Domain-specific Interactions between the
p185neu and Epidermal Growth Factor Receptor
Kinases Determine Differential Signaling Outcomes*
Xiaolan
Qian
§,
Donald M.
O'Rourke¶,
Zhizhong
Fei
,
Hong-Tao
Zhang
,
Chih-Ching
Kao**, and
Mark I.
Greene

From the
Department of Pathology and Laboratory
Medicine, and the ¶ Department of Neurosurgery, University of
Pennsylvania School of Medicine, and the ** Department of Pathology,
University of Pennsylvania School of Veterinary Medicine, Philadelphia,
Pennsylvania 19104
 |
ABSTRACT |
We expressed the epidermal growth
factor receptor (EGFR) along with mutant
p185neu proteins containing the rat
transmembrane point mutation. The work concerned the study of the
contributions made by various p185neu
subdomains to signaling induced by a heterodimeric ErbB complex. Co-expression of full-length EGFR and oncogenic
p185neu receptors resulted in an increased
EGF-induced phosphotyrosine content of p185neu,
increased cell proliferation to limiting concentrations of EGF, and
increases in both EGF-induced MAPK and phosphatidylinositol 3-kinase
(PI 3-kinase) activation. Intracellular domain-deleted p185neu receptors (T691stop neu) were able to
associate with full-length EGFR, but induced antagonistic effects on
EGF-dependent EGF receptor down-regulation, cell
proliferation, and activation of MAPK and PI 3-kinase pathways.
Ectodomain-deleted p185neu proteins (T
5)
were unable to physically associate with EGFR, and extracellular
domain-deleted p185neu forms failed to augment
activation of MAPK and PI 3-kinase in response to EGF. Association of
EGFR with a carboxyl-terminally truncated
p185neu mutant (TAPstop) form did not increase
transforming efficiency and phosphotyrosine content of the TAPstop
species, and proliferation of EGFR·TAPstop-co-expressing cells in
response to EGF was similar to cells containing EGFR only. Thus,
neither cooperative nor inhibitory effects were observed in cell lines
co-expressing either T
5 or TAPstop mutant proteins. Unlike the
formation of potent homodimer assemblies composed of oncogenic
p185neu, the induction of signaling from
p185neu·EGFR heteroreceptor assemblies
requires the ectodomain for ligand-dependent physical
association and intracellular domain contacts for efficient intermolecular kinase activation.
 |
INTRODUCTION |
The ErbB family includes four members of homologous receptor
tyrosine kinases, the epidermal growth factor receptor
(EGFR1 or ErbB-1) (1),
ErbB-2·p185neu (2, 3), ErbB-3 (4), and ErbB-4
(5). ErbB family proteins are widely expressed in epithelial,
mesenchymal, and neuronal tissues, and play important roles in normal
growth and development (6-9). Aberrant expression of these ErbB
proteins is frequently observed in human malignancies (10).
The transmembrane mutation in rat p185neu (also
termed Tneu) (12) serves as a paradigm for receptor dimerization that
leads to constitutive kinase activation contributing to oncogenic
transformation (11-13). Additional support for this mechanism has come
from the identification of a naturally occurring activated EGFR
oncoprotein (
EGFR or EGFRvIII) in human tumors, which forms
constitutive dimers and confers increased tumorigenicity (14, 15). Gene amplification and overexpression of ErbB-2 have been observed in a high
frequency of human adenocarcinomas, including those of the breast and
ovary, and these features correlate with poor clinical prognosis (16,
17). Experimental support for this model is provided by in
vitro transformation assays using cell lines overexpressing either
protooncogenic rat p185c-neu or human ErbB-2 at
levels of 106 receptors/cell (18, 19). Biochemical and
biophysical analysis of baculovirus-expressed
p185neu proteins further support the notion of
receptor oligomerization as a mechanism of kinase activation of normal
holoreceptors (20, 21).
Heterodimeric interactions govern many signaling properties within the
ErbB receptor family. Co-expression of EGFR and
p185c-neu at modestly elevated levels
(105/cell) (but not either receptor independently) results
in synergistic transformation (22), due to increase of the ligand
binding affinity and catalytic kinase activity (23, 24).
Heterodimerization of EGFR and ErbB-2 has also been observed in human
breast tumor lines (25). Moreover, ligand treatment promotes the
assembly of an activated p185c-neu·EGFR kinase
complex in many cells (24), resulting in novel distinct cellular
signaling events (26). Therefore, the receptor tyrosine kinase ensemble
can be activated not only by homodimer formation, but also by
heterodimeric associations. In this regard, endodomain interactions
between p185neu and EGFR appear to influence
functional signaling outcomes (27).
In response to EGF or Neu differentiating factor/heregulin (a ligand
for ErbB-3 and ErbB-4) family ligands (28, 29), EGFR and ErbB-2 both
form heterodimers with ErbB-3 and ErbB-4 (30-34). Heterodimers between
p185neu·ErbB-2 and ErbB-3 are associated with
activated signaling and the transformed phenotype in primary human
cancer cells (35). Existence of an ErbB-3·ErbB-4 heterodimer has not
been convincingly demonstrated to date. More recent data support the
notion that p185neu·ErbB-2 is the preferred
heterodimerization partner of all ErbB receptors and a
mediator for divergent cellular signaling in many distinct cell types
(34, 36).
The structural basis for ErbB receptor heterodimerization has not been
completely defined and crystallographic information on dimerized ErbB
receptor kinases is currently unavailable. Previous work has revealed
that ectodomain interactions are sufficient to stabilize dimer
formation between p185neu and EGFR in
fibroblasts and transformed cells (5, 37, 38), which is supported by
observations showing that a partial deletion of the EGF receptor
ectodomain still allow dimer formation and receptor activation (14,
15). Although the transmembrane alone can stabilize the formation of
p185neu homodimers, the relative contributions
of the transmembrane region and the ectodomain have not been directly
compared regarding the formation of signaling heterodimers.
In this study, we have constructed various
p185neu deletion mutants in order to
specifically compare signaling events resulting from associations
between EGF receptors and either p185neu
ectodomain- or endodomain-derived mutant receptors. We have
co-expressed EGFR with low levels of
p185neu proteins, or their mutant derivatives,
to monitor p185neu-mediated enhancement of cell
growth and transformation in vitro and in vivo,
and to analyze the influence of EGF-induced heterodimeric receptor
interactions on downstream signaling effectors. Signaling resulting
from heterodimeric associations between full-length EGFR and mutant
p185neu proteins has revealed the functional
importance of p185neu subdomains in the
induction of Ras/extracellular signal-regulated kinase (ERK) and
phosphatidylinositol 3-kinase (PI 3-kinase) pathways contributing to
cell growth and transformation.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
As described previously (20, 39, 40), monoclonal
antibody 7.16.4, polyclonal antiserum
-Bacneu, and NCT are reactive with the ectodomain, intracellular domain, and carboxyl terminus of
p185neu, respectively. mAb 225 reactive with the
ectodomain of EGFR was obtained from Dr. John Mendelsohn (M. D. Anderson Cancer Center, Dallas, TX). A polyclonal rabbit antiserum
specifically against the COOH terminus of EGFR (termed CT) was provided
by Dr. Stuart Decker (40). The anti-phosphotyrosine monoclonal
antibody, PY20, was obtained from Santa Cruz Biotechnology (Santa Cruz,
CA.).
DNA Constructs--
All the deletion mutants were derived from
the rat oncogenic p185neu cDNA containing a
single point mutation (V664G) in the transmembrane region. The TAPstop
mutant, containing a 122-aa truncation of the COOH terminus was
prepared as described previously (41). A T691stop species was prepared
by site-directed mutagenesis and substitution of a stop codon for
Thr-691, resulting in a large cytoplasmic deletion (42, 43). The
ectodomain-deleted mutant T
5 neu protein was described previously
(27). These cDNAs encoding for mutant
p185neu forms were all cloned into the
pSV2neor/DHFR vector as described (44) for expression in
murine fibroblasts. These wild-type or mutant
p185neu cDNAs were also subcloned into
pcDNA3 vector for transient expression in COS7 cells.
pSR
EGFR/hygr vector (44) was used for full-length EGFR expression.
Transfection and Maintenance of Cell Lines--
Ten micrograms
of the p185neu constructs were transfected into
NR6 cells, a mouse fibroblast cell line devoid of endogenous EGF receptors (43), or NE91 cells expressing human EGFR (37) by calcium
phosphate precipitation. After 2-3 weeks of selection with Geneticin
(0.9 mg/ml), the established stable clones were screened and
characterized. Gene amplification by methotrexate was used to increase
the p185neu receptor level. Expression of
p185neu and its derivatives in resultant
subclones was examined by flow cytometric analysis following
anti-p185neu mAb 7.16.4 staining. Surface
expression of p185neu proteins was then
estimated by comparing the mean channel fluorescent intensity with that
of B104-1-1 cells, as the level of p185neu in
B104-1-1 cells was previously determined by 125I-labeled
anti-neu mAb binding assay (22). EGFR numbers in NE91 cells and mutant
p185neu co-transfected cells were determined by
Scatchard assays as described (37). These transfected clones were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
5% fetal bovine serum (FBS, HyClone) at 37 °C in a 5%
CO2 atmosphere.
Cross-linking, Immunoprecipitation, and Immunoblotting
Procedures--
Subconfluent cells in 10-cm dishes were washed and
starved in cysteine-free DMEM for 1 h, and grown in low
cysteine-containing 5% FBS-DMEM containing 55 µCi/ml
[35S]cysteine (Amersham Pharmacia Biotech) for 16 h
for metabolic labeling. Alternatively, the unlabeled cells were
cultured overnight in 10-cm Petri dishes. After treatment with or
without EGF, cells were washed twice with cold phosphate-buffered
saline (PBS) and treated with PBS containing 2 mM
membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate
(BS3, Pierce), for 30 min. After quenching the
cross-linking reaction with a buffer containing 10 mM
Tris-HCl (pH 7.6), 0.9% NaCl, and 0.1 M glycine, cells
were washed twice with cold PBS and solubilized with PI/RIPA buffer as
described (24). The immunocomplexes were washed and solubilized, then
separated by gradient SDS-PAGE gels (4-7.5%). Proteins from
metabolically labeled cells were analyzed by autoradiography. Proteins
from unlabeled cells were transferred onto nitrocellulose and then
immunoblotted with anti-phosphotyrosine mAb PY20, anti-EGFR CT, or
anti-p185 antiserum as indicated in the figures. The protein signals
were identified by the binding of 125I-labeled protein A
(NEN Life Science Products), or by enhanced chemiluminase (ECL) using
ECL kit from Amersham Pharmacia Biotech.
Receptor Down-regulation Studies--
Cells (1 × 105) were plated in a six-well dish with DMEM containing
5% FBS overnight. Cells were then treated with EGF (50 ng/ml) for 0-4
h and were harvested and washed with cold PBS containing 0.5% bovine
serum albumin and 0.1% sodium azide. Cell preparations were then
incubated with a saturating amount (0.5 µg/reaction) of anti-neu mAb
7.16.4 or anti-EGFR mAb 225, or an irrelevant mAB (such as 9BG5 against
the hemagglutinin of reovirus receptor), at 4 °C for 30 min,
restained with fluorescein isothiocyanate-conjugated anti-mouse IgG
(Sigma) for another 30 min after extensive washing. Cells were then
fixed with 2% paraformaldehyde and analyzed by flow cytometry
(FACScan, Becton Dickinson), as described previously (37). Briefly,
after subtracting the nonspecific background staining with 9BG5, the
mean channel values from each time point were used to determine the
percentage of surface expression of EGFR or
p185neu proteins at the various time points
after EGF treatment.
In Vitro and in Vivo Transformation
Assays--
Anchorage-independent growth ability was determined by
assessing the colony forming efficiency of cells suspended in soft agar
(15, 37). Cells (1000/dish) were suspended in 7% FBS-DMEM containing
0.18% agarose, and plated on 0.25% basal agar in each dish. Cells
were fed with DMEM supplemented with 7% FBS-DMEM, 20 mM
HEPES (pH 7.5). Colonies (>0.3 mm) were visualized at day 21 for all
cell lines after stained with p-iodonitrotetrazolium violet
(1 mg/ml). Each cell line was examined in triplicate samples for
separate experiments.
To analyze the tumor growth in athymic mice, cells (1 × 106) of each line were suspended in 0.1 ml of PBS and
injected intradermally in the mid-dorsum of NCR nude mice. PBS alone
was also injected as a control. Animals used in this study were
maintained in accordance with the guidelines of the Committee on
Animals of the University of Pennsylvania and those prepared by the
Committee on Care and Use of Laboratory Animals of the Institute of
Laboratory Animal Resource. Tumor growth was monitored twice a week up
to 10 weeks. Tumor size was calculated by this formula: 3.14/6 × (length × width × thickness) as described (27).
EGF-dependent Cell Proliferation Assay--
The
3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) (MTT)
assay for measuring cell growth has been described previously (38).
Briefly, cells (3000/well) of each cell line were seeded in 96-well
plates overnight in DMEM containing 5% FBS. Cells were starved in
serum-free ITS-DMEM for 48 h, then cultured in 100 µl of the
same medium plus various concentrations of EGF for another 48 h.
25 µl of MTT solution (5 µg/ml in PBS) were added to each well, and
after 2 h of incubation at 37 °C, 100 µl of the extraction
buffer (20% w/v SDS, 50% N,N-dimethyl formamide, pH 4.7)
was added. After an overnight incubation at 37 °C, the optical
density at 600 nm was measured using an enzyme-linked immunosorbent
assay reader. Each value represents a mean of four samples.
MAP Kinase and PI 3-Kinase Immune Complex Kinase
Assays--
COS7 cells were transiently transfected with
pcDNA3-HA-ERK2 (a gift from Silvio Gutkind, National Institutes of
Health, Bethesda, MD) and pSR
EGFR/hygr, along with
either empty vector or plasmids expressing wild-type or mutant
p185c-neu using LipofectAMINE (Life
Technologies, Inc.) according to the manufacturer's instructions and
assayed 48 h after transfection. Cells deprived of serum for
16-20 h were treated with or without EGF (50 ng/ml) for 5 min. For MAP
kinase assay, cells were lysed with RIPA buffer (25 mM
Tris-HCl (pH 7.5), 0.3 M NaCl, 1.5 mM MgCl2, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin). Protein concentrations were determined by the BCA kit
(Pierce). Equal amounts of protein (100 µg) from cell extracts were
immunoprecipitated with anti-HA (BabCo). After washing extensively, the
immunocomplexes were then incubated with 50 µl of reaction buffer (30 mM HEPES (pH 7.4), 10 mM MaCl2, 1 mM dithiothreitol, 5 µM ATP) containing 1 µCi of [
-32P]ATP (NEN Life Science Products) and 2 µg of myelin basic protein (Upstate Biotechnology Inc.). After
incubation for 20 min at 30 °C, kinase reactions were terminated by
the addition of 2× Laemmli sample buffer. The samples were then
resolved by SDS-PAGE, and the phosphorylated myelin basic protein
was visualized by autoradiography.
PI 3-kinase immune complex assays were carried out as described (45)
with slight modifications. Cells were lysed in Nonidet P-40 lysis
buffer (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,
10% glycerol, 1% Nonidet P-40, 1 mM sodium orthovanadate,
10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin). Equal amounts of protein (600 µg) from cell extracts were immunoprecipitated with anti-phosphotyrosine 4G10 (Upstate Biotechnology Inc.) for 3 h. Protein A-Sepharose was then
added and rotated at 4 °C for overnight. Immunocomplexes were washed
twice with lysis buffer; twice with 100 mM Tris (pH 7.4),
0.5 M LiCl, 0.2 mM sodium orthovanadate, plus
0.2 mM adenosine; and twice with reaction buffer (10 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl). The beads were resuspended in 40 µl of reaction buffer containing substrate mixture (phosphatidylinositol,
phosphatidylinositol 4-phosphate, and phosphatidylserine dispersed by
sonication in 10 mM HEPES (pH 7.5), 1 mM EGTA).
The tubes were incubated at room temperature for 10 min and reaction
were initiated by adding 5 µCi of [
-32P]ATP (NEN
Life Science Products) per reaction in 5 µl of 500 mM ATP
and terminated by addition of 80 µl of
CHCl3:CH3OH (1:1) after another 10 min.
Phospholipids were extracted, desiccated, and redissolved as described
(45). The samples were the chromatographed on thin layer chromatography
plates (precoated with potassium oxalate and baked at 100 °C for
1 h before use) in CHCl3:CHOH:2.5 M
NH4OH:H2O (45:35:2.7:7.3). Spots corresponding
to phosphatidylinositol 3-phosphate and phosphatidylinositol
3,4-bisphosphate were visualized after autoradiography. Unlabeled
phospholipid standards were included and were visualized by exposure to
iodine vapor.
 |
RESULTS |
Expression of EGFR and/or Mutant p185neu
Proteins--
Cell lines expressing EGFR and various
p185neu deletion mutant proteins derived from
full-length transforming p185neu were all
generated in the NR6 cell background (43). In addition, stable
transfectants derived from NR6 fibroblasts expressing human EGFR
(termed NE91 cells) were also generated. NE91 cells, as well as NR6
parental cells, were then transfected with various
p185neu cDNA constructs to express one of
the following mutant p185neu proteins with or
without EGFR, respectively (Fig. 1):
(a) Er/p185neu or
p185neu (full-length oncogenic
p185neu product), (b) Er/T691stop or
T691stop (lacking 591 aa from the carboxyl terminus), (c)
Er/TAPstop or TAPstop (a 122-aa truncation at carboxyl terminus), and
(d) Er/T
5 or T
5 (an ectodomain deleted p185neu product, also termed T
5). A schematic
representation of the oncogenic p185neu protein
and its mutant derivative species is shown in Fig. 1.

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Fig. 1.
Schematic representation of EGFR and mutant
p185neu proteins. Locations of
cysteine-rich subdomains (CRD), transmembrane region
(TM) containing the point mutation V664E (*), tyrosine
kinase domain (TK), and carboxyl-terminal region
(CT) are indicated. Tneu is the full-length
transforming rat p185neu. T691stop
contains a stop codon substituting for Thr-691 at the amino terminus to
the TK domain. TAPstop contains a 120-aa truncation within
the carboxyl terminus of p185neu.
T 5 is generated by the deletion of ectodomain
of p185neu but retains ~10 aa and the signal
sequence. These mutant p185neu proteins were
either expressed alone or co-expressed with EGFR in NR6 transfected
cells.
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B104-1-1 murine fibroblasts transformed by the expression of oncogenic
p185neu were used as a positive control, since
surface expression of p185neu, biochemical
features of p185neu homodimerization and
p185neu transforming potency have been
characterized previously (13, 22, 27). As shown in Table
I, relative expression levels of various
p185neu mutant proteins in selected clones were
estimated by a comparison with B104-1-1 cells, while the expression of
EGFR in these cells was estimated by Scatchard analysis. In order to
observe an enhancement of EGFR-mediated cellular signaling and
transformation, clone Er/p185neu expressing a
moderately low level of both receptors (~104/cell) was
chosen. In other subclones, the expression of EGFR and/or mutant
p185neu proteins was approximately
~105 receptors/cell.
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Table I
Transformation parameters and relative receptor expression levels of
cell lines
The number of EGFR on NE91 and other transfected cells was determined
by Scatchard assays. Cell surface expression of neu proteins were
estimated by comparing the mean channel fluorescent intensity with that
from B104-1-1 cells using flow cytometry analysis. p185neu on
B104-1-1 cells was originally determined by an 125I-labeled
anti-neu mAb binding assay (22). For the tumor growth assay, individual
clones (1 × 106 cells/site) were injected intradermally
into athymic mice. NT, no tumor after 10 weeks; ND, not determined.
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The Ectodomain of p185neu Is Required for
Heterodimerization with EGFR--
Stable cell lines expressing EGFR
and/or mutant p185neu proteins were used to
assess dimer formation using the chemical cross-linker BS3.
As shown in Fig. 2, B104-1-1 cells
expressing oncogenic p185neu contained
p185neu homodimers (~370 kDa) independent of
ligand stimulation (Fig. 2A, lane 1),
due to the activating transmembrane mutation (12). A cell line
expressing the ectodomain-derived T691stop neu alone was used as a
control to demonstrate the sizes of the monomer and dimer of this
truncated p185neu protein, which migrated at
approximately 115 kDa (Fig. 2A, lanes 2 and 3), and at ~230 kDa in the presence of a
chemical cross-linker (Fig. 2A, lane
3).

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Fig. 2.
Homodimerization and heterodimerization of
EGFR and p185neu proteins. A,
cells were labeled with [35S]cysteine overnight. Cell
lines expressing EGFR (lanes 4 and 5)
were then stimulated with EGF (200 ng/ml) at 37 °C for 10 min. All
cells (except lane 2) were treated with the
chemical cross-linker BS3 (2 mM). Cell lysates
were then immunoprecipitated with anti-neu mAb 7.16.4 or anti-EGFR
antiserum CT as indicated. Proteins were separated by 4-8% gradient
SDS-PAGE and analyzed by autoradiography. The estimated molecular
weight of monomers and dimers is indicated. B and
C, cell lines expressing EGFR (NE91, Er/T 5, and
Er/TAPstop) were stimulated with EGF. After BS3 treatment,
all the cells were lysed and subjected to immunoprecipitation with
either anti-neu (7.16.4 or NCT) or anti-EGFR (CT) antibodies, then
immunoblotted with either the anti-neu (NCT or -Bacneu) or anti-EGFR
probe (CT) as indicated.
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In the presence of EGF, the 170-kDa monomeric form and the 340-kDa
homodimer of EGFR were both detected in NE91 cells expressing EGFR
alone, and in Er/T691stop cells (Fig. 2A, lanes
4 and 5, respectively). An additional
intermediate band of ~285 kDa representing the heterodimer of EGFR
and T691stop was clearly detectable upon anti-EGFR immunoprecipitation
(Fig. 2A, lane 5). The 285-kDa
intermediate complex was similar to the heterodimer composed of EGFR
and truncated N691stop derived from proto-oncogenic
p185c-neu as described previously (44), except
that the heterodimeric EGFR·N691stop complex was even more
predominant than the EGFR homodimer in those studies. Notably, T691stop
is still able to complex with EGFR (lane 5) even
under conditions favorable for T691stop homodimerization
(lane 3). Densitometric analysis suggested that
at least 50% of the EGFR associated with T691stop neu in a
heterodimeric complex in Er/T691stop cells (Fig. 2A,
lane 5), further suggesting the strong preference
for EGFR·p185neu heterodimerization.
We have previously studied complex formation between the
p185neu and EGFR holoreceptors (22, 24, 37) and
heterodimerization between ectodomain p185neu
and either full-length (37, 44) EGFR or a form of EGFR that lacks the
majority of subdomains 1 and 2 (15) by immunoprecipitation and
immunoblotting using anti-receptor specific antibodies following EGF
and chemical cross-linker treatment.
In this next set of studies, we extended these observations to novel
species of p185neu. Er/Nneu cells expressing
higher levels of EGFR and normal p185c-neu
served as a positive control to examine the physical association of
EGFR with truncated mutant p185neu receptor
forms (Fig. 2C, lane 1). The
heterodimer between full-length p185neu with
EGFR in Er/neu cells could not be detected due to low expression levels
of each receptor (data not shown). Abundant levels of the EGFR monomer
and dimer were detected in Er/TAPstop cells by anti-EGFR immunoprecipitation and immunoblotting (Fig. 2C,
lane 3). Analysis of
anti-p185neu immunoprecipitates by
immunoblotting with anti-EGFR antisera indicated EGF-induced
heterodimerization of EGFR and TAPstop in Er/TAPstop cells (Fig.
2C, lane 2). As expected, the size of
this complex was slightly smaller when compared with the heterodimer of
EGFR and full-length normal p185neu in Er/Nneu
cells (Fig. 2C, lane 1). The control
cell line expressing TAPstop alone showed that TAPstop was only
recognized by an anti-neu antibody (Fig. 2C, lane
5), but not by anti-EGFR antibody CT (Fig. 2C,
lane 4).
Heterodimerization between EGFR and the ectodomain-deleted T
5
p185neu mutant was also analyzed. T
5 can be
recognized by either the
-Bacneu or anti-NCT polyclonal antisera
reactive with the intracellular domain or carboxyl terminus of the
p185neu protein, respectively. Immunoblotting
showed that the size of the T
5 neu mutant was approximately 95-97
kDa, and the detectable dimeric form was about ~200 kDa (Fig.
2B, lane 1). Er/T
5 cells express a
high level of EGFR and T
5, as homodimers of either form were clearly
detected in the presence of cross-linker (Fig. 2B,
lanes 2 and 3), when compared with
control cell lines NE91 and T
5 (Fig. 2B, lanes
1 and 4). However, unlike Er/T691stop and
Er/TAPstop cells, the heterodimer between EGFR and T
5 in Er/T
5
cells was undetectable following EGF and BS3 treatment
since the predicted intermediate size (~270 kDa) complex representing
EGFR and T
5 heterodimer was not observed (Fig. 2B, lanes 2 and 3). In an attempt to
identify the association of EGFR with this ectodomain-deleted T
5
protein, several alternative assays were performed, such as using the
membrane-permeable chemical cross-linker DSP (Pierce), or a mild
detergent digitonin lysis buffer. These methods were sensitive enough
to detect the complex formation between full-length
p185neu and T
5 (27). However, the association
of EGFR and T
5 was still undetectable (data not shown). Taken
together, these results strongly suggest that the ectodomain of the
p185neu receptor is necessary and sufficient for
heterodimerization with holoreceptor EGFR.
Tyrosine Kinase Activity in Living Cells--
It has been well
documented that EGF, in an EGFR-dependent manner,
stimulated phosphorylation of the p185c-neu and
c-ErbB-2 gene products with a concomitant increase in their tyrosine
kinase activities (46-49). Heterodimerization of p185 and EGFR
facilitates cross-phosphorylation (24, 25), since a full-length,
kinase-deficient p185neu mutant (K757M) is
trans-phosphorylated upon physical association with EGFR (37). We
next examined the tyrosine phosphorylation level of
p185neu derivatives in living cells in response
to EGF treatment. After the addition of EGF, oncogenic
p185neu and its derivatives were
immunoprecipitated by anti-neu antibodies, and receptor phosphotyrosine
content in vivo was detected by immunoblotting with an
anti-phosphotyrosine antibody (PY20) (Fig.
3). Full-length p185neu from control B104-1-1 fibroblasts
displayed constitutive kinase activity (Fig. 3A,
lane 1). Upon EGF stimulation, there was indeed an additional increase in tyrosine kinase activity of
p185neu in Er/neu cells expressing lower amounts
of the p185neu protein (Fig. 3A,
lanes 4 and 5), but not in cells
expressing p185neu alone (lanes
2 and 3). A weak tyrosine phosphorylation signal was detected in TAPstop cells (Fig. 3A, lane
6). EGF stimulation did not appreciably increase the
tyrosine phosphorylation of TAPstop in EGFR-co-expressing cells (Fig.
3A, lanes 7 and 8),
although the association of EGFR and TAPstop was evident (Fig.
2C). Truncation of the p185neu
carboxyl terminus, and deletion of at least three known critical tyrosine residues, was associated with the failure to
trans-phosphorylate the p185neu mutant protein.
Elimination of the ectodomain did not impair the intrinsic kinase
activity of p185neu-derived T
5, since the
T
5 mutant receptor was still a competent tyrosine kinase (Fig.
3C, lane 1). However, unlike the
full-length p185neu, no further increase in
tyrosine phosphorylation of T
5 was detected in Er/T
5 cells with
EGF stimulation (Fig. 3C, lane 2 and
3). In Er/T
5 cells, the EGFR was also immunoprecipitated
by the anti-Bacneu antisera and still autophosphorylated after EGF
treatment (Fig. 3C, lane 3). These
results correlated with failure to detect physical interactions between
EGFR and T
5 proteins (Fig. 2). Reprobing with anti-neu antibodies
(Fig. 3, A and C) confirmed equivalent protein
loading in paired samples with or without EGF treatment (Fig. 3,
B and D). These experiments indicated that the
full-length p185neu receptor, but not mutant
p185neu proteins with NH2-terminal
or distal COOH-terminal truncations, was able to interact with
activated EGFR functionally, resulting in trans-phosphorylation.

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Fig. 3.
Tyrosine phosphorylation of EGFR and mutant
p185neu proteins in living cells. Cells in
panels A, C, and E were
treated with or without EGF as indicated. Cells in panel
E were also treated with the chemical cross-linker
BS3 (2 mM). Cell lysates were then
immunoprecipitated with anti-neu antibodies, 7.16.4, -Bacneu, or
anti-EGFR CT as indicated. Proteins were separated by 6% (A
and C) or 4-8% (E) gradient SDS-PAGE followed
by immunoblotting with anti-phosphotyrosine mAb PY-20. After stripping
the PY20 signals presented in top panels, these
nitrocellulose membranes were reprobed with anti-neu NCT (B,
lanes 1-5, and D), -Bacneu
(B, lanes 6-8) or -EGFR CT
(F) to compare protein amounts used in each sample.
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We next analyzed tyrosine kinase activation in EGFR-positive NE91 cells
with or without T691stop neu co-expression. Treatment with EGF and a
chemical cross-linking reagent resulted in heavy tyrosine
phosphorylation of EGFR monomers and homodimers in NE91 cells (Fig.
3E, lane 1). No detectable tyrosine
phosphorylation of cytoplasmic domain-deleted T691stop neu was seen in
cells with or without EGFR co-expression (Fig. 3E,
lanes 2 and 3, respectively). In
addition, the tyrosine phosphorylation signal of an intermediate band
(~285 kDa) representing EGFR·T691stop heterodimeric complex was
also undetectable (Fig. 3E, lane 2),
although a significant portion of EGFR forms a heterodimer with
T691stop under these conditions (Fig. 2A, lane
5). Tyrosine kinase activation of full-length EGFR was thus
completely inhibited when EGFR was physically associated with the
T691stop neu mutant protein, which correlates with reduction of the
transformed phenotype of primary EGFR-positive glioma cells expressing
T691stop neu (42). Moreover, these results are consistent with the
observation from cells co-expressing EGFR with N691stop neu derived
from normal p185neu (37).
Re-probing the membrane with an anti-EGFR antibody (CT) showed total
EGFR levels in NE91 cells (Fig. 3F, lane
1), and confirmed the presence of the EGFR·T691stop
heterodimer (~285 kDa), since this complex was recognized by anti-neu
in immunoprecipitation and anti-EGFR in immunoblotting (Fig.
3F, lane 2). Lysates obtained from
T691stop neu-expressing cells did not react with the anti-EGFR CT probe
(Fig. 3F, lane 3). Although the
cytoplasmic domain deletion in T691stop did not impair
heterodimerization with EGFR, the undetectable phosphotyrosine content
of the intermediate heterodimer suggested that EGFR kinase activity was
reduced when associated with T691stop neu. These experiments further
support our model that the heteroreceptor assembly mediated primarily
by ectodomain interactions facilitates kinase trans-activation and
trans-phosphorylation caused by interactions between cytoplasmic
domains (15, 27, 37).
EGF-induced Receptor Down-regulation from the Cell
Surface--
Numerous studies indicate that ligand-mediated receptor
endocytosis and degradation is a kinase-dependent process
for many types of growth factor receptors (50). We found that the
efficiency of receptor down-regulation and degradation in cells
co-expressing EGFR and p185neu correlated well
with heterodimeric kinase activities (37). We used this method as an
alternative assay to examine the kinase activity of various heterodimers.
Cells were incubated with EGF (50 ng/ml) for various times prior to
cell surface staining with anti-neu mAb 7.16.4 or anti-EGFR mAb 225 followed by the staining with fluorescein isothiocyanate-conjugated anti-mouse-IgG. Cell surface expression of each receptor was analyzed using flow cytometric analysis. EGF treatment of NE91 cells (expressing EGFR only) resulted in a reduction of cell surface EGFR, and over 60%
of EGF receptors disappeared from the cell surface after 4 h of
treatment (Fig. 4A). Normal
EGFR down-regulation was not affected by the co-expression of T
5, as
the efficiency of EGFR down-regulation in Er/T
5 cells was very
similar to that seen in NE91 cells (Fig. 4A). A similar EGFR
down-regulation curve was observed in Er/neu and Er/TAPstop cells (Fig.
4, C and D, respectively), indicating that the
EGFR behaves as an active receptor kinase in these cells. Moreover,
about ~20% of p185neu or 25% TAPstop was
co-down-regulated with EGFR upon EGF stimulation (Fig. 4, C
and D). As illustrated above, the low expression of p185neu and EGFR in Er/neu cells was
insufficient to demonstrate the physical association of the two
receptors biochemically. The current assay was more sensitive in
determining EGF-mediated receptor interactions. Control cells
expressing TAPstop alone did not respond to EGF treatment, and the
surface expression of TAPstop remained unchanged within the period of
EGF treatment (Fig. 4D).

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Fig. 4.
EGF-mediated receptor down-regulation.
Cells were plated in six-well dishes overnight and treated with EGF (50 ng/ml) for 0-4 h at 37 °C. Cells were then washed with
fluorescence-activated cell sorting buffer and stained with anti-neu
mAb 7.16.4 or anti-EGFR mAb 425 as indicated. After subtracting the
background staining with irrelevant mAb 9BG5, the percentage of cell
surface receptor expression reflected by the mean fluorescent intensity
from each treated sample verses that from a non-treated sample was
plotted against EGF treatment time. A, NE91 and Er/T 5;
B, Er/T691stop; C, Er/neu; D, TAPstop
and Er/TAPstop.
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|
Analysis using an EGF-mediated pulse-chase assay showed that the
down-regulated EGFR and co-down-regulated TAPstop proteins efficiently
went into the degradation pathway (data not shown), similar to the
cells overexpressing EGFR and p185neu (37). Our
data suggested that EGFR and either p185neu or
TAPstop associated into an active kinase complex and that these
receptor assemblies exhibited comparable kinetics of receptor endocytosis.
However, co-expression of T691stop with EGFR resulted in diminished
EGF-induced down-regulation of EGFR. The maximal reduction of surface
EGF receptor was ~35% after 4 h. In addition, no detectable co-down-regulation of the cytoplasmic domain deleted T691stop was
observed in Er/T691stop cells (Fig. 4B), correlating with the observation of the inactive heterodimer of EGFR·T691stop (Fig. 3,
E and F). This finding supports the idea that
receptor down-regulation is coupled to receptor tyrosine kinase
activity. The formation of the inactive heterodimer between EGFR and
T691stop neu proteins influenced the overall kinetics of EGFR
down-regulation. Impairment of ligand-induced down-regulation of
holo-EGFR by T691stop neu has also been observed in primary human
cancer cells.2
Transforming Potency of Cells Expressing Mutant p185neu
Proteins with or without EGFR--
We and others have showed that the
transforming potency of p185neu requires not
only its intrinsic tyrosine kinase activity (13), but also the crucial
role of tyrosine phosphorylation of its carboxyl terminus, as the
oncogenicity of p185neu was greatly reduced by
alteration of several tyrosine residues (41) or large structural
deletions, such as seen with TAPstop (42). Transforming ability of
ectodomain-deleted T
5 in this system was less potent than
full-length p185neu, possibly due to the reduced
efficiency of forming active receptor complexes when compared with
full-length oncogenic p185neu (27) (see Fig.
2).
We examined whether co-expression of EGFR with
p185neu and its derivatives could enhance
transforming efficiency compared with cells expressing these mutant
p185neu proteins alone. Cell lines listed in
Table I (except kinase-deficient T691stop and Er/T691stop clones) were
able to form foci independent of ligand stimulation (data not shown).
Co-expression of EGFR with p185neu in Er/neu
cells increased the ability to form foci, both in density and absolute
number (by greater than 3-fold). However, co-expression of EGFR with
kinase-active truncated mutant TAPstop or T
5 did not enhance focus
formation efficiency in Er/TAPstop and Er/T
5 cells when compared
with TAP/stop and T
5 cells, respectively (data not shown).
The colony growth efficiency of these clones in soft agar is also
summarized in Table I. B104-1-1 cells expressing high levels of
p185neu served as a positive control, while
Er/T691stop clones served as a negative control and did not exhibit
transformed colonies under the same conditions. Compared with B104-1-1
cells, cells expressing lower levels of oncogenic
p185neu formed colonies less efficiently.
However, more colonies were observed in EGFR-co-expressing Er/neu
cells. Co-expression of EGFR with p185neu still
permits functional heterodimerization in addition to homodimerization of either receptor, resulting in elevated biological activity, contributing to increased transforming activity in vitro.
Cells expressing kinase-active truncated mutant TAPstop or T
5 mutant proteins alone displayed reduced colony growth efficiency in soft agar
when compared with control B104-1-1 cells, although the expression levels of p185neu variants in these cells were
similar. Critically, co-expression of EGFR with T
5 or TAPstop did
not increase colony growth efficiency in soft agar.
Tumorigenicity was studied by injection of these mutant clones
individually into athymic mice. Results are presented in Table I, which
summarizes receptor expression levels, tumor frequency, and tumor size.
B104-1-1 cells expressing oncogenic p185neu were
used as a positive control and tumors caused by those cells appeared
and grew quickly (with a latency of 5-7 days). No tumors were observed
with kinase-deficient mutant clones T691stop and Er/T691stop cells
(>10 weeks observation). Co-expression of EGFR and
p185neu, each at low levels, in Er/neu cells
greatly accelerated tumor appearance (~2 weeks), and the tumors grew
aggressively when compared with p185neu cells
that also expressed low level of oncogenic
p185neu (>4-5 weeks). Cooperative signaling
between EGFR and p185neu was thus also observed
in tumorigenicity assays in vivo. T
5 protein expression
was sufficient to induce tumors (latency period of 2-3 weeks), and
TAPstop mutant receptor expression also resulted in tumor formation
(latency of 4-5 weeks). Receptor expression levels for these two
mutant proteins was close to that in B104-1-1 cells. Notably,
co-expression of EGFR with these mutant proteins, i.e. in
Er/TAPstop and in Er/T
5, did not promote tumor growth.
The failure of distinct endodomain interactions between
p185neu and EGFR, caused by an ectodomain
deletion (T
5 mutant), or the lack of a functional COOH terminus
(TAPstop mutant), clearly impairs signaling needed for transformation.
EGF-dependent Cell Proliferation of Cell Lines
Co-expressing EGFR and Mutant p185neu--
To analyze
whether EGF-dependent heterodimerization conveys signals
leading to cooperative mitogenesis, we used the MTT assay to study
proliferation of various cell lines. NE91 cells expressing EGFR only
served as a positive control, and showed typical EGF induction of cell
growth. As expected, the maximal induction dosage of EGF was 10 ng/ml,
consistent with previous observations (44). However, the maximum
induction dosage of EGF in Er/neu cells was ~0.1 ng/ml, 2 orders of
magnitude less than that observed in NE91 cells (Fig.
5A). These data suggested that
p185neu sensitized the EGF receptor responding
to ligand.

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Fig. 5.
EGF-induced cell proliferation. Cells
were plated in 96-well plates (3000/well) overnight in DMEM containing
5% FBS. After starvation in serum-free media for 48 h, cells were
grown in the same media supplemented with various concentrations of EGF
as indicated for an additional 48-h period. Cell proliferation was
determined by the MTT assay as described under "Experimental
Procedures." The resultant OD600 was plotted against the
relevant EGF concentration. NE91 cells was used as a control for the
cell lines presented: A, Er/neu; B, Er/T691stop;
C, Er/TAPstop; D, Er/T 5.
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In contrast, the presence of T691stop in Er/T691stop cells suppressed
the proliferative response to EGF, and cell growth was dramatically
reduced (Fig. 5B). These results correlated with the
inhibition of EGFR kinase (Figs. 3E and 4B).
Interestingly, the EGFR in Er/TAPstop and Er/T
5 cells behaved
normally in EGF-dependent mitogenesis when compared with
that in NE91 cells, except that the basal growth level was higher (Fig.
5, C and D) due a more transformed phenotype
(data not shown). These data correlated with previous observations
(Figs. 2-4), suggesting that EGFR signaling is comparable in
Er/TAPstop and in Er/T
5 clones to that seen in NE91 cells,
i.e. neither enhanced nor suppressed. However, trans-receptor signaling was not observed due to either defective heterodimerization in Er/TAPstop cells or failure of heterodimerization in Er/T
5 cells.
EGF-dependent MAP Kinase and PI 3-Kinase
Activation--
To understand the mechanism underlying synergistic
proliferative and transforming signal propagated by heteroreceptor
interaction, we studied the EGF-induced MAP kinase and PI3 kinase
pathways signaling phenomena. The proto-oncogenic p185 (Nneu) and its
derivatives (N
5 or, N691stop) were co-expressed with EGFR, to
evaluate EGF-dependent activation of downstream kinases,
since p185neu and T
5 are both constitutively
active tyrosine kinases. An epitope-tagged HA-MAPK was also
co-expressed with the combination of receptors in COS7 cells to examine
downstream ERK activation.
Co-expression of p185c-neu, but not N
5, with
EGFR increased MAP kinase activity upon EGF stimulation. In contrast,
EGFR-mediated MAP kinase activity in N691stop-co-expressing cells was
suppressed when compared with cells expressing EGFR and an empty vector
control (Fig. 6A). Equivalent
protein expression levels of epitope-tagged HA-MAPK was also confirmed
in these studies (Fig. 6B). Ectopically expressed EGFR and
wild-type or mutant p185 forms were detected by immunoblot using
anti-receptor specific antisera (Fig. 6, C and
D). Since the intracellular domain-deleted N691stop could not be recognized by antiserum against the Nneu COOH terminus, the
expression of N691stop was independently confirmed using metabolic labeled cell extracts followed by anti-neu immunoprecipitation (Fig.
6D, lanes 7 and 8).

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Fig. 6.
EGF-induced MAP kinase activity. COS7
cells transiently expressing exogenous HA-MAPK, EGFR, and wild-type or
mutant p185c-neu were treated with or without
EGF (50 ng/ml) for 5 min as indicated. A, cells were then
lysed, and anti-HA immunocomplexes were washed and underwent kinase
reaction as described under "Experimental Procedures." The
phosphorylation level of myelin basic protein were shown after
autoradiography. B-D, equal amounts of cell extracts were
used for examining ectopically expressed proteins. Antibodies used in
immunoblot (IB) were indicated. Protein signals were
developed by ECL. Lanes 1-8 in these panels are
correspondent to those in panel A. D
(lanes 7 and 8), cells were
metabolically labeled with [35S]methionine and cell
extracts were immunoprecipitated (IP) with 7.16.4 and
analyzed in SDS-PAGE followed by autoradiography. Similar results were
obtained in other two independent experiments.
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Activation of PI-3-kinase requires phosphorylation of the Src homology
2-containing adapter p85 by receptor tyrosine kinases. Phosphatidylinositides are critical signaling intermediates and influence cell growth, differentiation, and adhesion (52). ErbB family
members, notably ErbB-3, have been shown to associate with the p85
subunit of PI 3-kinase (53). To examine the influence of wild-type or
mutant p185 on EGF-dependent activation of PI 3-kinase,
plasmids expressing EGFR with vector or p185 variants were transiently
expressed in COS7 cells. PI 3-kinase activity was examined in
serum-starved cells with or without EGF stimulation. We observed a
similar magnitude of the EGF-induced PI 3-kinase activity in cells
expressing EGFR only or Er/N
5. The PI 3-kinase activity was much
greater in Er/p185c-neu cells, and much weaker
in Er/N691stop cells (Fig. 7). Expression patterns of these receptor proteins were determined (Fig. 6,
C and D).

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Fig. 7.
EGF-induced PI 3-kinase activity. COS7
cells transiently expressing EGFR and wild-type or mutant
p185c-neu (as indicated) were treated with or
without EGF (50 ng/ml) for 5 min after serum starvation for 24 h.
Equal amounts of cell extracts were immunoprecipitated by anti-Tyr(P)
(4G10) and analyzed for PI 3-kinase activity as described under
"Experimental Procedures." Autoradiogram of thin layer
chromatography plate exposed overnight is shown. The positions of
origin (ori.), phosphatidylinositol 3-phosphate
(PIP), and phosphatidylinositol 3,4-bisphosphate
(PIP2) were indicated by arrows. Data shown are
representative of three individual experiments.
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The observed super PI 3-kinase activity in
Er/p185c-neu cells may arise through the
tyrosine phosphorylation of the p85 subunit by the heteroreceptor
complexes. We believe heteroreceptor complexes are more active since
truncated p185 proteins alone do not seem effective at interaction with
p85 (data not shown). Induced PI 3-kinase and MAPK activities therefore
paralleled the heterodimerization and trans-activation events depicted
in Figs. 2-4, and biological results obtained in Table I and Fig. 5.
Functional heterodimerization observed in Er/neu cells permits
cooperation and diversification of signaling, which contrasts with the
formation of signaling-defective complexes in Er/T691stop cells or the
failure of heterodimerization observed in Er/T
5 cells.
 |
DISCUSSION |
Using p185neu mutants, which retain the
capacity to homodimerize, we observed that EGF-induced
heterodimerization could occur. Heterodimerization was seen in cells
co-expressing EGFR with TAPstop or T691stop mutant receptors, but not
with the extracellular domain-deleted T
5 (Fig. 2), demonstrating
that the ectodomain of p185neu is necessary and
sufficient for heterodimerization with EGFR. Indeed, heterodimerization
of the EGFR and N691stop form derived from proto-oncogenic
p185neu has been observed to occur
preferentially to either p185c-neu or
EGFR·EGFR homodimerization (37).
Two alternative assays confirmed trans-activation of ErbB family
proteins following heterodimer formation. Anti-phosphotyrosine blotting
showed that enhancement of tyrosine phosphorylation in response to EGF
occurred only in cells co-expressing EGFR with the full-length
p185neu kinase, but not with the TAPstop or
T
5 mutant receptors. It appears that EGFR and the T691stop neu
mutant formed a kinase-inactive complex (Fig. 3), as described
previously for the N691stop form (37).
An analysis of EGF-induced receptor internalization, a
kinase-dependent event, also indicated that receptor
trans-activation is required for efficient internalization of the EGFR
found in these heteromers (Fig. 4). Full-length
p185neu and TAPstop proteins were modulated by
EGF and showed co-internalization with EGFR efficiently, indicating an
active heterodimer was formed. Co-expression of T
5 with EGFR did not
affect normal endocytosis of EGFR since T
5 could not associate with
EGFR, while T691stop neu expression interfered with normal EGFR
down-modulation.
Recent studies have shown that the signal adapter Grb2 is required for
efficient endocytosis of EGFR (54), and selective and regulated signal
transduction from activated receptor tyrosine kinases may continue
within the endosome (55). Interestingly, kinase-mediated activation of
ERKs may also involve endocytotic trafficking since inhibition of
clathrin-mediated endocytosis has been shown to impair rapid
EGF-stimulated activation of ERKs (56). Therefore, it is reasonable to
speculate that EGF-induced endocytosis of these receptor complexes
reflects both heterodimeric kinase activity and the efficiency of
activating downstream signaling components. Indeed, full-length
p185neu, but not other
p185neu-derived deletion mutants, displayed
increased coupling of the Src homology 2-containing signaling molecule
p85 to receptor activation (data not shown).
We previously reported that the co-expression of EGFR with
p185c-neu (22), but not with kinase-deficient
p185c-neu (44), synergistically transformed
rodent fibroblasts. EGFR and p185c-neu
associates into an active kinase complex (24) which up-regulates EGF
receptor function by increasing EGF binding affinity, ligand-induced DNA synthesis, and cell proliferation (23). In the current studies, when EGFR was co-expressed with oncogenic
p185neu at physiological levels
(~104 receptors/cell), we also observed enhancement of
tumor growth (4-fold) in vivo and anchorage-independent
growth (~2-fold) in vitro, compared with the cells
expressing p185neu alone (Table I). Deletion of
122 amino acid residues from the carboxyl terminus of
p185neu eliminates three known tyrosine
autophosphorylation sites (TAPstop mutant), and causes impaired
cellular signaling and transforming potency (41). Overexpression of
EGFR with the carboxyl-terminally truncated TAPstop mutant receptor,
although leading to an active heterodimeric complex, did not recover
the diminished transforming potency of TAPstop (Table I), indicating
that signaling propagation through the carboxyl terminus of
p185neu could not be restored by association
with full-length EGFR. These data emphasize that cooperative signaling
requires not only the formation of an active kinase complex, but also a
heteromeric functional carboxyl termini within the two receptor
endodomains that recruit various downstream molecules required to
generate signal to mediate cell growth and transformation.
The current results indicate that p185neu·EGFR
heterodimerization is greatly favored, even in the presence of the neu
transmembrane point mutation that facilitates
p185neu homodimerization (12). Together with the
observation that ErbB-2 is the preferred heterodimerization partner of
all ErbB members (36), these studies emphasize that Neu·ErbB-2 may
mediate signaling diversity through structural interactions governed by
particular ectodomain sequences. For instance, ErbB-3 is a less active
kinase than other ErbB proteins (57), but serves as a binding site for
Neu differentiating factor (28) and forms a potent heterodimer with
ErbB-2, consequently engaging various downstream substrates. Neu·ErbB-2 may not be required for ligand binding, but may
reconstitute signaling by laterally engaging other ErbB proteins in
some preferred, but not well understood manner.
Kinase phosphorylation increases the affinity of binding of Src
homology 2 and Src homology 3 domain-containing substrates, and
initiates a variety of cascades. The binding of Grb2·Sos complexes to
the active EGFR activates the Ras/Raf/MAP kinase cascade (58). Another
downstream effector whose importance in cell signaling and,
potentially, in tumorigenesis is becoming increasingly understood is PI
3-kinase (52). PI 3-kinase activation has also been shown to be
essential for induction of DNA synthesis by EGF (59). Current studies
have demonstrated that EGF-induced ErbB heterodimers activate both the
ERK and PI 3-kinase pathways. Functional wild-type heterodimers, but
not defective mutant heterodimers, efficiently induce both ERK and PI
3-kinase activities, which contribute to the synergistic effects on
mitogenesis and cellular transformation.
As depicted in Fig. 8, these results
further support the notion that cooperative signaling caused by
p185neu·EGF receptor ensembles requires the
ectodomain for ligand-mediated physical association, while the
intracellular domain provides contacts for efficient intermolecular
kinase activation. The phosphorylated carboxyl terminus is essential
for recruiting particular cellular substrates required for signal
diversification.

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Fig. 8.
The role of p185neu
subdomains in heterodimerization with EGFR and resultant
signaling consequences. Functional heterodimerization requires the
ectodomain for ligand-mediated physical associations, the endodomain
for kinase transactivation, and the carboxyl terminus for
cross-phosphorylation and combinatorial cellular signaling. Deletion of
each subdomain results in inefficient heterodimerization, preventing
kinase activation and defects in cooperative cellular signaling,
respectively. TD5, T 5.
|
|
In particular, specific ectodomain associations may therefore underlie
the combinatorial interactions within the ErbB family required for
signal diversification. These properties may be features that are used
by many receptor ensembles involved in enzymatic signaling in cells.
 |
FOOTNOTES |
*
This work was supported by a National Research Service Award
(to X. Q.); by grants from the Veterans Administration Merit Review Program, the Lucille Markey Charitable Trust, and the American Cancer Society (IRG-135P) (to D. M. O.); and by grants from
National Cancer Institute, the Lucille Markey Charitable Trust, the
United States Army, American Cancer Society, and the Abramson Institute for Cancer Research (to M. I. G.).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.
§
Present address: Laboratory of Cellular Oncology, NCI, National
Institutes of Health, Bethesda, MD 20892.
Present address: Cardiology Branch, NHLBI, National Institutes
of Health, Bethesda, MD 20892.

To whom correspondence should be addressed: 252 John Morgan
Bldg., Dept. of Pathology and Laboratory Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-898-2868; Fax: 215-898-2401; E-mail: greene{at}reo.med.upenn.edu.
The abbreviations used are:
EGFR, epidermal
growth factor receptor; EGF, epidermal growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; HA, hemagglutinin; PAGE, polyacrylamide
gel electrophoresis; MAP, mitogen-activated protein; MAPK, MAP kinase; mAb, monoclonal antibody; aa, amino acid(s); DMEM, Dulbecco's modified
Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered
saline; MTT, 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide); ERK, extracellular signal-regulated kinase; BS3, bis(sulfosuccinimidyl) suberate.
2
D. M. O'Rourke and M. I. Greene,
unpublished observations.
 |
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