From the
Heregulins (HRGs) are mosaic glycoproteins that bind to and
induce the tyrosine phosphorylation of the
HER4/p180
Heregulins (HRGs)
Although HRGs contain an EGF-like motif,
they do not bind to
EGFR/p170
As
a consequence of HRG/NDF binding to its receptor(s), human mammary
tumor cells have been shown to differentiate
(2, 17) and
up-regulate their expression of intercellular adhesion molecule-1
(ICAM-1)
(17) . The biological effects of HRGs are mediated
through receptors that possess an intrinsic tyrosine kinase activity
and are autophosphorylated upon HRG binding
(15) . Studies on
receptor tyrosine kinases such as the epidermal growth factor receptor,
the platelet-derived growth factor receptor, and the insulin receptor
have demonstrated a crucial role for receptor autophosphorylation in
intracellular signal transduction following ligand
binding
(18, 19) . It has been demonstrated that specific
autophosphorylation sites on receptor tyrosine kinases serve as
recognition structures for target molecules containing Src homology 2
(SH2) domains. SH2 domains are conserved noncatalytic sequences of
This report describes the
generation of versatile recombinant HRGs and the study of some of the
biological functions and intracellular signaling pathways that these
proteins trigger following receptor activation. Because the EGF-like
domain of HRG is sufficient for receptor
binding
(1, 16) , we cloned the cDNA fragments encoding
the EGF-like domain of HRG-
The EGF-like domains of
HRG-
Because MDA-MB-453
cells express HER3, HER4, and also high levels of HER2, the exact
identity of the 180-kDa phosphorylated band is unknown. HER4 and HER3
have both been identified as HRG
receptors
(13, 14, 15, 16) , making them
obvious candidates. However, since the samples were electrophoresed
under reducing conditions, the phosphorylated species might correspond
to a combination of monomeric forms of all three receptors, including
HER2. In fact, in response to HRG stimulation, HER2 is stimulated
indirectly through receptor transphosphorylation (15). In addition, a
recent study has indicated that a high affinity binding site for the
EGF-like domain of HRG-
The
experiment described above clearly demonstrates that the EGF-like
domains of rHRGs-T-Fc mediate the observed biological effects and that
these effects cannot be attributed to the Fc portion of the fusion
proteins.
We are grateful to Dr. S. Myrdal and T. Bailey for
expert assistance with confocal microscopy studies. We thank M.
Neubauer and the members of the DNA/peptide chemistry group for DNA
sequencing and oligonucleotide synthesis. We also thank Dr. D.
Hollenbaugh for helpful discussions and critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor. This work was
aimed at studying the biological effects induced by recombinant
epidermal growth factor (EGF)-like domains of HRGs as well as
identifying intracellular molecules involved in HER4 signaling. To this
end, we cloned the EGF-like domains of HRG-
, -
2, and -
3
into a eukaryotic expression vector in frame with sequences encoding a
thrombin cleavage site followed by the Fc portion of a human IgG1.
These chimeric genes directed the expression of recombinant fusion
proteins, rHRGs-T-Fc, which specifically stimulated the phosphorylation
of HER4/p180
. We also show that
rHRG-
-T-Fc bound to human breast cancer cells that express HER4
receptors and induced the expression of intercellular adhesion
molecule-1. After thrombin protease cleavage of rHRGs-T-Fc, their
EGF-like domains were purified and shown to stimulate protein
phosphorylation in HER4-expressing cells. Moreover, the rHRG-
2
EGF-like domain markedly induced the phosphorylation of Shc proteins on
tyrosine, suggesting a role for these adaptor molecules in HRG-mediated
signaling.
(
)(1) , neu differentiation factor (NDF)
(2, 3, 4) ,
glial growth factors
(5) , and acetylcholine receptor-inducing
activity
(6) are homologous multifunctional proteins. The HRG
isoforms originate from a single gene by alternative RNA splicing. HRG
cDNAs encode large transmembrane precursors with multiple domains,
including an immunoglobulin-like domain, a spacer domain with several
glycosylation sites, an EGF-like domain, a juxtamembrane domain of
variable length, a transmembrane region, and a cytoplasmic domain.
Soluble mature HRGs are most probably released from the cell surface by
proteolytic cleavage. Glial growth factors
(5) and acetylcholine
receptor-inducing activity
(6) , which were isolated from brain
tissues, also contain a kringle-like domain that is absent in HRGs and
NDFs. HRG
- and
-isoforms display sequence differences in the
third loop of the EGF-like domain and in the juxtamembrane domain. The
EGF-like domain of HRGs contains six cysteine residues that are
characteristic of the EGF family of growth factors, including
EGF
(7) , transforming growth factor-
(8) , vaccinia
virus growth factor
(9) , amphiregulin
(10) ,
heparin-binding EGF-like growth factor
(11) , and
betacellulin
(12) .
(1) . In fact, HRGs
bind to HER4/p180
, a recently
isolated member of the epidermal growth factor receptor
family
(13, 14, 15) . The
HER3/p180
receptor, another member
of this family, has also been reported to be a receptor for
HRGs
(16) . HRGs do not directly interact with the
HER2/p185
receptor, as originally
proposed
(1, 2) . However, it appears that
HER2/p185
indirectly participates
in HRG-mediated signaling through transphosphorylation or receptor
heterodimerization with HER4 and/or HER3
(15, 16) .
100 amino acids found in various signaling molecules and oncogenic
proteins
(20, 21) . SH2 domain-containing proteins bind
with high affinity to phosphotyrosine residues in the context of
specific flanking amino acids. For example, the p85 subunit of
phosphatidylinositol 3`-kinase, the p21
GTPase-activating protein, and phospholipase C-
have been
shown to contain SH2 domains. More recently, SH2 domain-containing
proteins that lack an apparent catalytic domain and that seem to
function as adaptors linking proteins involved in signal transduction
have been described (22-27). One of them, Shc, was identified and
cloned based on its homology to SH2 sequences from the human c-fes gene
(23) . The Shc cDNA is predicted to encode two proteins
of 46 and 52 kDa that contain a single C-terminal SH2 domain and a
collagen homologous region that is rich in glycine and proline. No
catalytic domain was identified in Shc. Anti-Shc antibodies have been
shown to recognize three proteins of 46, 52, and 66 kDa in a wide range
of mammalian cells. A variety of growth factors and cytokines have been
shown to induce the phosphorylation of Shc
proteins
(28, 29, 30, 31, 32, 33) .
Furthermore, overexpression of Shc proteins is associated with a
transformed phenotype in fibroblasts
(23) and neuronal
differentiation of PC12 cells
(34) , strongly suggesting that Shc
is involved in cell growth regulation.
, -
2, or -
3 into a eukaryotic
expression vector containing sequences encoding a thrombin cleavage
site followed by the Fc portion of a human IgG. The recombinant fusion
proteins generated, referred to as rHRGs-T-Fc, were used either as
chimeric proteins or as EGF-like domains (reHRGs) after thrombin
cleavage and removal of the Fc portion of the molecule. Herein, we
demonstrate that recombinant HRGs, in either form, bind to and activate
the HER4 receptor and show that the Shc proteins are
tyrosine-phosphorylated following HRG stimulation.
Antibodies
RC20 recombinant anti-phosphotyrosine
antibody (Transduction Laboratories) and PY20 anti-phosphotyrosine
antibody (ICN Biomedicals, Inc.) were used in Western blotting studies.
Polyclonal anti-Shc antibodies were purchased from Upstate
Biotechnology, Inc., and the monoclonal anti-Shc antibody was from
Transduction Laboratories. BBA 3, an anti-human ICAM-1 monoclonal
antibody, was from R& Systems.
Cell Lines
MDA-MB-453 human breast cancer cells
were obtained from the American Type Culture Collection. CHO/EGFR cells
were generated by Dr. B. Thorne (Bristol-Myers Squibb, Seattle, WA) as
follows. The complete recombinant human EGF receptor coding sequence
was inserted into a CDM8 expression vector containing the neomycin
resistance gene. The resulting construct was transfected into CHO-K1
cells. G418-resistant clones were analyzed for EGFR expression. Levels
of expression of functional EGFR in CHO/EGFR stable cells were assessed
by stimulating the cells with EGF, immunoprecipitating the EGFR, and
determining its phosphorylation level by phosphotyrosine Western
blotting as reported
(13) . CHO/HER4 cells expressing high levels
of recombinant human HER4 have previously been
described
(13, 14, 15) .
Construction of HRG-T-Fc Expression Plasmids
DNA
fragments encoding part of the spacer domain of human HRGs, the
EGF-like domains, the transmembrane domain, and a few residues of the
cytoplasmic domain were amplified by reverse transcription-PCR from
total RNA isolated from HepG2 cells. The oligonucleotide primers were
designed based on the sequence of human HRG-
(1) . The PCR
primers used were 5`-GTGTCTTCAGAGTCTCCCATTAGA-3` (forward) and
5`-CTTGGTTTTGCAGTAGGCCAC-3` (reverse). Amplification was performed with
Taq DNA polymerase (Perkin-Elmer) using 35 cycles, with each
cycle being composed of a denaturing step for 1 min at 95 °C, an
annealing step for 1 min at 65 °C, and an extension step for 30 s
at 72 °C. The PCR products were blunt-ended using the Klenow
fragment of Escherichia coli DNA polymerase I and subcloned
into an SmaI-digested pBluescript II vector (Stratagene), and
the nucleotide sequences of individual clones were determined by the
dideoxy-mediated chain termination reaction.
, -
2, and -
3 were generated by PCR using HRG-
or -
2 template plasmids generated as described above. The
oligonucleotide primers described below were designed to place an
SpeI site at the 5`-end and a BamHI site at the
3`-end of the amplified products for cloning purposes. The epidermal
growth factor-like domain of human HRG-
was amplified using the
following primers: 5`-GAGACTAGTAGCCATCTTGTAAAATGTGCG-3` (forward) and
5`-CCGTGGATCCTTCTGGTACAGCTCCTCCGC-3` (reverse). PCR conditions
consisted of 40 cycles of 30 s at 94 °C, 1 min at 55 °C, and 2
min at 72 °C using Pfu polymerase and reagents recommended
by the vendor (Stratagene). The PCR product encoded complementary
sequences corresponding to residues 177-241 of HRG-
. The
epidermal growth factor-like domains of human HRG-
2 and -
3
were amplified using an HRG-
2 clone as a template. The forward
primer was described above. The HRG-
2 reverse primer had the
sequence 5`-CCGTGGATCCTTCTGGTACAGCTCCTCCGCCTT-3`. Amplification was
performed with Pfu polymerase using the same temperature
program as that used for HRG-
. This PCR product encoded sequences
corresponding to residues 177-238 of HRG-
2. The HRG-
3
reverse primer contained a silent point mutation introducing a
HindIII site for diagnostic purposes and had the following
sequence:
5`-CCGTGGATCCTCAGGCAAGCTTAGAAAGGGAGTGGACGTACTGTAGAAGC-TGGCCATTAC-3`.
PCR conditions consisted of 40 cycles of 1 min at 94 °C, 2 min at
50 °C, and 3 min at 72 °C using Pfu polymerase. The
PCR product encoded sequences corresponding to residues 177-241
of HRG-
3. All PCR products were digested with BamHI and
SpeI and ligated to a BamHI-SpeI-cut
CDM7-derived vector containing cDNA sequences coding for the CD5 signal
peptide 5` of the cloning site for proper secretion of the expressed
proteins as well as cDNA sequences encoding a thrombin cleavage site
(amino acid sequence: DPGGGGGRLVPRGFGTG) and cDNA sequences encoding
the hinge and constant regions of a human IgG1 3` of the cloning
site.
(
)
All constructs were sequenced by the
dideoxy-mediated chain termination reaction to confirm the sequence of
the EGF-like domains as well as to verify that their sequences were in
frame with the thrombin and Fc coding sequences. Constructs were
transfected into COS cells as described previously
(35) , and the
resulting fusion proteins were recovered from culture supernatants
using protein A-Sepharose (Repligen). Purified proteins were visualized
on 8% SDS-polyacrylamide gel under reducing and nonreducing conditions.
Protein concentrations were determined using a protein assay kit
(Bio-Rad).
Thrombin Cleavage
Fusion proteins were incubated
for 30 min at room temperature with human thrombin (Sigma) at a 1:50
(w/w) thrombin/fusion protein ratio. Cleaved proteins were then loaded
on a protein A-Sepharose column. Column flow-through fractions
containing the recombinant EGF-like domains of HRGs were stored at
-20 °C until further use.
Detection of Tyrosine-phosphorylated Proteins by Western
Blotting
CHO/HER4 cells (5 10
), CHO/EGFR
cells (2
10
), and MDA-MB-453 cells (4
10
) were seeded in 48-well plates. 24 h later, cells were
serum-starved for 8 h and then stimulated with various samples for 10
min at 37 °C. Supernatants were discarded, and cells were lysed by
adding boiling electrophoresis sample buffer. Lysates were subjected to
SDS-PAGE on 8% polyacrylamide gels (Novex) and then electroblotted onto
nitrocellulose. PY20 monoclonal anti-phosphotyrosine antibody and
horseradish peroxidase-conjugated goat anti-mouse IgG F(ab`)
(Cappel) were used as primary and secondary probing reagents,
respectively. Immunoreactive bands were visualized using enhanced
chemiluminescence (Amersham Corp.).
Immunoprecipitation
CHO/HER4 cells were seeded in
100-mm dishes. 80-90% confluent monolayers were washed and
incubated with various recombinant HRGs for 10 min at 37 °C.
Monolayers were washed with ice-cold PBS and solubilized for 10 min on
ice in PBSTDS lysis buffer (10 mM sodium phosphate, pH 7.3,
150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%
SDS) containing 1 mM EDTA, 2 mM phenylmethylsulfonyl
fluoride, 1 mM NaVO
, 20 µg/ml
aprotinin, 20 µg/ml leupeptin, and 20 µg/ml pepstatin. The
protein concentrations of the clarified extracts were determined using
a bicinchoninic acid protein assay kit (Pierce). Lysates (1
mg/immunoprecipitation) were incubated overnight at 4 °C with a
rabbit anti-Shc antibody (Upstate Biotechnology, Inc.). Immune
complexes were precipitated by adding protein G Plus-/protein A-agarose
(Oncogene Science Inc.) to the suspensions. After 1 h of incubation at
4 °C, the immunoprecipitates were washed three times with PBSTDS
lysis buffer and then resolved on 8% polyacrylamide gels under reducing
conditions. Proteins were electroblotted onto nitrocellulose and probed
with RC20 recombinant anti-phosphotyrosine antibody or a monoclonal
anti-Shc antibody. Immunoreactive bands were visualized using enhanced
chemiluminescence.
Immunohistochemical Staining
MDA-MB-453 cells were
plated on 8-well borosilicate-chambered slides (Lab-Tek, Nunc). For
receptor binding visualization, after a 48-h culture period, the cells
were placed on ice for 10 min, washed twice with ice-cold binding
buffer (Dulbecco's modified Eagle's medium supplemented
with 44 mM sodium bicarbonate, 50 mM Bes, pH 7.0,
0.1% bovine serum albumin), and then incubated on ice for 2 h with
rHRG--T-Fc or, as a negative control, an irrelevant fusion protein
consisting of the extracellular domain of the Tek receptor
(36) fused to the thrombin cleavage site followed by the Fc
region of an IgG1, as in rHRGs-T-Fc.
(
)
The
reagents, cloning vector, and mammalian cells used to construct and to
generate the Tek-Fc fusion protein were identical to the ones used to
make rHRGs-T-Fc. The cells were washed twice and incubated for 45 min
on ice with a fluorescein-conjugated goat anti-human IgG F(ab`)
fragment (Tago, Inc.). The cells were rinsed twice with PBS and
fixed for 20 min in PBS, 2% formaldehyde. For ICAM-1 expression
studies, after a 24-h culture period, the cells were incubated for 3
days with 50 ng/ml rHRG-
-T-Fc, p45
(14) , Tek-Fc fusion
protein as a negative control, or culture medium alone. Staining was
then performed on live cells. The cells were washed and incubated for 1
h on ice with an anti-ICAM-1 antibody diluted 1:500 in binding buffer.
The cells were washed and incubated for 45 min on ice with a
fluorescein-conjugated goat anti-mouse IgG F(ab`)
fragment
(Tago, Inc.). The cells were rinsed and fixed as described above. The
levels of receptor staining and ICAM-1 expression were analyzed using a
Leica confocal microscope.
Construction of rHRGs-T-Fc
Our objective was to
generate versatile recombinant HRGs to study various aspects of the
biology of the HER4/HRG receptor/ligand pair. Because the EGF-like
domain of HRG-1 had previously been shown to be sufficient for
receptor binding
(1) , we constructed three chimeric genes that
encode soluble proteins consisting of the EGF-like domain of HRG-
,
-
2, or -
3 linked to a thrombin cleavage site followed by the
hinge, CH2, and CH3 regions of a human IgG1 antibody, with secretion of
the proteins directed by the signal sequence of CD5 (see
``Experimental Procedures'' for details). The EGF-like domain
of HRG-
corresponded to residues 177-241 of the mature
protein, while those of HRG-
2 and -
3 corresponded to residues
177-238 and residues 177-241, respectively. The three
fusion proteins (rHRGs-T-Fc) were prepared by transient expression in
COS cells, purified from culture supernatants on protein A-Sepharose,
and gave yields in the range of 350-1900 µg/liter. The CD5 signal
peptide allowed efficient processing and secretion of rHRGs-T-Fc. All
three fusion proteins were secreted as disulfide-linked homodimers
similar to immunoglobulins and therefore were each capable of
presenting two HRG EGF-like domains. To establish that rHRGs-T-Fc were
able to bind and to activate the HER4 receptor, we examined their
potential to induce the phosphorylation of HER4 as well as
morphological changes and up-regulation of ICAM-1 expression.
Activation of the HER4 Receptor by rHRGs-T-Fc
For
these experiments, we used CHO/HER4 cells, which express high levels of
recombinant human HER4/p180 and
have previously been shown to respond to HRG
(13, 14) .
rHRG-
-T-Fc, -
2-T-Fc, and -
3-T-Fc were added to CHO/HER4
cells at 50 and 200 ng/ml for 10 min at 37 °C. Cells were lysed,
and then the pattern of tyrosine-phosphorylated proteins was analyzed
by anti-phosphotyrosine Western blotting as compared with untreated
cells. As shown in Fig. 1A, all three rHRGs-T-Fc induced
the hyperphosphorylation of the HER4 receptor. Ligand activation not
only resulted in receptor autophosphorylation, but also in the tyrosine
phosphorylation of several substrates, including an M
100,000 band, yet to be identified (Fig. 1A). When
tested on CHO/EGFR cells, which express high levels of recombinant
human EGFR, rHRGs-T-Fc (200 ng/ml) failed to activate the EGFR
(Fig. 1B). As expected, EGF (200 ng/ml) markedly induced
the phosphorylation of the EGFR in CHO/EGFR cells
(Fig. 1B, lane2). These experiments
indicate that rHRGs-T-Fc are active molecules and are able to
specifically induce HER4 tyrosine phosphorylation.
Figure 1:
Tyrosine autophosphorylation of the
HER4 receptor following rHRGs-T-Fc stimulation. A, CHO/HER4
cells were incubated in the absence (lane1) or
presence of rHRG--T-Fc (lanes2 and 3),
rHRG-
2-T-Fc (lanes4 and 5), and
rHRG-
3-T-Fc (lanes6 and 7) at 50 ng/ml
(lanes2, 4, and 6) or 200 ng/ml
(lanes3, 5, and 7). Cells were
lysed, and proteins were separated by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with anti-phosphotyrosine antibodies.
Horseradish peroxidase-conjugated goat anti-mouse IgG antibodies and
chemiluminescence reagents were used to visualize the bound antibodies.
B, CHO/EGFR cells were incubated in the absence (control
(c); lane1) or presence of EGF (lane2), rHRG-
-T-Fc (lane3),
rHRG-
2-T-Fc (lane4), and rHRG-
3-T-Fc
(lane5) at 200 ng/ml. Cells lysates were processed
as described for A. The positions of HER4 and EGFR are
indicated.
Binding of rHRG-
After demonstrating that the three fusion proteins are
activators of HER4, we further characterized rHRG--T-Fc to HER4-expressing
Cells
-T-Fc and
checked its ability to bind to HER4-expressing cells. MDA-MB-453 cells,
which are known to express the HER4 receptor
(13) as well as the
related receptors HER2 and HER3
(37, 38) , were used in
this assay. These cells were incubated on ice with 1 or 10 µg/ml
rHRG-
-T-Fc. Bound fusion proteins were detected by adding
fluorescein-conjugated anti-human IgG antibodies, which recognized the
human Fc portion of rHRG-
-T-Fc. As shown in Fig. 2,
rHRG-
-T-Fc bound to MDA-MB-453 cells (Fig. 2, B, 10
µg/ml; and D, 1 µg/ml). Fluorescence, as analyzed by
confocal microscopy, was localized at the periphery of the cells, which
is consistent with the fact that the staining was performed on live
cells kept on ice. When no fusion protein was added, the
fluorescein-conjugated anti-human IgG showed no detectable binding to
the MDA-MB-453 cells (Fig. 2A). Minimal background
staining was observed when an irrelevant Tek-Fc fusion protein was used
at 10 µg/ml (Fig. 2C). This experiment indicates
that rHRG-
-T-Fc binds to HER4-expressing cells and can be used to
detect cells expressing HRG-binding proteins in a manner similar to
monoclonal antibodies. Thus, rHRGs-T-Fc might represent an interesting
alternative to antibodies for cell staining.
Figure 2:
Binding of rHRG--T-Fc to MDA-MB-453
cells. Cells were plated on 8-well Lab-Tek chamber slides at 2
10
cells/well. After 2 days, the cells were placed on ice
and stained with rHRG-
-T-Fc at 10 µg/ml (B) and 1
µg/ml (D) or with an irrelevant fusion protein used at 10
µg/ml (C). No fusion proteins were added in the experiment
shown in A. Fluorescein-labeled goat anti-human Fc antibodies
were used to visualize the bound fusion proteins. Fluorescent staining
was analyzed by confocal microscopy.
Induction of ICAM-1
NDF, the rat homologue of HRG,
has been shown to induce morphological changes in AU565 mammary tumor
cells
(2) as well as the expression of ICAM-1
(17) . We
have previously used the human breast cancer cell line MDA-MB-453 in
differentiation assays to monitor the purification of an HRG isoform,
p45
(14) . These cells can also be induced to differentiate when
treated with rHRG--T-Fc, -
2-T-Fc, and -
3-T-Fc at similar
concentrations (data not shown). To examine the ability of
rHRG-
-T-Fc to induce the expression of ICAM-1 at the surface of
MDA-MB-453 cells, these cells were treated for 3 days with a 50 ng/ml
concentration of the fusion protein and stained with an anti-ICAM-1
antibody. Bound anti-ICAM-1 antibodies were detected using a
fluorescein-conjugated anti-human IgG antibody. As shown in
Fig. 3B, rHRG-
-T-Fc induced a clear up-regulation
of ICAM-1 expression in MDA-MB-453 cells as compared with untreated
cells (Fig. 3A) and with cells treated with an
irrelevant Tek-Fc fusion protein (Fig. 3C). p45, an HRG
isoform purified from conditioned medium from HepG2 cells (14), was
used at 50 ng/ml as a positive control and as expected induced
up-regulation of ICAM-1 (Fig. 3D). In conclusion,
rHRGs-T-Fc elicited biological responses similar to those elicited by
natural HRGs in breast carcinoma cells expressing the HER4 receptor and
thus can be used to study the biological consequences of HRG binding
to such cells.
Figure 3:
Induction of ICAM-1 expression in response
to rHRG--T-Fc. MDA-MB-453 cells were cultured for 24 h on 8-well
Lab-Tek chamber slides. Cells were treated with 50 ng/ml
rHRG-
-T-Fc (B), irrelevant fusion protein (C),
or p45 (14) (D) or were left untreated (A). Following
an additional 3 days of incubation, the cells were stained with an
anti-ICAM-1 monoclonal antibody. Fluorescein-labeled goat anti-mouse Fc
antibodies were used to visualize the bound anti-ICAM-1 antibodies.
Staining was analyzed by confocal
microscopy.
Release of EGF-like Domains of HRGs after Thrombin
Cleavage of Fusion Proteins
We have constructed CDM7-derived
vectors containing sequences encoding a thrombin cleavage site upstream
and in frame with the Fc portion of a human IgG1 antibody. The addition of a thrombin cleavage site in the expression vector
was based on a system developed by Hakes and Dixon
(39) for
recombinant protein expression in bacteria. The presence of a thrombin
cleavage site in rHRGs-T-Fc allowed for separation of the two
functional domains of the fusion proteins. Following thrombin cleavage,
the purified EGF-like domains would be recovered as monomeric proteins
since the thrombin site is located upstream of the hinge region of the
Fc domain of the fusion proteins. rHRG-
2-T-Fc and -
3-T-Fc
were incubated with human thrombin. reHRGs were then separated from the
Fc portion of the molecules by protein A-Sepharose chromatography and
recovered in the column flow-through fractions. Fc portions were
recovered from protein A-Sepharose by acid elution.
Fig. 4
(lane1) shows a silver-stained
polyacrylamide gel of rHRG-
3-T-Fc before thrombin cleavage. The
intact fusion protein displays an apparent molecular mass of 40 kDa
under reducing conditions, corresponding to its monomeric form. After
cleavage but before protein A-Sepharose chromatography (lane2), a 34-kDa band, corresponding to the Fc portion of the
fusion protein, and a 6-kDa band, corresponding to the EGF-like domain
of HRG-
3, were identified. The two fragments were separated by
protein A-Sepharose chromatography. The 6-kDa EGF-like domain of
HRG-
3 (reHRG-
3) was recovered in the column flow-through
fraction (lane3), and the 34-kDa Fc domain of the
fusion protein was acid-eluted from the column (lane4).
Figure 4:
Thrombin cleavage of rHRG-3-T-Fc. The
fusion protein was incubated with human thrombin at room temperature
for 30 min and loaded on a protein A-Sepharose column. reHRG-
3 was
recovered in the column flow-through fraction, while the Fc portion of
the fusion protein was eluted from the column. The resulting products
were analyzed by SDS-PAGE and silver-stained. Lane1,
untreated rHRG-
3-T-Fc; lane2, rHRG-
3-T-Fc
after thrombin cleavage; lane3, protein A-Sepharose
column flow-through fraction (reHRG-
3); lane4,
protein A-Sepharose column eluate (Fc portion of the fusion
protein).
Stimulation of Protein Phosphorylation in Response to
reHRGs
The purified reHRG-2, reHRG-
3, and Fc domains
of rHRG-
2-T-Fc and -
3-T-Fc were tested for their ability to
stimulate protein phosphorylation in MDA-MB-453 cells. As expected,
rHRG-
2-T-Fc and -
3-T-Fc (Fig. 5, lanes3 and 6, respectively) were potent stimulators of the
tyrosine phosphorylation of a 180-kDa protein as compared with
background levels of phosphorylation observed in the absence of
treatment (lane1) or following EGF treatment
(lane2). reHRG-
2 (lane4) and
reHRG-
3 (lane7) elicited an increase in the
phosphorylation level of the 180-kDa protein similar to that obtained
with rHRGs-
-T-Fc, whereas the Fc domains of rHRG-
2-T-Fc and
-
3-T-Fc failed to induce protein phosphorylation (lanes5 and 8, respectively).
Figure 5:
Stimulation of protein phosphorylation in
response to reHRGs. MDA-MB-453 cells were incubated in the absence
(lane1) or presence of EGF (lane2), rHRG-2-T-Fc (lane3),
reHRG-
2 (lane4), the Fc portion of
rHRG-
2-T-Fc (lane5), rHRG-
3-T-Fc (lane6), reHRG-
3 (lane7), and the Fc
portion of rHRG-
3-T-Fc (lane8) at 200 ng/ml.
Cells were lysed, and proteins were separated by SDS-PAGE, transferred
to nitrocellulose membrane, and blotted with an anti-phosphotyrosine
antibody. Immunoreactive bands were visualized with enhanced
chemiluminescence reagents.
Following cleavage of
the fusion proteins, several amino acid residues from the glycine-rich
region of the thrombin cleavage site remain at the carboxyl terminus of
reHRGs. Cleavage occurs at the Pro-Arg recognition sequence of the
thrombin cleavage site (see Ref. 39 for details). These additional
residues did not affect the properties of reHRGs, as demonstrated
above. In fact, among the multiple HRG/NDF isoforms, the region
proximal to the EGF domain (referred to as the juxtamembrane region in
the HRG/NDF precursor forms) can be absent, e.g. HRG-2/NDF-
2, or comprise up to 26 amino acids, e.g. NDF-
4
(1, 4) . A truncated form of NDF-
that lacks the juxtamembrane region displays the same receptor binding
affinity as the full-length NDF
-isoform, implying that this
region proximal to the EGF domain is not involved in receptor
binding
(4) . We conclude that, after thrombin cleavage, reHRGs
retain the activity displayed by rHRGs-T-Fc.
1 can be reconstituted by coexpression of
HER2 and HER3 in COS-7 cells and that binding of HRG results in the
tyrosine phosphorylation of both HER2 and HER3
(40) .
Phosphorylation of Shc upon HER4 Receptor
Activation
Activation of receptor tyrosine kinases, such as the
EGF receptor, the insulin receptor, and the platelet-derived growth
factor receptor, results in the phosphorylation of a number of
intracellular signaling molecules
(18, 19) . In a
preliminary attempt to analyze the molecules that might be involved in
HRG signaling, we stimulated MDA-MB-453 cells with or without 200 ng/ml
reHRG-2. Cell lysates were immunoprecipitated with the following
antibodies: anti-GTPase-activating protein, anti-phospholipase
C-
1, anti-phosphatidylinositol 3`-kinase, and anti-Shc.
Precipitated proteins were separated by SDS-PAGE and then immunoblotted
with anti-phosphotyrosine antibodies. Shc and, to a lesser degree,
phosphatidylinositol 3`-kinase immunoprecipitates displayed enhanced
patterns of protein phosphorylation following HRG stimulation (data not
shown). We decided to further analyze Shc phosphorylation. Shc proteins
are ubiquitously expressed proteins containing a single SH2 domain.
Three structurally related Shc proteins, p46
,
p52
, and p66
, have been
described as adaptor molecules that are implicated in Ras
activation
(23, 34) . CHO/HER4 and MDA-MB-453 cells were
exposed to reHRG-
2 and lysed. Equivalent amounts of cell lysates
were immunoprecipitated with an anti-Shc antibody and blotted with
either anti-Shc (Fig. 6A) or anti-phosphotyrosine
(Fig. 6B) antibodies. Fig. 6A shows that
equal amounts of proteins from stimulated and unstimulated cell lysates
were loaded per lane and that MDA-MB-453 cells (lanes1 and 2) express only p46
and
p52
(p66
was not detected
in our assay), whereas CHO/HER4 cells (lanes3 and
4) express all three Shc isoforms. p66
is translated from a different transcript than the other two Shc
isoforms and is not expressed in every cell type; for example, it is
absent in human hematopoietic cell lines
(23) . As seen in
Fig. 6B, reHRG-
2 induced the hyperphosphorylation
of Shc in both cell types. In MDA-MB-453 cells, reHRG-
2
stimulation resulted in the tyrosine phosphorylation of both
p46
and p52
(lanes1 and 2). Following reHRG-
2 stimulation,
the phosphorylation of p52
was markedly
increased in CHO/HER4 cells (lanes3 and 4).
p46
appeared to display a relatively high
endogenous level of phosphorylation in those cells and was only
marginally affected following HRG treatment. A longer exposure time of
the blot shown in Fig. 6B (lanes3 and
4) resulted in a loss of resolution between the
p46
and p52
bands, but
revealed that p66
was phosphorylated in response
to reHRG-
2 (lane6) as compared with
unstimulated cells (lane5). We were not able to
show, by immunoprecipitation, ligand-induced association of Shc with
HER4 in CHO/HER4 cells (data not shown), an interaction that has been
reported to take place between Shc and the EGFR
(23) , the
HER2/p185
receptor
(41) , or
the platelet-derived growth factor receptor
(29) . Further
studies will be required to assess the ability of HER4 to recruit Shc.
The possibility remains that Shc might indirectly bind to HER4 via
another adaptor molecule, such as
Grb2
(22, 24, 25, 26, 27) .
Figure 6:
Tyrosine phosphorylation of Shc proteins
upon HER4 activation. MDA-MB-453 cells (lanes1 and
2) and CHO/HER4 cells (lanes 3-6) were treated
with (+) or without (-) 200 ng/ml reHRG-2 for 10 min at
37 °C and solubilized. Cell lysates containing equal amounts of
protein (1 mg) were precipitated with a polyclonal rabbit anti-Shc
antibody. Immune complexes were washed, separated by SDS-PAGE, and
transferred to nitrocellulose. A, Shc proteins were detected
by immunoblotting using a monoclonal anti-Shc antibody; B, the
tyrosine phosphorylation of Shc proteins was analyzed by immunoblotting
using anti-phosphotyrosine antibodies (Anti-Ptyr). The
positions of the three Shc isoforms are indicated. IP,
immunoprecipitation.
In
summary, we have generated recombinant EGF-like domains of HRG-,
-
2, and -
3 fused to a thrombin cleavage site followed by the
Fc domain of a human IgG1. These reagents can be used in in vitro assays as fusion proteins or as a source of truncated recombinant
HRGs. We have demonstrated in this study that both forms can activate
the HER4 receptor and elicit known HRG biological responses. We have
shown for the first time that, following HRG stimulation, Shc proteins,
which have been implicated in the Ras activation pathway, are
phosphorylated on tyrosine. The availability of recombinant HRGs will
allow us to further dissect the mechanism of HRG receptor signaling as
well as to compare the HER4 substrates with those of other members of
the EGFR family of tyrosine kinases.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.