(Received for publication, June 19, 1996, and in revised form, November 7, 1996)
From the a Tumor Growth Factor Section, Laboratory of Tumor Immunology and Biology, NIC, National Institutes of Health, Bethesda, Maryland 20892, the b ICRF Oncology Unit, Hammersmith Hospital, London, United Kingdom, the c Department of Pathology, BML-350, Yale University School of Medicine, New Haven, Connecticut 06520-8023, the e Friedrich Meischer-Institut, CH-4002 Basel, Switzerland, the f Istituto Internazionale di Genetica e Biofisca, Naples, Italy, the g Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, D. C. 20007, the h Oncologia Sperimentale d Istituto Nazionale Per lo Studio e La Cura, Dei Tumori-Fondazione Pascale, Naples, Italy, and the j Cattedra di Oncologia Medica, II Facoltá di Medicina e Chirurgia, Universitá degli Studi di Napoli Federico II, 80131 Naples, Italy
Cripto-1 (CR-1), a recently discovered protein of
the epidermal growth factor (EGF) family, was found to interact with a
high affinity, saturable binding site(s) on HC-11 mouse mammary
epithelial cells and on several different human breast cancer cell
lines. This receptor exhibits specificity for CR-1, since other
EGF-related peptides including EGF, transforming growth factor ,
heparin-binding EGF-like growth factor, amphiregulin, epiregulin,
betacellulin, or heregulin
1 that bind to either the EGF receptor or
to other type 1 receptor tyrosine kinases such as erb B-3
or erb B-4 fail to compete for binding. Conversely, CR-1
was found not to directly bind to or to activate the tyrosine kinases
associated with the EGFR, erb B-2, erb B-3, or
erb B-4 either alone or in various pairwise combinations
which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte
cells. However, exogenous CR-1 could induce an increase in the tyrosine
phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5
min) increase in the tyrosine phosphorylation of the SH2-containing
adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11
epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer
cells. CR-1 was also found to promote an increase in the association of
the adaptor Grb2-guanine nucleotide exchange factor-mouse son of
sevenless (mSOS) signaling complex with tyrosine-phosphorylated
Shc in HC-11 cells. Finally, CR-1 was able to increase p42erk-2
mitogen-activated protein kinase (MAPK) activity in HC-11 cells within
5-10 min of treatment. These data demonstrate that CR-1 can function
through a receptor which activates intracellular components in the
ras/raf/MEK/MAPK pathway.
The human cripto-1 (CR-1)1 gene (also known as teratocarcinoma-derived growth factor-1 (TDGF-1)) is located on chromosome 3p21·3 and codes for a 28-36-kDa glycoprotein of 188 amino acids. CR-1 possesses an epidermal growth factor (EGF)-like consensus sequence that contains six cysteine residues in a region of approximately 37 amino acids (1). However, unlike other peptides within this family of growth factors that have a three-looped EGF-motif (designated A, B, and C) which are formed by three intramolecular disulfide bonds, the EGF-like repeat in CR-1 lacks an A loop and the B loop is truncated (2, 3). In addition, unlike most growth factors in the EGF family, the human CR-1 protein lacks a conventional hydrophobic signal peptide and a classical hydrophobic transmembrane domain (2). Nevertheless, recombinant human CR-1 protein is secreted when transiently expressed in Chinese hamster ovary cells (3). Refolded CR-1 peptides that correspond to the EGF-like repeat within the human CR-1 protein are mitogenic for nontransformed and malignant human and mouse mammary epithelial cells (3). Human CR-1 can also function as a dominant transforming gene in vitro in mouse NIH-3T3 fibroblasts and in mouse NOG-8 mammary epithelial cells (1, 4). CR-1 mRNA and immunoreactive CR-1 protein are differentially expressed in several human breast cancer cell lines, in approximately 80% of primary human infiltrating breast tumors and in 50% of ductal carcinomas in situ (5, 6).
Peptide growth factors that are members of the EGF family include
transforming growth factor (TGF
), amphiregulin (AR), heparin-binding EGF-like growth factor, betacellulin (BTC), epiregulin, and the neuregulin subfamily that consists of various isoforms of
and
heregulin (HRG), gilial cell growth factors, and acetycholine receptor inducing activity (7-10). The different neuregulins are derived by alternative splicing from a single gene. Peptides in the EGF
family bind to and activate members of the erb B family of
type 1 receptor tyrosine kinases. EGF, TGF
, AR, heparin-binding EGF-like growth factor, and epiregulin bind exclusively to the epidermal growth factor receptor (erb B/EGFR), whereas the
neuregulin subfamily of peptides bind to c-erb B-3/HER-3 or
c-erb B-4/HER-4 that can then heterodimerize and activate
c-erb B-2/HER-2 following transphosphorylation (11-15). BTC
can bind to either the EGFR or c-erb B-4. Since
ligand-dependent activation of the EGF receptor can also
lead to heterodimerization with c-erb B-2, c-erb
B-3, or c-erb B-4, this suggests that different pairs of
heterodimers within the type 1 receptor tyrosine kinase family may
contribute to the array of responses to various EGF-like ligands in a
cell-specific and possibly ligand-specific manner by the recruitment of
different combinations of intracellular signaling proteins (14). This apparent redundancy of different ligands and receptors may therefore contribute to signal diversification and amplification. One major pathway that is activated by a several different EGF-like ligands through these type 1 receptor tyrosine kinases is the
ras/raf/mitogen-activated protein kinase (MAPK)
signal transduction pathway (16).
The HC-11 mouse mammary epithelial cell line is a clonal isolate
originally derived from the COMMA-1D cell line, which was established
from a midpregnant Balb/c mouse mammary gland (17). These cells express
a number of mammary epithelial markers such as -casein and express
several distinct type 1 receptor tyrosine kinases, including the EGFR,
c-erb B-2, and c-erb B-3 (17-20). In this
context, HC-11 cells have been useful as an in vitro model system in which to define the intracellular signaling pathways that are
engaged by EGF-related peptides that utilize these type 1 receptor
tyrosine kinases and are involved in regulating proliferation and
differentiation in mammary epithelial cells (19, 20). In the present
study, we have attempted to define the intracellular signaling pathway
that might be activated in HC-11 cells and in several different human
breast cancer cell lines by a 47-mer refolded CR-1 peptide that
corresponds in sequence to the EGF-like repeat of the human CR-1
protein (3). The CR-1 related peptide was found to bind to a unique
specific, high-affinity receptor that is not the EGFR, erb
B-2, erb B-3, or erb B-4. CR-1 was also found to
enhance the tyrosine phosphorylation of the SH2-adaptor protein, Shc,
and to promote the association of Grb2-mSOS intracellular signaling
complex with tyrosine-phosphorylated Shc. These events were
subsequently related to the downstream activation of the p42erk-2 MAPK isoform.
HC-11 mouse mammary epithelial
cells were routinely grown in RPMI 1640 medium containing 8%
heat-inactivated fetal bovine serum, 5 µg/ml bovine insulin
(Sigma), and 10 ng/ml EGF (Collaborative Research,
Watham, MA). Human breast cancer cell lines, MCF-7, ZR-75-1, T-47D,
MDA-MB-231, MDA-MB-453, and SKBr-3 were cultured in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum and insulin
(10 µg/ml) as described previously (21). The Ba/F3 mouse pro-B
lymphocyte cell line and clonal derivatives that express individual or
pairs of type 1 receptor tyrosine kinases were generated as described
previously (22, 23). Cells were treated with different concentrations
of either refolded p47 CR-1 peptide that corresponds in sequence to the
EGF-like motif in human CR-1 (3), recombinant human TGF (Bachem),
recombinant human AR, recombinant human HB-EGF, recombinant human BTC
(R & D Systems, Inc.), recombinant mouse epiregulin (generously
supplied by Toshi Komuraski, Taisho Pharmaceutical Co., Saitama,
Japan), or recombinant human HRG
1177-244 (generously
supplied by Mark Sliwkowski, Genentech, Inc., South San Francisco,
CA).
Cells were grown until they were subconfluent and then propagated in serum-free medium containing human transferrin (10 µg/ml) and type 1V Pedersen fetuin (1 mg/ml) for 48 h. Cells were treated in serum-free medium with different concentrations of p47 CR-1 for various times and then lysed in 20 mM Tris-HCl (pH 7.5) containing 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. In some cases the clarified protein lysates were either immunoprecipitated (0.5 mg/sample) with 2 µg of a rabbit anti-Shc antibody (Transduction Laboratories, Lexington, KY) or with 2 µg of anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc.). Crude protein lysates (25 µg/sample) or immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 2% dry milk in Tris-buffered saline with 0.05% Tween 20, and incubated with a 1:1000 dilution of anti-Shc, anti-Grb-2, or anti-mSOS monoclonal antibodies (Transduction Laboratories) or a 1:2000 dilution of the anti-phosphotyrosine monoclonal antibodies 4G10 and/or PY-20 (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The bound mouse monoclonal antibodies were detected using a 1:5000 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (Amersham). Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham). erb B receptors were immunoprecipitated from recombinant Ba/F3 clonal derivatives using monospecific antisera as described previously (22).
MAPK AssaysA rabbit anti-MAPK antibody (Upstate
Biotechnology, Inc., Lake Placid, NY) at a 1:1000 dilution that
recognizes both extracellular signal-regulated protein kinase
erk-1 and erk-2 MAPK was used to detect
activation of immunoreactive MAPK proteins by means of band shift
following SDS-PAGE of crude cellular lysates as described previously
(24). Bands were detected by the colorimetric NBT/BClP system
(Kirkegaard Perry Labs, Gaithersburg, MD). Alternatively, cells were
lysed in buffer containing 1% Nonidet P-40, 0.5% deoxycholate, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM sodium orthovanadate, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysates
(100 µg/sample) were immunoprecipitated with an anti-erk-1
rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After
2 h, the immunoprecipitates were washed twice with lysis buffer
followed by a final wash in kinase buffer containing 30 mM
HEPES (pH 7.4), 10 mM MgCl2, 10 mM
MnCl2, and 1 mM dithiothreitol. After the final
wash, the immunoprecipitates were suspended in 30 µl of kinase buffer
containing 5 µg of myelin basic protein (MBP) and 5 µCi of
[-32P]ATP (1000 Ci/mmol, Amersham) and incubated for
30 min at 37 °C. The reaction was stopped with 2 × SDS sample
buffer. The samples were then run on a 10% Tricine gel followed by
autoradiography. The MBP bands were quantified with a densitometer
(Molecular Dynamics) (19).
125I-p47 CR-1 peptide was
prepared using IODO-GEN (Pierce Chemical Co.) in which 5 µg of
peptide was labeled with 1 mCi of Na125I to a specific
activity of 5-10 µCi/µg. Monolayers of HC-11 cells or human breast
cancer cells in 12-well cluster dishes were washed in serum-free RPMI
1640 or Dulbecco's modified Eagle's medium containing 20 mM HEPES (pH 7.4) and 0.1% bovine serum albumin and
treated in the same medium. Cells were incubated at 23 °C for 2 h in 1 ml of binding buffer containing 3 × 105 cpm of
125I-p47 CR-1 in the absence or presence of increasing
concentrations of unlabeled p47 CR-1 or with other EGF-related
peptides. Cells were washed twice with phosphate-buffered saline and
then solubilized in 1 ml of buffer containing 10 mM
Tris-HCl (pH 8.0) and 0.5% SDS and counted in a -counter. The
binding data were analyzed using the Ligand program to determine the
Kd value as described (25).
A 47-mer CR-1 refolded peptide that contains
the EGF-like motif of human CR-1 is able to stimulate the proliferation
and to inhibit -casein expression in mouse HC-11 mammary epithelial cells.2 To determine if a high-affinity,
specific binding site is expressed on HC-11 mammary epithelial cells
that might mediate the biological effects of CR-1, HC-11 cells were
incubated with 125I-p47 CR-1 in the absence or presence of
increasing concentrations of unlabeled p47 CR-1. As illustrated in Fig.
1A, there is specific binding of the
125I-p47 CR-1 peptide to HC-11 cells with a
Kd of approximately 96 nM (Fig.
1A, inset). Various concentrations of EGF, TGF
, AR, or
HRG
1 were unable to compete for binding with 125I-p47
CR-1 on HC-11 cells (Fig. 1B). Likewise, heparin-binding EGF-like growth factor, epiregulin, or BTC were ineffective in blocking
the binding of the 125I-p47 CR-1 peptide (data not shown).
This potential receptor is not unique to HC-11 mouse mammary epithelial
cells since 125I-p47 CR-1 also binds specifically and with
high affinity to several estrogen receptor positive (MCF-7, T47-D, and
ZR-75-1) and to estrogen receptor negative (MDA-MB-231, MDA-MB-453 and
SKBr-3) human breast cancer cell lines that exhibits comparable
specificity for CR-1 binding as in mouse HC-11 cells (Table
I). Moreover, blocking the EGFR receptor on MDA-MB-231
human breast cancer cells with 25 µg/ml of the 528 anti-EGFR
monoclonal blocking antibody which is sufficient to impede
125I-EGF binding (26) does not lead to any change in the
specific binding of 125I-p47 CR-1 to these cells (Fig.
1C), confirming the observation that CR-1 does not directly
bind to the EGFR (3). In addition, the binding of 125I-p47
CR-1 peptide to MDA-MB-231 cells or SKBr-3 cells (data not shown) is
unaffected by the anti-erb B-2 TAb 250 blocking monoclonal antibody.
|
Parental Ba/F3 mouse
pro-B lymphocytes express low levels of endogenous erb B-3
but do not express EGFR, erb B-2, or erb B-4. Ba/F3 clones have been genetically engineered to ectopically express either EGFR, erb B-2, erb B-3, or erb
B-4 type 1 receptor tyrosine kinases either alone or in different
pairwise combinations (22, 23). These recombinant cell lines have been
particularly useful in defining different homodimeric and heterodimeric
combinations of the type 1 receptor kinases that can bind to various
ligands within the EGF family of peptides (23). Since CR-1 is
structurally related in part to this family of peptides, the 47-mer
CR-1 refolded peptide and an Sf9-derived recombinant human GST-CR-1
fusion protein3 were tested for their
ability to directly bind and stimulate receptor tyrosine
phosphorylation in single recombinant Ba/F3 derivatives that are
expressing different erb B family members (Fig.
2). p47 CR-1 was unable to significantly modify EGFR
(Fig. 2A), erb B-2/neu/HER-2 (Fig.
2B), erb B-3/HER-3 (Fig. 2D), or erb B-4/HER-4 (Fig. 2C) tyrosine phosphorylation
in Ba/F3 clones that were expressing these tyrosine kinases. In
contrast, BTC was able to stimulate EGFR and erb B-4
phosphorylation (Fig. 2, A and C) while HRG1
was able to indirectly stimulate erb B-2 phosphorylation
(Fig. 2B) by binding to endogenous erb B-3 (22). p47 CR-1 was also ineffective in modulating the tyrosine
phosphorylation of these different type 1 receptor tyrosine kinases in
Ba/F3 cells that were expressing different pairs of erb B
receptors demonstrating that CR-1 does not directly bind to
heterodimers within this family of receptor tyrosine kinases (data not
shown). Similar negative results on tyrosine receptor phosphorylation
in either single or double erb B expressing recombinant
Ba/F3 clones were obtained after treatment of these cells with the
full-length Sf9 recombinant GST-human CR-1 fusion protein.
CR-1 Stimulates Tyrosine Phosphorylation of Shc and Activates MAPK in HC-11 Mammary Epithelial Cells and in Human Breast Cancer Cells
Although CR-1 does not directly activate any known type 1 receptor erb B tyrosine kinase, its receptor may still
consist of a membrane-associated tyrosine kinase or alternatively the
receptor may associate with a another cytoplasmic or membrane bound
tyrosine kinase to generate a signal. To determine if the p47 CR-1
peptide could modulate the tyrosine phosphorylation of other proteins in HC-11 cells, serum-starved cells were treated with p47 CR-1 for 5 min and cell lysates were immunoprecipitated with 4G10 mouse monoclonal
anti-phosphotyrosine antibody. Immunoprecipitates were then
electrophoresed and probed by Western blot analysis with the PY-20
mouse monoclonal anti-phosphotyrosine antibody (Fig. 3A). An increase in tyrosine phosphorylation
of a 185-kDa protein was observed. A similar CR-1-induced increase in
the tyrosine phosphorylation of a 185-kDa protein was also observed in
other cells that were capable of binding 125I-p47 CR-1 such
as MDA-MB-453, SKBr3, and T47-D human breast cancer cells after
immunoprecipitation and Western blotting with anti-phosphotyrosine antibodies (data not shown). The identity of the p185 phosphoprotein has not yet been identified.
Shc is an SH-2 containing adaptor protein that exists as three distinct
isoforms of 66, 52, and 46 kDa and that becomes tyrosine-phosphorylated after ligand activation of several different type 1 receptor tyrosine kinases (27-29). Cell lysates from p47 CR-1-treated serum-starved HC-11 cells exhibited a time-dependent increase in the
tyrosine phosphorylation of the p66 Shc and p46 Shc isoforms, which
could be detected following immunoprecipitation with an anti-Shc
antibody and subsequent screening in Western blot analysis with
anti-phosphotyrosine antibodies (Fig. 3, B andC).
In HC-11 cells which had been sufficiently serum-starved, the increase
in phosphorylation of p46 Shc (Fig. 3B) and p66 Shc (Fig.
3C) in response to p47 CR-1 was transient with an 8-fold
increase in p46 Shc phosphorylation being observed after 5-7 min,
which decreased to control levels after 10 min of treatment (Figs.
3B and 5B). No significant changes in the phosphorylation of the p52 isoform of Shc were detected in HC-11 cells.
A less dramatic but measurable increase in the tyrosine phosphorylation
of either p52 and/or p46 Shc could also be detected in either
MDA-MB-453 or SKBr-3 human breast cancer cells after treatment with
either the p47 CR-1 peptide (Fig. 3C) or the GST-CR-1 recombinant fusion protein (data not shown). Proteins of 185 and 120 kDa that were also tyrosine-phosphorylated in a transient manner were
detected in the Shc immunoprecipates after probing the Western blots
with a mixture of two different anti-phosphotyrosine monoclonal
antibodies (Fig. 3C). These proteins were found to co-immunoprecipitate with Shc after stimulation of HC-11, MDA-MB-453, or SKBr-3 cells with p47 CR-1. The identity of these two proteins has
not been determined.
Shc is an adaptor protein that can bind to specific consensus sequences
which contain phosphotyrosine residues in the COOH terminus of several
different tyrosine kinase receptors through either an SH2 domain or a
phosphotyrosine-binding domain (29). In turn, binding of phosphorylated
Shc to the SH2 domain of Grb2 through the tyrosine-phosphorylated
collagen homology domain of Shc can link a number of different growth
factor receptor tyrosine kinases to the
ras/raf/MEK/MAPK signaling pathway since Grb2 is intrinsically complexed with the ras guanine nucleotide
exchanger, SOS (27, 29). To ascertain if p47 CR-1 could facilitate the association of the Grb2-mSOS complex with phosphorylated Shc in mammary
epithelial cells, HC-11 cell lystates were immunoprecipated with the
Shc antibody and immunoblotted with either an anti-Grb2 (Fig.
4A) or an anti-SOS (Fig. 4B)
antibody. In both cases, p47 CR-1 induced a time-dependent
increase in the association of Grb2 and mSOS with phosphorylated Shc.
Activation of p21ras by SOS can ultimately lead to the
downstream activation of mitogen-activated, erk-activating
kinase (MEK) through raf and the subsequent stimulation of
MAPK activity (16, 30). To ascertain if p47 CR-1 could stimulate MAPK
activity, serum-starved HC-11 cells were treated for different times
with the peptide, and cell lysates were electrophoresed and probed with
an anti-MAPK antibody that detects both MAPK isoforms, p44erk-1
and p42erk-2 (Fig. 5A).
Alternatively, cell lysates were immunoprecipitated with an anti-MAPK
antibody and utilized in an in vitro kinase assay with MBP
as a substrate to quantify total MAPK activity (Fig. 5B).
The p47 CR-1 peptide produced a rapid tyrosine phosphorylation of
p42erk-2 within 3-5 min that could be detected by the presence
of a slower migrating band that represents the phosphorylated form of
p42erk-2 (Fig. 5A). Phosphorylation of this species
of p42erk-2 peaked at 7 min and subsequently declined. A nearly
4-fold increase in phosphorylation of the MAPK substrate, MBP, was also
observed and found to have identical kinetics in response to p47 CR-1
treatment and was delayed relative to the increase in p46 Shc
phosphorylation (Fig. 5B).
EGF, TGF, AR, and HRG
1 stimulate the growth and regulate the
differentiation of normal and malignant mouse and human mammary epithelial cells in vitro (7, 11, 31-34). However, the role of other EGF-related peptides in regulating the growth and
differentiation of mammary epithelial cells has not been fully
explored. This may be particularly important since only a subset of
peptides within the EGF family bind exclusively to the EGFR. Additional proteins in this family, such as the neuregulin subfamily which include
the HRGs, bind to other members of the type 1 receptor tyrosine kinase
family of receptors such as c-erb B-3 or c-erb B-4, that can then heterodimerize and activate c-erb B-2
following transphosphorylation (3-15, 31, 34-36). Finally, BTC
can equally activate either the EGFR or c-erb B-4 (23).
Since ligand-dependent activation of the EGF receptor can
also lead to heterodimerization with c-erb B-2,
c-erb B-3, or c-erb B-4, this suggests that
different combinatorial pairs of heterodimers within the type 1 receptor tyrosine kinase family may contribute in a hierarchical
fashion through signial diversification to the array of responses that are produced to various EGF-like ligands in a cell-specific manner (14,
15, 35, 36).
The present study is the first to demonstrate that a refolded peptide which corresponds in sequence to the EGF-like motif of the human CR-1 protein can bind to a unique receptor that can modify the tyrosine phosphorylation of different proteins which are components in the ras/raf/MEK/MAPK pathway. CR-1 is a newly discovered member of the EGF family of peptides that is structurally unique within this family as CR-1 lacks an A loop and possesses a truncated B loop (2, 3, 7). Since conserved amino acid residues in the A loop in conjunction with residues in the C loop are necessary for peptide binding to the EGFR receptor (37) and since the 47-mer refolded CR-1 related peptide does not directly compete with EGF for binding to the EGFR (3), this demonstrates that CR-1 cannot bind to this receptor. Nevertheless, CR-1 is able to stimulate the proliferation and differentiation of HC-11 cells2 and the growth of several different nontransformed human mammary epithelial and breast cancer cell lines suggesting that a receptor exists for this peptide (3). In this context, the synthetic CR-1 peptide interacts with a high-affinity binding site on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines that exhibit specificity for the CR-1 47-mer peptide since other EGF-related peptides that bind either to the EGFR or c-erb B-3 and c-erb B-4 fail to compete for binding with the labeled p47 CR-1 peptide. This suggests that the CR-1-binding site is unique. Conversely, the CR-1 peptide or recombinant GST-CR-1 fusion protein does not directly activate the tyrosine kinase of either the EGFR or other members of the type 1 receptor tyrosine kinase family either alone or in various heterodimeric combinations in mouse Ba/F3 cells (22, 23). Nevertheless, the ability of 47-mer CR-1 peptide to induce the tyrosine phosphorylation of 185- and 120-kDa proteins in HC-11 mouse mammary epithelial cells and MDA-MB-453 or SKBr-3 human breast cancer cells suggests that CR-1 binds to a potential receptor that has either an intrinsic tyrosine kinase activity or that associates with a tyrosine kinase. In this context, it is known that various members of the type 1 receptor tyrosine kinase family can heterodimerize following ligand binding or associate with p60c-src. Heterodimerization might facilitate the activation of different signaling proteins thereby contributing to signal amplification and diversification in response to different type 1 receptor ligands (14, 15, 36). The present experiments do not formally exclude the possibility that the CR-1 receptor can also heterodimerize with one of these type 1 receptor tyrosine kinases or with other soluble src-related tyrosine kinases and that these interactions may be essential for propagation of an intracellular signal.
The present data also demonstrates that p47 CR-1 treatment of HC-11,
MDA-MB-453, or SKBr-3 cells can lead to a rapid increase in tyrosine
phosphorylation of the p66, p52, and p46 isoforms of Shc which can then
associate with the Grb2-mSOS signaling complex. Since this is one
possible mechanism by which other growth factor receptor tyrosine
kinases can couple to the MEK/MAPK pathway through ras and
raf (27-30, 38), these results may be functionally
significant with respect to defining components in the intracellular
signal transduction pathway that are activated by the CR-1 receptor. Moreover, the results demonstrate that CR-1 can activate
p42erk-2 MAPK by rapidly inducing the tyrosine phosphorylation
of this MAPK isoform. Since activation of MAPK by various growth
factors appears in certain cells to be obligatory for cell
proliferation and/or for differentiation (16), then this same situation
may also be applicable to CR-1 in its ability to stimulate growth and
modulate the expression of -casein and whey acidic protein in HC-11
cells and in primary mouse mammary epithelial cells in response to
lactogenic hormones such as prolactin.2 Identification and
characterization of the CR-1 receptor following chemical cross-linking
of its ligand should clarify some of these issues. In this respect,
attempts to chemically cross-link the 125I-p47 CR-1 peptide
have been unsuccessful. However, we have recently expressed a
full-length, refolded, biologically active human CR-1 protein in
Escherichia coli and in Sf9 insect cells with a baculovirus expression vector.3 Cross-linking of these proteins to
appropriate target cells such as HC-11 should now be feasible.