cervix (34), prostate (35), and pancreas
(26, 27).
The EGFR is an 1186-amino acid residue protein that consists of an
extracellular ligand binding domain, a transmembrane domain, an
intracellular tyrosine kinase domain, and a COOH-terminal region that
contains autophosphorylation sites at tyrosines 992, 1068, 1086, 1148, and 1173 (1, 36). The COOH-terminal tail of the EGFR is believed to
regulate access of substrates to the catalytic domain and, when
phosphorylated, to provide docking sites for Src homology 2 (SH2)
domain-containing proteins (37, 38). Paradoxically, several studies
have shown that this EGFR region is not required for EGF-stimulated
mitogenesis or transformation (39-44) but is critical to the cell
motility that occurs in response to EGF (40).
With the exception of EGF, little is currently known regarding the
structural requirements within the EGFR that are critical to signaling
by the various EGFR ligands. Previous work has demonstrated a
requirement for EGFR kinase activity but not for COOH-terminal autophosphorylation sites in ligand-stimulated activation of signal transducers and activators of transcription (STAT) 1, 3, and 5 by
either AR or EGF, and no significant differences were observed between
the ligands (45). In contrast to those findings we report here that the
COOH-terminal region of the EGFR is critical for AR-induced mitogenic
signaling, whereas it appears to be expendable for EGF-mediated
mitogenesis. The purpose of this work was to identify the molecular
basis for this difference. The results revealed that this differential
requirement for the cytoplasmic tail of the EGFR in AR and EGF
mitogenic signaling can be attributed to ErbB2.
Cell Culture--
Either human wild type (WT) or truncated EGFR
corresponding to the amino-terminal 1000 residues (c'1000) was
expressed in EGFR-devoid NR6 fibroblasts (46) via retrovirus-mediated
transduction (39, 40). These polyclonal cell lines have been
shown to express comparable levels of EGFR and bind EGF with very
similar affinities (40). The cells were maintained in culture as
described previously (40).
Antibodies, Reagents, and cDNAs--
Antibodies to the EGFR
(Ab-5) and ErbB2 (c-neu, Ab-3) were obtained from Oncogene
Science (Uniondale, NY), and biotinylated PY-20 antibody was purchased
from ICN Biomedical, Inc. (Costa Mesa, CA). The human-specific antibody
against ErbB2 (Ab-10) was obtained from NeoMarkers (Union City, CA),
and antibodies to SHC, SHP-2 (PTP1D), mitogen-activated protein kinase
(MAPK) (anti-pan ERK) were obtained from Transduction Laboratories
(Lexington, KY). The biotinylated 4G10 and anti-EGFR LA22 monoclonal
antibodies were purchased from Upstate Biotechnology Inc. (Lake Placid,
NY). Antibodies directed against the phosphorylated activated form of
p44 mitogen-activated protein kinase were obtained from New England
Biolabs (Beverly, MA). GRB2-glutathione S-transferase (GST)
fusion protein were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). The 87-amino acid residue form of recombinant human AR was
generated as described previously (12). Recombinant human EGF was
obtained from PeproTech, Inc. (Rocky Hill, NJ). The pFR-Luc and
pFA2-Elk-1 plasmids were obtained from Stratagene (La Jolla, CA), and
the pcDNA 3.1/His/lacZ plasmid was purchased from
Invitrogen (Carlsbad, CA). An XhoI fragment encoding
full-length human ErbB2 cDNA was generously provided by Jacalyn
Pierce (National Cancer Institute, National Institutes of Health) and
subcloned into pcDNA 3.1 (Invitrogen). The cDNA encoding
ErbB2-(1-813) was generated by digesting the full-length ErbB2
construct with SacII and HindIII, blunting the
ends with the large Klenow fragment of DNA polymerase I and ligating
the ends of the plasmid. The sequence of this construct was confirmed
by DNA sequencing.
DNA Synthesis Assay--
Cells were grown to confluency in
96-well plates, serum-starved for 3 h, and treated with serum-free
medium containing EGF or AR. After 6 h, the cells were pulsed for
an additional 16 h with [3H]thymidine (2 mCi/well;
Amersham Pharmacia Biotech). DNA was harvested, and the incorporation
of [3H]thymidine into newly synthesized DNA was
quantitated as described previously (23).
Elk-1 Transactivation Assays--
Subconfluent cells in 6-well
plates were incubated with 1 ml of Pfx-6 transfection lipid
(Invitrogen) in Opti-Mem (Life Technologies, Inc.) containing 0.5 µg
of pFR-Luc, 50 ng of pFA2-Elk-1, and 100 ng of pcDNA
3.1/His/lacZ for 4 h at 37 °C. In some experiments cells were also transfected with 1.35 µg of pcDNA 3.1 or
pcDNA 3.1 encoding either ErbB2 or ErbB2-(1-813). The cells were
then allowed to recover overnight in serum-containing medium,
serum-starved, and stimulated for 6 h with growth factor. Cells
were lysed, and luciferase and Receptor Binding Analysis--
Cells were grown to confluency in
12-well plates, washed in binding buffer (Eagle's minimal essential
medium containing 0.1% bovine serum albumin and 25 mM
Hepes, pH 7.2), and incubated in binding buffer for 1 h at
4 °C. One ml of ice-cold binding buffer containing 100 pM of 125I-EGF (Amersham Pharmacia Biotech) was
added to each well in the absence or presence of unlabeled EGF or AR
ranging in concentration from 0.1 to 200 nM. After 3 h
at 4 °C the cells were washed twice with 1 ml of ice-cold binding
buffer and lysed in 1 ml of 1 N NaOH, and radioactivity was
quantitated. Specific binding was defined as the quantity of
125I-EGF that bound in the presence of excess (200 nM) unlabeled EGF subtracted from that which bound in the
absence of unlabeled EGF. Specific binding of 125I-EGF was
greater than 96% in all experiments performed. The IC50 was defined as the concentration of unlabeled ligand that inhibited specific binding by 50%.
Whole Cell Lysates--
Cell monolayers were serum-starved for
3 h and treated with serum-free medium containing EGF or AR for
the indicated times at 37 °C, washed with phosphate-buffered saline,
and lysed in ice-cold 1% Triton X-100, 10% glycerol, 50 mM Hepes, 100 NaCl, 1 mM sodium orthovanadate,
5 mM Immunoprecipitations--
One µg of antibody was added to 0.5 mg of whole cell lysate and incubated for 1 h at 4 °C, and 5 µl of protein G-agarose was added. After 1 h at 4 °C, the
immune complexes were pelleted at 15,000 × g for 2 min
and washed three times with ice-cold lysis buffer. Bound proteins were
released by boiling in SDS-PAGE sample buffer for 4 min.
Binding of Proteins to GRB2-Glutathione S-Transferase Fusion
Protein--
Whole cell lysates (0.5 mg) were incubated with 1 µg of
GRB2-glutathione S-transferase (GST) fusion protein bound to
glutathione-agarose for 12 h at 4 °C. The glutathione-agarose
resin was pelleted at 15,000 × g for 2 min and washed
three times with ice-cold lysis buffer. Bound proteins were released by
boiling in SDS-PAGE sample buffer for 4 min.
Western Blotting--
Proteins were separated on SDS-PAGE gels
and transferred to polyvinyl difluoride (Novex). Membranes were probed
with 0.3 µg/ml primary antibody, and detection was performed using
the Vectastain ABC Elite Kit (Vector Laboratories) and enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Characterization of NR6 Cells Expressing Wild Type or c'1000
EGFRs--
Either wild type (WT) or truncated EGFR lacking the
COOH-terminal 186 amino acid residues (c'1000) was expressed in
EGFR-devoid NR6 fibroblasts (46) via retrovirus-mediated transduction
(40). The WT and c'1000 polyclonal cell lines possess comparable
numbers of EGF-binding sites with similar affinities as described
previously (40). Both WT and c'1000 receptors undergo
ligand-dependent endocytosis with rates of 0.080 and 0.042 per min, respectively.2 WT
and c'1000 cells express detectable levels of murine ErbB2 (see below)
but do not respond to heregulin The COOH-terminal Region of the EGFR Is Critical to AR-induced DNA
Synthesis--
To understand better the role of the COOH-terminal
region of the EGFR in mitogenic signaling, we assessed the ability of
various concentrations of AR and EGF to stimulate DNA synthesis in
serum-starved parental NR6, WT, and c'1000 cells (Fig.
1). As expected, neither AR nor EGF had
any effect on DNA synthesis in NR6 parental cells (Fig. 1, top
panel), whereas cells expressing WT EGFR acquired the ability to
respond to both AR or EGF (middle panel). In WT cells the
approximate concentration of growth factor necessary to achieve
one-half of the maximal DNA synthesis response (EC50) was
~0.12 and ~0.30 nM for EGF and AR, respectively. The
EC50 for DNA synthesis by EGF in c'1000 cells was ~0.12
nM (Fig. 1, bottom panel) demonstrating that
removal of the 186 amino acid residues from the COOH terminus of the
receptor had no significant effect on the ability of EGF to stimulate
DNA synthesis. However, in striking contrast to EGF, AR was impaired in
its ability to stimulate DNA synthesis in c'1000 cells such that,
relative to WT cells, the EC50 was shifted 67-fold to ~20
nM (bottom panel). Furthermore, no significant
increase in DNA synthesis occurred in c'1000 cells in response to AR
until the concentration was escalated to 3.3 nM, whereas a
significant response was observed in WT cells at the lowest AR dose
tested (41 pM). In c'1000 cells the EC50 for
growth factor-stimulated DNA synthesis was 167-fold higher for AR than
EGF. These results demonstrate that the COOH-terminal region of the WT
EGFR is critical to the ability of AR to generate a strong mitogenic
signal in NR6 cells.
Truncation of the EGFR Has No Significant Effect on AR
Binding--
The simplest explanation for the defect in the ability of
AR to stimulate DNA synthesis effectively in c'1000 cells is that the
truncated EGFR does not bind AR with the same affinity as the WT
receptor. To test this hypothesis we studied the ability of AR and EGF
to inhibit the binding of 125I-EGF to intact cells at
4 °C (Table I). In these experiments the concentration of unlabeled ligand that was necessary to inhibit the
specific binding of radiolabeled EGF by 50% (IC50) was
determined in each of the two cell lines. This analysis demonstrated
that truncation of the cytoplasmic domain of the EGFR had no
significant effect on the binding of AR to the extracellular domain of
the receptor (Table I). As expected, WT and c'1000 receptors bound EGF
with comparable affinities consistent with previous observations (40).
Thus, the impaired ability of AR to drive DNA synthesis in c'1000 is
not due to a defect in the binding of AR to the truncated receptor.
COOH-terminal Truncation of the EGFR Impairs the Ability of AR to
Elicit Transactivation of Elk-1--
Activation of mitogen-activated
protein kinase (MAPK, extracellular signal-regulated kinase) plays a
critical role in mitogenic signaling by receptor tyrosine kinases such
as the EGFR (47). MAPK-mediated phosphorylation of the transactivation
domain of the ternary complex factor Elk-1 results in binding of Elk-1
to the serum response element in the promoters of growth factor-induced genes and the stimulation of transcription (48, 49). To assess the
ability of the two ligands to transactivate Elk-1, cells were cotransfected with a plasmid that constitutively expresses a fusion protein consisting of the DNA binding domain of the yeast protein Gal4
and the transactivation domain of Elk-1, along with a reporter plasmid
that expresses luciferase under the control of five tandem repeats of
the Gal4-binding element. The WT and c'1000 cells were stimulated with
various concentrations of AR or EGF for 6 h and lysed, and
luciferase activity was quantitated (Fig.
2). Both ligands were potent stimulators
of Elk-1 transactivation in WT cells with EC50 values of
~0.1 nM for EGF and ~0.4 nM for AR (Fig. 2,
top panel). The EC50 for EGF-stimulated Elk-1
transactivation in c'1000 cells was found to be ~0.2 nM
and thus was comparable to WT cells (Fig. 2, bottom panel).
However, the EC50 value for AR in c'1000 cells, increased
to ~7 nM. Little or no expression of the luciferase
reporter occurred in response to AR in these cells except at the
highest concentration tested (10 nM, bottom panel). In general, the ability of the two growth factors to
stimulate Elk-1 transactivation in WT and c'1000 cells (Fig. 2)
correlated well with the ability of the ligands to stimulate DNA
synthesis in these cells (Fig. 1). These results indicate that the
COOH-terminal region of the EGFR plays a critical role in the
capability of AR to elicit transcriptional activation that is mediated
by Elk-1.
The EGFR COOH-terminal Tail Is Essential to Sustained MAPK
Activation by AR but Not by EGF--
To determine whether the defect
in the ability of AR to stimulate DNA synthesis and Elk-1
transactivation in c'1000 cells could be explained by a defect in MAPK
activation, we performed a time course experiment to monitor the
enzymatic activation of MAPK. To reveal a potential defect in AR
signaling in c'1000 cells, we used a concentration of ligand (1 nM) that was mitogenic for EGF but not for AR (Fig. 1,
bottom panel). Cells were lysed at various time points, and
lysates were analyzed by Western blotting using antibodies that
specifically detect the phosphorylated activated form of MAPK (Fig.
3). As expected, both 1 nM AR
and EGF strongly activated MAPK in cells expressing WT EGFR, and this
stimulation was still evident 7 h after exposure to the growth
factors (lanes 2-11 and 13-22). In c'1000
cells, however, a relatively strong but transient MAPK activation was
elicited by AR (lanes 24-33), whereas EGF generated a
robust and much more persistent activation of MAPK (lanes
35-44). This transient activation of MAPK by AR in c'1000 cells
was also observed when MAPK activity was measured in immune complex
kinase assays (data not shown). Thus, unlike EGF, AR requires the
presence of the COOH-terminal region of the WT EGFR for sustained
enzymatic activation of MAPK.
AR- and EGF-stimulated Tyrosine Phosphorylation of Cellular
Proteins in WT and c'1000 Cells--
As seen in Fig.
4, both AR and EGF evoked a
concentration-dependent tyrosine phosphorylation of an
~170-kDa protein as detected in whole cell lysates (Fig. 4A,
lanes 1-9), and anti-EGFR immunoprecipitations performed on
lysates derived from WT cells exposed to either 10 nM AR or
EGF confirmed this protein to be the WT EGFR (Fig. 4A, lanes
10-15). Anti-phosphotyrosine analysis of lysates from c'1000 cells revealed a concentration-dependent phosphorylation of
~150-, ~185-, and ~210-kDa proteins in response to EGF (Fig.
4B, lanes 6-9). AR induced tyrosine phosphorylation of the
~185-kDa protein, and a very low level of phosphorylation of the
~150- and ~210-kDa proteins was also observed (Fig. 4B, lanes
1-5). Anti-EGFR immunoprecipitations demonstrated EGF-stimulated
tyrosine phosphorylation of c'1000, whereas no c'1000 tyrosine
phosphorylation was detected in response to AR under these conditions
(Fig. 4B, lanes 10-15). The c'1000 receptor contains one
minor autophosphorylation site at Tyr-992 (36) which becomes
tyrosine-phosphorylated in an EGF-dependent manner when
expressed in NR6 cells (40). Co-migration of the immunoprecipitated
c'1000 receptor (Fig. 4B, lanes 12-15) with the ~150-kDa
phosphoprotein detected in lysates (lanes 8 and
9) suggests that the ~150-kDa lysate protein is the c'1000
receptor.
The Role of the COOH-terminal Region of the EGFR in Ligand-induced
Coupling of GRB2 to Signaling Molecules--
A major pathway
implicated in mitogenic signaling by the EGFR involves the recruitment
of the adaptor protein GRB2 and the guanine nucleotide exchange factor
Son of sevenless to the plasma membrane where activated Ras initiates a
kinase cascade which proceeds downstream to activate MAPK (50). Due to
the fact that the adaptor protein GRB2 plays such a crucial role in the
activation of MAPK and mitogenic signaling by the EGFR, we performed a
time course experiment to monitor the association of GRB2 with a number of signaling molecules. As before, to reveal potential defects in AR
signaling in c'1000 cells, we used a concentration of ligand (1 nM) that was mitogenic for EGF but not for AR. Cells were
lysed at various time points after treatment and probed with a GRB2-GST fusion protein (Fig. 5A).
Analysis by Western blotting of proteins that bound to GRB2-GST
demonstrated that AR or EGF induced binding of GRB2 to the EGFR only in
cells expressing the full-length receptor (lanes 1-9),
whereas no association between GRB2 and c'1000 receptor was detected
(lanes 10-18). Furthermore, no coupling of the EGFR-related tyrosine kinase ErbB2 to GRB2 was detected in WT cells in response to
either ligand (lanes 2-9), and coupling was also not
observed when the dose of AR and EGF was increased to 10 nM
(data not shown). AR was not able to induce an interaction between
ErbB2 and GRB2 in c'1000 (lanes 11-14), whereas EGF
stimulated the association of ErbB2 and GRB2 in c'1000 cells that
persisted for at least 30 min in the presence of ligand (lanes
15-18). Therefore, the EGF-activated c'1000 receptor drives the
association of ErbB2 and GRB2 into a protein complex.
Previous work has demonstrated that the catalytic activity and
both SH2 domains of the protein-tyrosine phosphatase SHP-2 are
essential for MAPK activation by the entire ErbB family of receptors
(51), and EGF stimulates the recruitment of SHP-2 into a protein
complex with GRB2 via the COOH-terminal Src homology 3 (SH3) domain of
GRB2 (52). The presence of the COOH-terminal region of the EGFR was
found to be essential for AR-induced coupling of SHP-2 to GRB2, since
AR was only able to elicit a significant association in WT cells
(lanes 2-5) but not in c'1000 cells (lanes 11-14). However, EGF stimulated the association between GRB2 and SHP-2 in both cell lines (lanes 6-9 and 15-18).
We also evaluated the interaction between GRB2 and SHC that occurs via
an interaction between the SH2 domain of GRB2 and
tyrosine-phosphorylated SHC. Interestingly, removal of the
COOH-terminal region of the EGFR had little effect on this interaction
in response to AR since levels of SHC (p46 and p52) which bound to
GRB2-GST were comparable in both cell lines (lanes 2-5 and
11-14). For EGF, enhanced association of GRB2 and SHC was
observed in c'1000 relative to WT cells (lanes 6-9 and
15-18), and in particular, coupling of the p66 SHC isoform was significantly elevated (lanes 15-18). Taken together,
the data presented in Fig. 5A revealed that truncation of
the cytoplasmic tail of the EGFR resulted in defective AR-induced GRB2
adaptor function.
EGF Stimulates Association of ErbB2 with SHC in c'1000
Cells--
To provide further insight into the potential role of ErbB2
in signaling by WT and c'1000 receptors, we analyzed ligand-induced association of ErbB2 and SHC. Both AR and EGF were capable of stimulating ErbB2 tyrosine phosphorylation in WT and c'1000 cells, but
no striking differences were observed (data not shown). SHC interacts
with and is an excellent substrate for ErbB2 and appears to play an
important role in mitogenic signaling through this receptor tyrosine
kinase (53). We therefore assessed the ability of SHC to associate with
ErbB2 in a ligand-dependent manner in WT and c'1000 cells.
In these experiments, cells were exposed to the growth factors, SHC was
immunoprecipitated, and the immunoprecipitates were probed for the
presence of SHC, ErbB2, and phosphotyrosine-containing proteins by
Western blotting (Fig. 5B). No induced coupling of ErbB2 to
SHC could be detected in WT cells (lanes 2-4). Western blotting analysis for phosphotyrosine-containing proteins did demonstrate association of SHC with the highly tyrosine-phosphorylated ~170-kDa WT EGFR in response to AR and EGF (Fig. 5B, lanes
2-4). In c'1000 cells, it was possible to demonstrate EGF- but
not AR-induced association between SHC and ErbB2, albeit at barely
detectable levels (lanes 7 and 8).
Anti-phosphotyrosine analysis confirmed the presence of a protein in
the SHC immunoprecipitates that co-migrated exactly with ~185-kDa
ErbB2 (lanes 7 and 8). Furthermore, EGF elicited
the complex formation between SHC and additional
phosphotyrosine-containing proteins of ~150 and ~210 kDa
(lanes 7 and 8). Reprobing of this blot with
antibodies directed against the EGFR extracellular domain was not able
to confirm that the ~150-kDa protein was the c'1000 receptor (data
not shown). Thus, in the absence of the COOH-terminal region of the WT
EGFR, EGF stimulates the generation of a complex that contains both
ErbB2 and SHC.
Transient Overexpression of ErbB2 Results in
Ligand-independent Transactivation of Elk-1 in c'1000 Cells--
To
test the hypothesis that ErbB2 was responsible for the strong
EGF-induced signaling that we observed in c'1000 cells, we generated
two cytomegalovirus promoter-driven cDNA constructs that encode
either full-length wild type human ErbB2 (WT ErbB2) or a truncated
ErbB2 consisting of residues 1-813 (ErbB2-(1-813)). ErbB2-(1-813)
possesses the extracellular and transmembrane domains of ErbB2 but
lacks the kinase domain and COOH-terminal tail that contains the
tyrosine phosphorylation sites. Cells were transfected with the ErbB2
cDNAs, and as seen in Fig.
6A transient expression of WT
ErbB2 in c'1000 cells resulted in a strong activation of Elk-1 that
occurred in the absence of any exogenous growth factor. In contrast,
expression of WT ErbB2 in the NR6 parent or WT cells only modestly
increased Elk-1 transactivation demonstrating that the strong Elk-1
activation observed in c'1000 cells by WT ErbB2 was due to the presence
of the c'1000 receptor in these cells. Expression of ErbB2-(1-813) had
no significant effect on ligand-independent Elk-1 transactivation in
the three cell lines (Fig. 6A) indicating that the ErbB2
kinase activity and/or COOH-terminal region was required for the
constitutive Elk-1 activity by the c'1000 EGFR. To confirm consistent
expression of the ErbB2 structures and overexpression of the ErbB2s
relative to endogenous murine ErbB2, pooled lysates derived from the
experiments shown in Fig. 6, A and C, were
analyzed for ErbB2 expression by Western blotting with two different
monoclonal antibodies (Fig. 6B). The lower panel
of Fig. 6B was probed with an antibody that cross-reacts
with both human and mouse ErbB2 and is directed against the COOH
terminus of rat ErbB2 (c-neu) (amino acid residues 1242-1255). This
analysis demonstrated that the transfected WT ErbB2 was expressed at
comparable levels in all three cell lines and confirmed that WT ErbB2
was overexpressed relative to endogenous murine ErbB2. The upper
panel of Fig. 6B was probed with an antibody that is
specific to the extracellular domain of human ErbB2 and does not detect
mouse ErbB2. These data demonstrated that both WT ErbB2 (lanes 2, 5, and 8) and ErbB2-(1-813) (lanes 3, 6, and 9) were expressed at comparable levels in each of the
cell lines confirming that the ligand-independent transactivation of
Elk-1 by WT ErbB2, but not by ErbB2-(1-813), in c'1000 cells was
specific and not due to differential levels of expression of the two
ErbB2 proteins. Taken together, these results reveal that when ErbB2 is
overexpressed in the presence of the c'1000 receptor, downstream
signaling can occur in the absence of ligand and suggests that c'1000,
relative to WT EGFR, has an enhanced ability to signal through
ErbB2.
Overexpression of ErbB2-(1-813) Inhibits Elk-1 Transactivation by
EGF but Not by Activated Ras--
Since the data suggested that the
EGF-activated c'1000 EGFR was signaling through ErbB2, we tested the
ability of the ErbB2-(1-813) to inhibit ligand-induced Elk-1
transactivation and to thus act as a dominant-negative structure.
Overexpression of ErbB2-(1-813) or WT ErbB2 in WT EGFR cells had no
significant effect on Elk-1 activation which occurred in response to 10 nM AR or EGF (Fig. 6C). Conversely, expression
of ErbB2-(1-813) in c'1000 cells inhibited EGF-stimulated Elk-1
transactivation by ~54% (Fig. 6C). In addition, ErbB2-(1-813) also modestly inhibited (~29%) the weaker Elk-1 activation that was observed in response to 10 nM AR (Fig.
6C). To confirm the specificity of the ErbB2-(1-813)
dominant-negative effect and to demonstrate that ErbB2-(1-813)
functioned upstream of Ras, we tested the ability of it to influence
activated Ras-mediated Elk-1 transactivation (Fig. 6C,
bottom). In these experiments the cells were also transfected with
either 50 ng of a constitutively activated form of Ras (Q61L) or empty
vector. As seen in Fig. 6C neither WT ErbB2 nor residues
1-813 had any significant effect on Elk-1 activation by Ras Q61L
in either NR6-WT or c'1000 cells. These results confirm that the
ErbB2-(1-813) dominant-negative effect in c'1000 cells is specific and
is consistent with an interference of EGF-activated c'1000 signaling
between the c'1000 receptor and Ras. In conclusion, these findings
demonstrate that endogenous ErbB2 plays a critical role in
ligand-induced signaling by the c'1000 but not by the WT EGFR in NR6 cells.
In the present study we have investigated the mitogenic
signaling by AR and EGF in EGFR-devoid NR6 fibroblasts that ectopically express either WT EGFR or a COOH-terminal truncated EGFR (c'1000) that
lacks the three major sites of autophosphorylation. Here we demonstrate
that the last 186 amino acid residues in the cytoplasmic tail of the
EGFR are critical to and play an important role in AR-mediated
mitogenic signaling. It should be noted that AR can elicit some
signaling when very high, non-physiological concentrations of AR were
used (i.e. 10 nM). However, when compared with
the WT EGFR, the ability of AR to stimulate DNA synthesis, generate a
sustained MAPK activation, and to induce Elk-1 transactivation through
the c'1000 receptor was weak and defective. Furthermore, the data imply
that defective GRB2 adaptor function may form the molecular basis for
the weak AR-induced signaling observed in cells expressing the
truncated EGFR. Conversely, removal of the COOH-terminal region of the
EGFR had no significant effect on mitogenic signaling by EGF consistent
with the previous findings of others (39-44). The goal of this study
was to attempt to understand the molecular basis for this differential
signaling by AR and EGF. The fact that, unlike EGF, AR requires the
presence of heparan sulfate proteoglycan for receptor binding and full
bioactivity (16) cannot explain the defective AR signaling in c'1000,
because no significant difference in ligand binding to receptors on WT and c'1000 cells was observed. EGF-stimulated tyrosine phosphorylation of Tyr-992 in c'1000 cells has been demonstrated previously (40). In
contrast to EGF, we observed no significant AR-induced tyrosine phosphorylation of the c'1000 receptor. Regardless, tyrosine
phosphorylation of Tyr-992 has been shown not to be required for
EGF-mediated mitogenesis but is essential for phospholipase C- The most striking signal that was generated by EGF in c'1000, but not
in WT cells, was the association of GRB2 and SHC with the EGFR-related
tyrosine kinase, ErbB2. Unfortunately, attempts to detect chemically
cross-linked ligand-induced c'1000 and ErbB2 heterodimers was not
successful (data not shown), most probably due to the relatively low
levels of ErbB2 in NR6 cells and/or the inefficiency of the
cross-linking process. It has long been known that EGFR ligands can
induce tyrosine phosphorylation of ErbB2 (11, 56-59) and that EGFR and
ErbB2 can cooperate in signaling and malignant transformation of cells
via heterodimerization (60, 61). A possible role for ErbB2, in
EGF-induced MAPK activation and mitogenic signaling by EGFR mutants,
has been proposed previously (43, 62). However, the role of ErbB2 in WT
EGFR signaling is somewhat controversial. The present data indicate
that ErbB2 is critical for signaling from EGFRs lacking the
COOH-terminal tail but suggest that ErbB2 is not required for
full-length WT EGFR signaling. This finding is consistent with previous
studies demonstrating that ectopic expression of WT EGFR in
hematopoietic 32D or BAF3 cells, which do not express ErbB2, results in
the acquisition of EGF-mediated mitogenic signaling by these cells (63,
64).
Since SHC and GRB2 appear to be involved in the activation of Ras by
ErbB2 (53, 65, 66), we hypothesized that the EGF-stimulated interactions of GRB2 and SHC with ErbB2 in c'1000 cells may supplant the requirement for GRB2-SHC interactions with the COOH-terminal region
of the EGFR in WT cells. To demonstrate a causal relationship between
ErbB2 and EGF mitogenic signaling in c'1000 cells, we transiently
expressed either WT ErbB2 or a truncated ErbB2-(1-813) in the WT and
c'1000 cells and studied ligand-dependent and -independent Elk-1 activation. Greene and co-workers (67) have used an ErbB2 molecule truncated at residue 691 to suppress cellular transformation mediated by oncogenic ErbB2 (neu), as well as the malignant phenotype of human glioblastoma cells (68). In our study overexpression of
full-length ErbB2 resulted in ligand-independent signaling and
transactivation of Elk-1 in c'1000 but not in parental or WT NR6 cells.
This indicated that removal of the COOH-terminal region of the EGFR
results in a receptor (c'1000) that has a propensity to interact with
ErbB2 and supports the notion that the cytoplasmic tail of the EGFR
functions to block the inappropriate interaction of the EGFR with ErbB2
(i.e. in the absence of ligand).
The most convincing result to demonstrate that the potent EGF
stimulated signaling by c'1000 receptor was occurring through ErbB2 was
our finding that expression of ErbB2-(1-813), which lacks the kinase
domain and COOH-terminal phosphorylation sites, functions as a
dominant-negative and specifically inhibited EGF signaling in c'1000
but not WT cells. Western blotting analysis of lysates demonstrated
that NR6 cells express relatively low to moderate levels of ErbB2 when
compared with recombinant cell lines engineered to express ErbB2 or
human breast or ovarian carcinoma cells such as SK-Br-3 or
SK-OV-3.3 WT cells express ~100,000 EGFRs per cell (40).
Thus, the fact that ErbB2-(1-813) does not inhibit either AR or EGF
signaling in WT cells is most likely due to the fact that the truncated ErbB2 molecule is not expressed at a high enough level to compete effectively with ligand-induced homodimerization of full-length EGFRs.
Conversely, ErbB2-(1-813) can compete with the relatively low to
moderate levels of endogenous ErbB2 in NR6 cells, and the EGF-activated
c'1000 receptor needs to interact with endogenous ErbB2 for strong
signaling. We cannot rule out the possibility that endogenous ErbB2 is
involved in AR and EGF signaling in WT cells, but ErbB2 clearly plays
an important role in EGF mitogenic signaling by the truncated EGFR,
c'1000.
The differential requirement for the COOH-terminal region of the EGFR
in AR and EGF signaling implies a certain degree of ligand-specific
diversity in the signal transduction mechanisms of the EGFR. Clearly,
the COOH-terminal region of the EGFR is essential for efficient
mitogenic signaling by AR, and removal of this region results in a
number of signaling defects that have a molecular basis
(i.e. GRB2 adaptor function). Furthermore, our findings
suggest that receptor homo-oligomerization is a fundamental requirement
for AR function. The fact that AR requires HSPGs to activate the EGFR
(16) suggests that HSPGs may be involved in preferentially guiding the
AR-bound EGFR into homo-oligomers relative to hetero-oligomers. On the
other hand, EGF mitogenic signaling does not require the EGFR
cytoplasmic tail, and in the absence of this region, the EGF-activated
truncated EGFR finds an additional mechanism to signal, and this
signaling necessitates ErbB2 function. This may reflect a greater
degree of redundancy in the ability of EGF to activate specific
mitogenic signaling pathways and linked protein-protein interactions.
Our results also suggest that EGF, when compared with AR, has a greater
tendency to elicit hetero-oligomerization of the EGFR with ErbB2. The
findings that we have presented here support the concept that studies
involving EGFR mutants and different EGFR ligands may provide important
insights into the mechanisms that the EGFR utilizes to evoke downstream
signaling events.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(8, 9), amphiregulin (AR) (10-12),
heparin-binding EGF-like growth factor (13), betacellulin (14), and
epiregulin (15). In the case of AR and heparin-binding EGF cell-surface heparan sulfate proteoglycan (HSPG) is essential for ligand-induced activation of the EGFR (16-18). The biological purpose of the apparent redundancy in ligands that can activate the EGFR is not fully understood but may represent a mechanism of receptor activation and
signaling that can be modulated by expression of the various ErbBs and HSPGs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-galactosidase activity were measured
with the Luciferase Assay system (Promega) and Galacto-Light Plus
System (Tropix), respectively.
-glycerophosphate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2 µg/ml aprotinin, pH 7.2 (lysis buffer). The
lysate was vortexed, incubated on ice for 10 min, and clarified by
centrifugation at 16,000 × g for 15 min at 4 °C.
Protein concentration of lysates were determined using the Bio-Rad
detergent-compatible protein assay.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or
indicating that these cells
do not express significant quantities of the EGFR-related receptors,
ErbB3 or ErbB4.3 The WT and
c'1000 cell lines express equivalent levels of GRB2, ErbB2, SHP-2, SHC,
and ERK 2 as confirmed by Western blotting of whole cell lysates (data
not shown).
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Fig. 1.
Effect of AR and EGF on DNA synthesis in NR6
parent, WT, and c'1000 cells. NR6 parental cells or those
expressing either human wild type (NR6-WT) or truncated EGFR
corresponding to amino acid residues 1-1000 (NR6-c'1000)
were grown to confluence in 96-well plates, serum-starved and
stimulated with various concentrations of AR (open circles)
or EGF (closed circles). After 6 h, the cells were
treated with [3H]thymidine for an additional 16 h.
DNA was harvested, and the incorporation of [3H]thymidine
into newly synthesized DNA was quantitated as described previously (23)
and expressed as cpm/well. Data points represent the means ± S.E.
of triplicate experiments.
Analysis of AR and EGF Binding to WT and c'1000 Receptors
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Fig. 2.
Ligand-induced transcriptional activation of
Elk-1 in NR6-WT and NR6-c'1000 cells. NR6-WT (upper
panel) or c'1000 cells (lower panel) were transfected
with 0.5 µg of a reporter plasmid that expresses luciferase under the
control of 5 tandem repeats of a Gal4-binding element (pFR-Luc), 50 ng
of a plasmid that constitutively expresses a fusion protein consisting
of the transactivation domain of Elk-1 and the DNA binding domain of
Gal4 and 100 ng of cytomegalovirus promoter-driven -galactosidase
(pcDNA 3.1/His/lacZ). The serum-starved cells were
stimulated with various concentrations of AR (open circles)
or EGF (closed circles) for 6 h, cells were lysed, and
luciferase and
-galactosidase activities were measured. Luciferase
activity is normalized to
-galactosidase activity and data points
represent the means ± S.E. of duplicate experiments.
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Fig. 3.
Kinetic analysis of
ligand-dependent MAPK activation. Cell monolayers of
NR6-WT and c'1000 cells were serum-starved and stimulated with 1 nM either AR or EGF for the appropriate time at 37 °C.
Whole cell lysates were generated and protein concentrations
quantitated. Fifteen µg per lane of whole cell lysate protein was
separated in an 8% gel and transferred to polyvinyl difluoride
membrane. The activation of MAPK was monitored by Western blotting
(WB) using antibodies directed against the phosphorylated
form of ERK 1 (phosphoMAPK) which detect both activated ERK
1 (p44) and ERK 2 (p42). Consistent protein loading of each lane was
confirmed by stripping the blot and reprobing for MAPK using an
anti-pan ERK antibody (WB: MAPK). It should be noted that
the predominant form of MAPK in NR6 cells is ERK 2, and thus, in the
Western blotting analysis for MAPK protein, it is only possible to
detect ERK 2 protein directly in the analysis of whole cell
lysates.
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Fig. 4.
Ligand-stimulated tyrosine phosphorylation of
proteins in NR6-WT and c'1000 cells. Cell monolayers of NR6-WT
(A) and c'1000 cells (B) were serum-starved and
stimulated with increasing concentrations of either AR or EGF for 5 min
at 37 °C. Whole cell lysates were generated, protein concentrations
were quantitated, and 10 µg of lysate was fractionated in an 8%
SDS-PAGE gel and analyzed by Western blotting (WB) for the
presence of phosphotyrosine using a mixture of biotinylated 4G10 and
PY20 antibodies (WB: PY; lanes 1-9). WT and
c'1000 EGFR were immunoprecipitated (IP) from 1 mg of cell
lysate derived from cells treated with either 10 nM AR
(A) or EGF (E) using Ab-5 antibody directed
against the extracellular domain. Immunoprecipitated receptors were
analyzed by WB for the presence of phosphotyrosine (lanes
10-12), and consistent immunoprecipitation of receptors was
confirmed by WB using the anti-EGFR ECD antibody, LA22 (lanes
13-15). Numbers shown to the right denote the position
in the gel and size in kilodaltons of molecular mass markers
proteins.
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Fig. 5.
Analysis of ligand-induced coupling of
signaling molecules to the adaptor proteins, GRB2 and SHC, in NR6-WT
and c'1000 cells. Cell monolayers were serum-starved and
stimulated with 1 nM of either AR or EGF for 0, 5, 15, 30, or 60 min at 37 °C. Whole cell lysates were generated and protein
concentrations determined. A, 1 µg of GRB2-glutathione
S-transferase fusion protein (GRB2-GST) bound to
glutathione-agarose was added to 0.5 mg of cell lysate and incubated
for 12 h at 4 °C. After thorough washing of the GRB2-GST resin,
bound proteins were released by boiling in SDS-PAGE sample buffer. The
binding of EGFR, ErbB2, SHP-2, and SHC was analyzed by Western blotting
(WB). In all instances, no binding of these proteins was
detected in lysates probed with a GST control protein (data not shown).
B, cells were stimulated for 5 min with 0, 1, or 10 nM of AR or EGF. SHC was immunoprecipitated (IP)
from 1 mg of cell lysate, and co-immunoprecipitation of ErbB2
and PY-containing proteins was analyzed by WB. Consistent
immunoprecipitation of SHC was confirmed by WB using an anti-SHC
antibody. The arrow on the right side of the
middle panel (WB: PY) denotes the position of
ErbB2 in the anti-phosphotyrosine blot, and numbers denote
the position in the gel and size in kilodaltons of molecular mass
markers proteins.
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Fig. 6.
The effect of transient overexpression of
ErbB2 or truncated ErbB2 on ligand-independent and
-dependent Elk-1 transactivation in NR6-WT and c'1000
cells. Parental mouse NR6 (parent), NR6-WT, or
NR6-c'1000 cells were transfected with 0.5 µg of pFR-Luc, 50 ng of
pFA2-Elk-1, 100 ng of pcDNA 3.1/His/lacZ, and 1.35 µg
of pcDNA 3.1 (empty vector) or pcDNA 3.1 encoding
either full-length human ErbB2 (WT) or ErbB2-(1-813). The
cells were allowed to recover overnight in serum-containing medium and
were serum-starved. A, cells were lysed without growth
factor treatment, and luciferase and -galactosidase activities were
measured. B, pooled lysates derived from identically
transfected dishes were analyzed for ErbB2 expression by Western
blotting (WB) using either a monoclonal antibody that is
specific to the extracellular domain of human ErbB2 or a monoclonal
antibody that detects the COOH terminus (amino acid residues
1242-1255) of both mouse and human ErbB2. C, serum-starved
cells were stimulated for 6 h with 10 nM AR or EGF,
lysed, and luciferase and
-galactosidase activities were measured.
In the bottom bar graph of C, cells were also
transfected with 50 ng of a cDNA construct encoding a
constitutively active form of Ras (Q61L) (+) or
empty vector (
), and cells were lysed without growth factor
treatment. Luciferase activity is normalized to
-galactosidase
activity, and data points represent the means ± S.E. of duplicate
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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activation, motility (40, 54), and attenuation of EGFR mitogenic
signaling by a phospholipase C-
/protein kinase C feedback mechanism
(55).
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FOOTNOTES |
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* This research was supported in part by an appointment (to T. B. D.) to the Postgraduate Research Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U. S. Department of Energy and the U. S. Food and Drug Administration and by NIGMS Grant R01-54739 (to A. W.) from National Institutes of Health.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: Division of
Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bldg. 29A, Rm. 3B-16, 8800 Rockville Pike,
Bethesda, MD 20892. Tel.: 301-827-1770; Fax: 301-402-1659; E-mail:
JohnsonG{at}CBER.FDA.Gov.
2 A. Wells, unpublished observations.
3 L. Wong, T. B. Deb, S. A. Thompson, A. Wells, and G. R. Johnson, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
EGFR, epidermal
growth factor receptor;
EGF, epidermal growth factor;
AR, amphiregulin;
TGF-, transforming growth factor-
;
BTC, betacellulin;
HSPG, heparan sulfate proteoglycan;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
SH2 domain, Src homology 2 domain;
GST, glutathione S-transferase;
WT, wild type;
PAGE, polyacrylamide gel electrophoresis;
Ab, antibody.
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