Cripto-1 Indirectly Stimulates the Tyrosine Phosphorylation of
erb B-4 through a Novel Receptor*
Caterina
Bianco
,
Subha
Kannan§,
Marta
De Santis
,
Masaharu
Seno¶,
Careen K.
Tang
,
Isabel
Martinez-Lacaci
,
Nancy
Kim
,
Brenda
Wallace-Jones
,
Marc E.
Lippman
,
Andreas D.
Ebert
**,
Christian
Wechselberger
, and
David S.
Salomon

From the
Tumor Growth Factor Section, Laboratory of
Tumor Immunology and Biology, NCI, National Institutes of Health,
Bethesda, Maryland 20892, § MacMaster University, Hamilton,
Ontario, L8S 4K1 Canada, ¶ Department of Bioscience and
Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530 Japan, and
Lombardi Cancer
Center, Department of Biochemistry, Georgetown University Medical
Center, Washington, D. C. 20007-2197
 |
ABSTRACT |
Cripto-1 (CR-1) is a recently discovered protein
of the epidermal growth factor family that fails to directly bind to
any of the four known erb B type 1 receptor tyrosine
kinases. The present study demonstrates that CR-1 indirectly induces
tyrosine phosphorylation of erb B-4 but not of the
epidermal growth factor-related receptors erb B-2 and
erb B-3 in different mouse and human mammary epithelial
cell lines. In addition, down-regulation of erb B-4 in
NMuMG mouse mammary epithelial cells and in T47D human breast cancer
cells, using an anti-erb B-4 blocking antibody or a
hammerhead ribozyme vector targeted to erb B-4 mRNA,
impairs the ability of CR-1 to fully activate mitogen-activated protein
kinase. Finally, chemical cross-linking of 125I-CR-1 to
mouse and human mammary epithelial cell membranes results in the
labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor
structurally unrelated to any of the known type I receptor tyrosine
kinases. In conclusion, these data demonstrate that CR-1, upon binding
to an unknown receptor, can enhance the tyrosine kinase activity of
erb B-4 and that a functional erb B-4 receptor
is required for CR-1-induced MAPK activation.
 |
INTRODUCTION |
The human CR-1 gene encodes for an
EGF1-related peptide that was
isolated and sequenced from a human NTERA2/D1 embryonal carcinoma cDNA expression library (1). A homologous gene has also been identified in the mouse from an F-9 mouse embryonal carcinoma cDNA
expression library (2). More recently, additional Cripto-related genes
have been identified in Xenopus laevis
(FRL1), in mouse embryonic stem cell-derived mesoderm cells
(Cryptic), and in Zebrafish (One-eyed pinhead)
(3-5). Based on the strong sequence similarities, CR-1, FRL1, Cryptic,
and One-eyed pinhead represent a new family of growth factor-like
molecules named CFC (CR-1, FRL1, and Cryptic) family. These genes share
a potential N-terminal leader sequence, a modified EGF-like domain, a
second cysteine-rich region, and a C-terminal hydrophobic domain (5,
6). The modified EGF-like domain found in the CFC family is highly
conserved. All EGF-like motifs contain six cysteines, which form three
disulfide bonds in the case of EGF and the other related peptides in
this family. In CR-1, FRL1, Cryptic, and One-eyed pinhead, the EGF-like
domain is quite unusual. In fact, the first two cysteines are adjacent, thereby eliminating the A loop that is normally found between these
residues. In addition, the spacing between the third and fourth
cysteines is reduced relative to other EGF-like repeats, resulting in a
smaller B loop (6). Because these three disulfide-linked loops are
important in the ability of these proteins to assume a specific
secondary structure that is essential for binding to specific
erb B type I receptor tyrosine kinases, it has been proposed that the presence of an unusual EGF-like domain in the CFC family may
indicate that this subfamily of peptides binds to a unique receptor. In
fact, we have previously shown that CR-1 does not bind directly to the
epidermal growth factor receptor (EGFR/erb B-1) or to
erb B-2, erb B-3, or erb B-4 type 1 receptor tyrosine kinases that have been ectopically expressed in Ba/F3
mouse pro-B lymphocytes or in 32-D mouse myeloid cells either alone or
in various pairwise combinations (7). CR-1 binds to a high affinity, saturable receptor in HC-11 mouse mammary epithelial cells and in
several different human breast cancer cell lines. This receptor is
specific for CR-1, because it does not bind other EGF-related peptides,
such as EGF, heparin binding EGF-like growth factor, transforming
growth factor
, amphiregulin, betacellulin, or heregulin
1
(HRG
1) (7).
The erb B family is characterized by extensive
receptor-receptor interactions leading to an enormous degree of signal
diversification through ligand-activated dimerization (8, 9). By
binding to the extracellular domain of their respective receptors,
EGF-related peptides induce receptor homodimerization and the
subsequent stimulation of the intrinsic tyrosine kinase, which leads to
the phosphorylation of specific tyrosine residues in the intracellular
domain of the receptor (10). This process generates docking sites for
cytoplasmic signaling molecules, such as the adaptor proteins Shc and
Grb2 and the p85 subunit of the phosphatidylinositol 3-kinase, which link receptor tyrosine kinases to intracellular signal transduction pathways (9). Moreover, ligand binding induces not only receptor homodimers but also heterodimers between different erb B
receptors. The first evidence for the ability of these receptors to
form heterodimers was derived from the observation that erb
B-2 undergoes transactivation and phosphorylation by both EGF and
neu differentiation factor/heregulins, which do not bind
directly to this receptor but bind to either the EGFR or erb
B-3 or erb B-4 receptors, respectively (11-13). In
addition, erb B-2 appears to be the preferred
heterodimerization partner with the other erb B molecules
(14). Another example of heterodimerization is the interaction with
erb B-3. Erb B-3 has an impaired tyrosine kinase
activity, but it can undergo transactivation and phosphorylation by
other erb B members (15, 16). The extensive receptor
interactions within the erb B family raise the possibility that although CR-1 does not directly bind to any of the erb
B receptors, it may be able to transactivate members of the
erb B receptor family by triggering heterodimerization and
transphosphorylation through another receptor. In the present study, we
demonstrate that CR-1 specifically activates erb B-4 in
several mouse and human mammary epithelial cell lines, whereas it does
not increase the tyrosine phosphorylation of the other three
erb B members. Furthermore, down-regulation of
erb B-4 expression in T47D human breast carcinoma cells
expressing a specific hammerhead ribozyme targeted to erb
B-4 mRNA results in a reduction in the ability of CR-1 to fully
activate MAPK. In addition, a specific erb B-4-blocking antibody can also interfere with the capacity of CR-1 to activate MAPK
in mouse and human mammary epithelial cell lines. These data suggest
that erb B-4, although not a direct receptor for this growth
factor, is required for the activation of the signal transduction cascade by CR-1. Finally, chemical cross-linking of
125I-CR-1 to several cell lines identifies a novel receptor
with properties that are different from the other erb B
receptors. In fact, the presence of two specific bands of 60 and 130 kDa suggests the possibility that this receptor has a multicomponent structure that renders the CR-1 receptor unique among the other erb B receptors.
 |
MATERIALS AND METHODS |
Cell Culture and Growth Factors--
NMuMG mouse mammary
epithelial cells, human breast carcinoma cell lines, MDA-MB-453, T47D,
and SKBr-3, the human glioblastoma cell line A172, and NIH 3T3 cells
overexpressing erb B-4 (kindly provided by Dr. C. Arteaga,
Vanderbilt University, Nashville, TN) were cultured in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. The T47D
cells transfected with the ribozyme vector (Rz6) targeted to
erb B-4 mRNA were generated as described previously
(17). Recombinant human CR-1 protein was expressed in Escherichia
coli and purified as described previously (18). Recombinant human
EGF and HRG
1 were purchased from Collaborative Research and R & D
Systems, respectively.
Immunoprecipitation and Western Blotting--
Cells were grown
until they reached 70-80% confluence and then switched to serum-free
Dulbecco's modified Eagle's medium containing human transferrin (10 µg/ml) and type IV Pedersen fetuin (1 mg/ml) for 24 h. Cells
were treated with EGF, HRG
1, or CR-1 at 100 ng/ml for various times.
To block erb B-4 activation, the cells were pretreated for
30 min with a mouse monoclonal anti-erb B-4 blocking antibody (Neo marker Ab-3) and then stimulated with HRG
1 or CR-1 for
5 min. The cells were lysed in a buffer containing 20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 5 mM MgCl2, 2 µg/ml aprotinin,
2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 20 mM sodium
fluoride. Lysates were immunoprecipitated (0.5 mg/sample) with the
following antibodies: rabbit polyclonal anti-EGFR antibody (1005, Santa Cruz), rabbit polyclonal anti-erb B-2 antibody (neu C-18,
Santa Cruz), rabbit polyclonal anti-erb B-3 antibody (C-17,
Santa Cruz), or rabbit polyclonal anti-erb B-4 antibody (Neo
marker Ab-1). Crude protein lysates (30 µg/sample) or
immunoprecipitates were separated by SDS-PAGE, transferred to
nitrocellulose, blocked in 5% dry milk in 20 mM
Tris-buffered saline with 0.05% Tween 20 and incubated overnight with
one of the following antibodies: 1:1000 dilution of
anti-phosphotyrosine mouse monoclonal antibody PY20 (Transduction
Laboratories), 1:1000 dilution of a rabbit polyclonal anti-phospho MAPK
antibody (Biolab Laboratories), or a 1:1000 dilution of rabbit
polyclonal anti-EGFR antibody (1005, Santa Cruz), rabbit polyclonal
anti-erb B-2 (neu C-18, Santa Cruz), rabbit polyclonal
anti-erb B-3 antibody (C-17, Santa Cruz), or rabbit
polyclonal anti-erb B4 antibody (C-18, Santa Cruz). The bound rabbit or mouse antibodies were detected using a 1:2000 dilution
of either goat-anti-rabbit or goat-anti-mouse IgG conjugated to
horseradish peroxidase (Amersham Pharmacia Biotech). Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). Densitometric analysis was performed using NIH Image program
1.58.
Chemical Cross-linking--
125I-CR-1 protein was
prepared using IODOGEN (Pierce) as described previously (7). Cell
membranes were prepared from NMuMG or MDA-MB-453 cells after lysis in
hypotonic buffer containing 20 mmol/liter HEPES, pH 7.4, 1 mmol/liter
EGTA, 1 mmol/liter MgCl2, 1 mmol/liter sodium
orthovanadate, 1 mmol/liter phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin, pepstatin, and leupeptin. Lysates were centrifuged
at 1,000 × g for 10 min at 4 °C. After discarding the pellet, 1% Triton X-100 was added, and the membrane fraction was
isolated by centrifuging the samples at 100,000 × g
for 30 min at 4 °C. Cell membranes were incubated at room
temperature for 2 h with 125I-CR-1 (1 × 106 cpm) in the absence or in the presence of an excess of
unlabeled CR-1 protein (1 µg). The chemical cross-link reagent
bis(sulfosuccinimdyl)suberate (BS3 (Pierce)) was then added
(1 mM) for 30 min on ice. Cell membranes were lysed and
resolved on a 6% SDS-PAGE gel or 4-12% SDS-PAGE gradient gel.
Cross-linked bands were visualized by autoradiography.
 |
RESULTS |
CR-1 Specifically Stimulates Tyrosine Phosphorylation of erb
B-4--
We have previously shown that CR-1 does not directly activate
any of the four known erb B receptor tyrosine kinases that
have been ectopically expressed in Ba/F3 pro-B lymphocyte cells or in
32-D mouse myeloid cells, either alone or in various pairwise combinations (7). These results do not formally exclude the possibility
that the CR-1 receptor cannot heterodimerize with or indirectly
transactivate one or several of the type I receptor tyrosine kinases.
In fact, the generation of heterodimeric receptor complexes represents
a model for signal diversification and amplification in response to
different EGF-like type I receptor ligands (19, 20). NMuMG mouse
mammary epithelial cells express moderate levels of all four
erb B receptors and mitogenically respond to CR-1 with an
increase in the phosphorylation of Shc and MAPK (18). Therefore, the
effect of CR-1 on the tyrosine phosphorylation of the EGFR,
erb B-2, erb B-3, and erb B-4 was
examined in NMuMG cells. Treatment of serum-starved NMuMG cells with
CR-1 resulted in a rapid tyrosine phosphorylation of erb B-4
but not of the EGFR, erb B-2, or erb B-3, as
determined by immunoprecipitation with receptor monospecific polyclonal
antibodies and Western blot analysis with anti-phosphotyrosine antibody
PY20 (Fig. 1). A nearly 1.5-fold increase
in phosphorylation of erb B-4 was observed within 5 min of
stimulation. As an internal control, treatment with EGF was found to
stimulate a 3-fold increase in the tyrosine phosphorylation of the
EGFR, and HRG
1 induced a 4-fold increase in the phosphorylation of
erb B-2, a 0.8-fold increase in the phosphorylation of
erb B-3, and a 2-fold increase in the phosphorylation of
erb B-4, indicating that the other three erb B
receptors are functionally able to respond to ligand stimulation in
these cells. Each Western blot was stripped and reprobed with the
respective anti-erb B receptor antibodies to ensure that
equal amounts of the immunoprecipitates had been loaded (Fig. 1).

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Fig. 1.
Modulation of erb B-4
receptor tyrosine phosphorylation by CR-1 in NMuMG mouse mammary
epithelial cells. Serum-starved NMuMG cells were treated without
or with EGF (100 ng/ml), HRG 1 (100 ng/ml), or CR-1 (100 ng/ml) for 5 min. Cell lysates were immunoprecipitated (IP) with
anti-EGFR antibody (A), anti-erb B-2 antibody
(B), anti-erb B-3 antibody (C), or
anti-erb B-4 antibody (D) and analyzed by Western
blot (WB) analysis with anti-phosphotyrosine antibody PY20
(Transduction Laboratories). The same blots were stripped and reprobed
with the monospecific anti-erb B receptor antibodies to
demonstrate that equal amounts of immunoprecipitates were present in
all lanes.
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To exclude the possibility that this phenomenon might be unique to
NMuMG cells, several additional cell lines were tested. CR-1
stimulation of erb B-4 tyrosine phosphorylation was observed in the human breast cancer cell line, MDA-MB-453. Activation of the
erb B-4 receptor was observed at 3 min, with a 2-fold
increase in phosphorylation of erb B-4, which remained high
even after 10 min of stimulation (Fig.
2). HRG
1 induced a 3-fold increase of
the tyrosine phosphorylation of erb B-4. No activation of
the other three erb B receptors could be detected. To
determine whether functional erb B receptors were expressed
in these cells, MDA-MB-453 cells were treated with either EGF or
HRG
1. EGF was found to stimulate a 3-fold increase in the tyrosine
phosphorylation of the EGFR (21), and HRG
1 could enhance a 2.2-fold
erb B-2 and 3.1-fold erb B-3 increase in tyrosine
phosphorylation. Western blot analysis with monospecific anti-receptor
antibodies showed that equal amounts of protein were loaded in the
various experiments (Fig. 2). A 2-fold increase of erb B-4
phosphorylation by CR-1 could also be observed in another human breast
cancer cell line, SKBr-3 (Fig.
3A), which expresses very low
levels of this receptor (21), and in A172 human glioblastoma cells
(Fig. 3B). The effect of CR-1 on the tyrosine
phosphorylation of erb B-4 in NIH 3T3 cells transfected with
an expression plasmid encoding for erb B-4 (NIH 3T3
erb B-4) was then ascertained (22). NIH 3T3 parental cells
have no detectable erb B-4 expression, whereas the
transfected cells express high levels of erb B-4 as
determined by immunoprecipitation and Western blot using an
anti-erb B-4 antibody (Fig.
4A). CR-1 induced a 4-fold
increase in the tyrosine phosphorylation of erb B-4 in NIH
3T3 erb B-4 cells, as does the natural ligand of this receptor, HRG
1 (Fig. 4B). It has previously been
demonstrated that CR-1 does not bind directly to erb B-4.
Therefore, the CR-1-induced tyrosine phosphorylation of erb
B-4 is mediated by an additional receptor for CR-1 or a tyrosine kinase
in trans. We have been unable to demonstrate a physical
association between erb B-4 and this receptor. Attempts to
chemically cross-link recombinant E. coli-derived
125I-CR-1 protein followed by immunoprecipitation with
several different anti-erb B-4 antibodies have proven
unsuccessful in identifying a specific binding component for this
protein.

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Fig. 2.
Modulation of erb B-4
receptor tyrosine phosphorylation by CR-1 in MDA-MB-453 human breast
cancer cells. Serum-starved MDA-MB-453 cells were treated without
or with 100 ng/ml of EGF, HRG 1, or CR-1 for 5 min. Cell lysates were
immunoprecipitated (IP) with anti-EGFR antibody
(A), anti-erb B-2 antibody (B),
anti-erb B-3 antibody (C), or anti-erb
B-4 antibody (D) and analyzed by Western blot
(WB) analysis with anti-phosphotyrosine antibody PY20
(Transduction Laboratories). The same blots were stripped and reprobed
with the monospecific anti-erb B receptor antibodies.
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Fig. 3.
Erb B-4 phosphorylation by CR-1 in SKBr-3
human breast cancer cells and in A172 glioblastoma cells.
Serum-starved SKBr-3 (A) and A172 (B) cells were
treated without or with 100 ng/ml CR-1 or HRG 1 for 5 min. Cell
lysates were immunoprecipitated (IP) with
anti-erb B-4 antibody, and Western blot (WB)
analysis was performed using anti-phosphotyrosine antibody PY20.
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Fig. 4.
Tyrosine phosphorylation of erb
B-4 by CR-1 in NIH 3T3 erb B-4 cells. In
A, NIH 3T3 and NIH 3T3 erb B-4 cell lysates were
run on 4-12% SDS-PAGE gradient gel and probed with an
anti-erb B-4 antibody (C-17 Santa Cruz). In B,
NIH 3T3 erb B-4 cells were stimulated without or with 100 ng/ml HRG 1 or CR-1 for 5 min. Cell lysates were immunoprecipitated
(IP) with anti-erb B-4 antibody (Neo marker
AB-1), and Western blot (WB) analysis was performed using an
anti-phosphotyrosine antibody PY20.
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Inactivation of erb B-4 Blocks CR-1-induced MAPK
Activation--
The effect of either a blocking anti-erb
B-4 antibody or a hammerhead ribozyme vector directed to erb
B-4 mRNA was tested for the ability to potentially inhibit MAPK
activation that was induced by CR-1 in T47D human breast carcinoma
cells and in NMuMG normal mouse mammary epithelial cells. CR-1 can
function through a receptor that activates intracellular components in
the Ras/Raf/MAPK pathway (7). In fact, treatment of several mouse and
human mammary epithelial cell lines with CR-1 can lead to a rapid
increase in the tyrosine phosphorylation of the p66, p52, and p46
isoforms of Shc, which can then associate with the Grb2-mSOS-signaling complex. CR-1 can then subsequently activate MAPK by rapidly inducing the phosphorylation of p42 and p44 isoforms of MAPK (7, 18). Because
erb B-4 is specifically tyrosine-phosphorylated in response to CR-1, we investigated whether MAPK activation by CR-1 requires prior
erb B-4 activation. We used two different approaches to inactivate erb B-4. First a monoclonal blocking
anti-erb B-4 antibody that is able to block the ability of
HRG
1 to bind to its receptor and that impairs erb B-4
dimerization was tested in NMuMG cells and in T47D human breast
carcinoma cells to evaluate its effect on CR-1-stimulated MAPK
activity. Pretreatment with the blocking anti-erb B-4
antibody in both cell lines reduced by 50% the phosphorylation of MAPK
induced by CR-1 (Fig. 5). As expected,
the blocking anti-erb B-4 antibody impairs the ability of
HRG
1 to activate MAPK in NMuMG cells with a 40% reduction of MAPK
phosphorylation observed. However, treatment with the blocking
anti-erb B-4 antibody does not significantly interfere with
the activation of MAPK by HRG
1 in T47D cells. However, in T47D
cells, pretreatment with the blocking anti-erb B-4 antibody
was able to reduce by 25% or by 70% erb B-4 receptor
tyrosine phosphorylation that was induced by HRG
1 or CR-1,
respectively (Fig. 6). The second
strategy was to inactivate erb B-4 by utilizing T47D breast
cancer cells that had been transfected with a specific hammerhead
ribozyme targeted to erb B-4 mRNA. Expression of this
functional erb B-4 ribozyme in T47D breast carcinoma cells
leads to a 70% down-regulation of erb B-4 expression as
determined by fluorescence-activated cell sorter analysis, a decrease
in erb B-4 mRNA levels, and a significant reduction in
anchorage-independent colony formation (17). CR-1 is able to activate
MAPK in the parental T47D cell line, with 2.4-fold increase of MAPK
phosphorylation, whereas HRG
1 induced a 3.3-fold increase in the
phosphorylation of MAPK (Fig. 7). In the
erb B-4 ribozyme expressing T47D cells, a 50% reduction in
MAPK activation could be detected following CR-1 treatment (Fig. 7). In
contrast, there was no reduction of MAPK activation after treatment
with HRG
1 in the T47D cells expressing the specific
anti-erb B4 ribozyme.

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Fig. 5.
MAPK phosphorylation in NMuMG and T47D cells
treated with a blocking anti-erb B-4 antibody.
Serum-starved NMuMG (A) and T47D (B) cells were
stimulated for 5 min with HRG 1 (100 ng/ml) or CR-1 (100 ng/ml) or
pretreated for 30 min with a blocking anti erb B-4 antibody
( erb B-4) (Neo marker AB-3) (5 µg/ml) and then
stimulated with HRG 1 or CR-1. Cell lysates were run on a 10%
SDS-PAGE gel and probed with an anti-phospho MAPK antibody (Biolab
Laboratories) that recognizes the activated phosphorylated forms of
MAPK (p44 and p42).
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Fig. 6.
Inhibition of erb B-4
phosphorylation by HRG 1 and CR-1 in the
presence of a blocking anti-erb B-4 antibody in T47D
cells. Serum-starved T47D cells were stimulated for 5 min with 100 ng/ml HRG 1 or CR-1 for 5 min or pretreated with a blocking
anti-erb B-4 antibody ( erb B-4) (Neo marker
AB-3) (5 µg/ml) for 30 min and then stimulated with the growth
factors. Cell lysates were immunoprecipitated (IP) with an
anti-erb B-4 antibody (Neo marker AB-1), run on a 6%
SDS-PAGE gel, and probed with anti-phosphotyrosine antibody PY20.
WB, Western blot.
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Fig. 7.
MAPK phosphorylation in T47D cells and in
T47D expressing a specific hammerhead ribozyme targeted to
erb B-4 mRNA. Serum-starved T47D cells (first
three lanes) and erb B-4 ribozyme-expressing T47D
cells (last three lanes) were stimulated for 5 min with
HRG 1 or CR-1 at 100 ng/ml. Cell lysates were run on a 10% SDS-PAGE
gel and probed with an anti-phospho MAPK antibody (Biolab Laboratories)
that recognizes p44- and p42-phosphorylated forms of MAPK.
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Chemical Cross-linking of CR-1 to Mammary Epithelial
Cells--
Chemical cross-linking was utilized to identify and
characterize a CR-1 binding component. Chemical cross-linking of
125I-CR-1 to NMuMG and MDA-MB-453 cell membranes with
BS3 results in the specific labeling of two bands at 150 and 80 kDa (Fig. 8). These two species do
not resemble any of the erb B receptors in size, because the
molecular weight of the erb B receptors range from 170 to
185 kDa. In fact, the two bands identified as possible binding
components of CR-1 have an expected size of 130 and 60 kDa, after
accounting for the size of bound iodinated CR-1 protein. This result
demonstrates that, although CR-1 is an EGF-related peptide, the
receptor for this growth factor seems to be quite different from the
other erb B receptors.

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Fig. 8.
Chemical cross-linking of
125I-CR-1 to NMuMG and MDA-MB-453 cell membranes.
NMuMG and MDA-MB-453 cell membranes were incubated with
125I-CR-1 in the presence or absence of cold CR-1 (1 µg)
for 2 h at room temperature followed by cross-linking with
BS3. The proteins were run on a 4% SDS-PAGE gel
(A) or on a 4-12% SDS-PAGE gradient gel (B).
Lane 1, NMuMG cross-linked to 125I-CR-1.
Lane 2, NMuMG cross-linked to 125I-CR-1 in the
presence of an excess of cold CR-1 (1 µg). Lane 3,
MDA-MB-453 cross-linked to 125I-CR-1. Lane 4,
MDA-MB-453 cross-linked to 125I-CR-1 in the presence of an
excess of cold CR-1 (1 µg).
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 |
DISCUSSION |
Cripto-related proteins including FRL-1 in Xenopus,
One-eyed pinhead in Zebrafish, and Cryptic in the mouse possess a
modified EGF-like motif. This modified EGF motif is unique and
consequently distinguishes these proteins from other classical EGF-like
peptide growth factors, because the modified EGF-like motif in the
Cripto-related proteins lacks an A loop, possesses a truncated B loop,
and has a complete C loop. This suggests that peptides that contain
this modified EGF-like domain probably do not directly bind with high affinity to any of the known type I erb B receptor tyrosine
kinases, because conserved amino acid residues in the A loop of the
N-terminal portion are obligatory for high affinity receptor binding of
either HRG, EGF, or transforming growth factor
to either
erb B-3, erb B-4, or the EGFR, respectively (23,
24). This is the case because in mouse Ba/F3 lymphoid cells or in 32-D
myeloid cells that are ectopically expressing different members of the
erb B tyrosine kinase family either individually or in
different pairwise combinations, CR-1 does not directly bind to any
homodimers or heterodimers within this family (7). However, the results
of the present study demonstrate that CR-1 can indirectly transactivate erb B-4 by enhancing tyrosine phosphorylation of this
receptor. CR-1 treatment increases the tyrosine phosphorylation of
erb B-4 in several mouse and human mammary epithelial cell
lines. This effect is specific for erb B-4 because CR-1 was
unable to enhance the tyrosine transphosphorylation of the EGFR,
erb B-2, or erb B-3. The transactivation of
erb B-4 by CR-1 apparently is not influenced by the level of
erb B-4 receptor expression because cell lines with high
density erb B-4 receptor expression (MDA-MB-453, T47D, and
NIH3T3 erb B-4 cells) as well as with low levels of erb B-4 expression (NMuMG and SKBr-3 cells) respond equally
to CR-1. The transactivation of erb B-4 by CR-1 may be
because of heterodimerization between the CR-1 receptor and
erb B-4 by the ability of CR-1 to bind with low affinity to
erb B-4. In this respect, EGF, heparin binding EGF-like
growth factor, and HRG can bind through their C terminus to a low
affinity site on erb B-2, whereas betacellulin and
epiregulin can bind to an analogous low affinity site on erb
B-4 even though these receptors are not the primary high affinity
receptors for these peptides (24-26). In fact, recent evidence
suggests that ligands in the EGF family are bivalent because a high
affinity binding site is present in the N-terminal A loop of these
peptides which can bind to the primary receptor and can then facilitate
through a low affinity binding site in the C-terminal loop weak binding
to a different secondary receptor, resulting in the simultaneous
binding of one ligand molecule to two different erb B
receptors in a 1:2 stoichiometry (26). Because binding to the secondary
receptor is generally 1000-fold weaker in affinity, attempts to
identify these partners through conventional cross-linking have been
limited, especially in circumstances where the primary receptor is
expressed at low to moderate levels (26). This may account for our
inability to potentially immunoprecipitate 125I-CR-1 that
has been chemically cross-linked to either NMuMG or MDA-MB-453 cells
using several different monoclonal or polyclonal anti-erb
B-4 antibodies. If a heterodimeric complex is formed between the CR-1
receptor and erb B-4, it is probably unstable. An
alternative explanation that could account for the ability of CR-1 to
stimulate erb B-4 tyrosine phosphorylation may be the capacity of CR-1 to indirectly stimulate a soluble src-like
or Jak-like cytoplasmic tyrosine kinase, which would then be capable of
stimulating erb B-4 tyrosine phosphorylation in
trans without physical association between these two receptors.
Irrespective as to the mechanism by which this tyrosine
transphosphorylation of erb B-4 occurs, we have demonstrated
that erb B-4 is an effective mediator of CR-1-induced MAPK
stimulation as assessed by phosphorylation of MAPK. Pretreatment of
cells with a blocking anti-erb B-4 antibody that interferes
with the binding of HRG
1 to erb B-4 and that can also
attenuate the interaction between erb B-4 and other
erb B-related tyrosine kinases is also able to significantly
inhibit the ability of CR-1 to stimulate MAPK phosphorylation. In
addition, down-regulation of erb B-4 expression in T47D
cells that are expressing a specific hammerhead ribozyme directed
against erb B-4 mRNA also impairs the ability of CR-1 to
activate MAPK. Surprisingly, inhibition of erb B-4 in T47D
cells using either a blocking anti-erb B-4 antibody or a
specific hammerhead ribozyme vector targeted to erb B-4
mRNA does not significantly interfere with the ability of HRG
1
to stimulate MAPK phosphorylation in these cells, as was observed in
the mouse NMuMG mammary epithelial cells. This anomaly is probably because of the fact that in T47D cells, erb B-3 and
erb B-2 are also expressed at elevated levels. Because
erb B-3 can also function as a viable receptor for HRG
1
through its heterodimerization with erb B-2 (14, 15), MAPK
activity could be unaffected in cells where erb B-4 activity
has been compromised (17). In addition, because of the relatively high
levels of erb B-4 expression in T47D cells, the
anti-erb B-4 blocking antibody was only partially capable of
muting HRG
1-induced tyrosine phosphorylation of erb B-4
in these cells. Nevertheless, these results demonstrate that erb B-4 is involved in mediating a signal transduction
pathway that is activated by CR-1 and that involves the downstream
activation of MAPK. This pathway may be the Ras/Raf/MAPK pathway,
because previous results have shown that CR-1 can stimulate the
tyrosine phosphorylation of Shc and the subsequent association of Shc
with Grb 2 and with SOS (7). In addition, impairment of p21ras activity can significantly inhibit the ability of CR-1 to modulate
-casein expression in response to lactogenic hormones in HC-11 mammary epithelial cells (27).
Transactivation of erb B-4 by an unrelated receptor that
binds CR-1 may not be an isolated phenomenon because transactivation of
the EGFR or erb B-2 has been demonstrated in response to
ligand stimulation of structurally unrelated receptors. For example, G-protein-coupled receptors such as the thrombin receptor may be
transactivated by the EGFR, and reciprocally, the EGFR may be
tyrosine-phosphorylated in response to thrombin stimulation through
src kinase (28, 29). In addition, functional
heterodimerization between different receptor families may also occur
that could account for interfamily receptor transactivation. In this
respect, the EGFR can form a complex with the platelet-derived growth
factor
receptor and can enhance the tyrosine phosphorylation of the platelet-derived growth factor
receptor after EGF treatment (30).
In addition, cytokine receptors such as the growth hormone and
prolactin receptors can increase the tyrosine phosphorylation of the
EGFR through Jak2 by forming a complex with the EGFR. (31, 32).
Recently, it has also been shown that interleukin-6 (IL-6) can enhance
the tyrosine phosphorylation of erb B-2 and erb
B-3 in a human prostate cancer cell line by inducing the formation of a
functional complex between the gp130 subunit of the IL-6 receptor and
erb B-2 (33). Inhibition of erb B-2 tyrosine
kinase activity using either a specific tryphostin inhibitor of the
erb B-2 kinase activity or by using a single chain
monoclonal antibody against erb B-2 that entraps
erb B-2 in the endoplasmic reticulum results in abrogation
of IL-6-induced MAPK stimulation, demonstrating that activation of MAPK
by IL-6 is dependent upon a functional erb B-2 tyrosine
kinase. Because erb B-4 and erb B-2 are not the receptors for CR-1 and IL-6, respectively, these data demonstrate that
these two erb B-related growth factor receptor tyrosine
kinases are essential components of a signal transduction pathway which involves MAPK that is activated by two unrelated peptides.
These present data may be biologically significant because
erb B-4 and CR-1 have a similar role during mouse embryonic
development. In fact, both erb B-4 and CR-1 are involved in
cardiac development because they are both expressed in the myocardium
of the developing heart (2, 34). Gene disruption of erb B-4
or CR-1 in knockout mice results in cardiac malformations because of
the aborted development of cardiac muscle (35). Homozygous embryos for
erb B-4 die in utero at embryonic day 10 because
of an absence of heart trabeculae. Similarly, gene disruption of CR-1
in embryonic stem cells by homologous recombination results in the
defective differentiation of cardiomyocytes because of the absence of
contractile muscle protein expression (36). In addition, embryos that
have had the CR-1 gene disrupted also die in utero because
of cardiac defects (37). Collectively, these data demonstrate that both
erb B-4 and CR-1 are involved in cardiac development,
supporting the present biochemical results of an interaction between
this growth factor and the erb B-4 receptor.
Chemical cross-linking of 125I-CR-1 to NMuMG and MDA-MB-453
cell membranes identified two specific bands at 130 and 60 kDa. These data demonstrate that the CR-1 receptor is different in size from the
other four known erb B receptors, which range in size from 170 to 185 kDa (8-15). We have previously demonstrated that CR-1 induces the tyrosine phosphorylation of two unknown proteins of 185 and
120 kDa in HC-11 mouse mammary epithelial cells and in MDA-MB-453 and
SKBr-3 human breast cancer cells (7). The phosphorylated band at 120 kDa may correspond to the 130-kDa band that can be cross-linked to
125I-CR-1, whereas the 185-kDa band may be erb
B-4. The other band at 60 kDa that was identified through chemical
cross-linking may represent an additional binding component of the CR-1
receptor. This band was not observed in the Western blots using an
anti-phosphotyrosine antibody, suggesting that it is not
tyrosine-phosphorylated in response to CR-1 treatment. The presence of
these two specific bands at a molecular weight that is unusual for the
other four erb B receptors renders the CR-1 receptor unique.
In fact, the presence of two binding components resembles some other
cytokine receptor families such as the
glycosyl-phospatidylinositol-linked cell surface receptor for glial
cell line-derived neurotrophic factor (GDNF). GDNF binds to a
multisubunit receptor complex in which the GDNF receptor
binds GDNF
and mediates the activation of the associated ret tyrosine
kinase (38). Interestingly, chemical cross-linking of
125I-GDNF to 293T rat retinal cells transiently expressing
GDNF receptor
identifies two species at 60 and 130 kDa (39).
In summary we have shown that CR-1, although it does not directly bind
to any of the type I receptor tyrosine kinases, does enhance the
specific tyrosine phosphorylation of erb B-4. Inhibition of
erb B-4 activity results in abrogation of CR-1-induced MAPK activation, demonstrating that erb B-4 is essential for
activation of MAPK by CR-1. Finally, the two binding components for
CR-1 that were observed after chemical cross-linking indicate the
presence of a potentially novel receptor for CR-1. Additional studies
are necessary to further characterize this receptor. Although the CR-1
receptor is different from the other erb B receptor in size and subunit composition, the transactivation of erb
B-4 by CR-1 demonstrates a functional link between the
erb B tyrosine kinase family and this unknown receptor.
 |
FOOTNOTES |
*
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.
**
Supported by DFG Grant Eb152/4-1.

To whom correspondence and reprint requests should be
addressed: Bldg. 10, Room 5B39, Tumor Growth Factor Section, Laboratory of Tumor Immunology and Biology, NCI, National Institute of Health, Bethesda, MD 20892. Tel.: 301-496-9536; Fax: 301-402-8656; E-mail: davetgfa{at}helix.nih.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
EGF, epidermal
growth factor;
EGFR, EGF receptor;
HRG
1, heregulin
1;
MAPK, mitogen-activated protein kinase;
PAGE, polyacrylamide gel
electrophoresis;
IL-6, interleukin 6;
GDNF, glial cell line-derived
neurotrophic factor.
 |
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