(Received for publication, November 8, 1994; and in revised form, January 20, 1995)
From the
Heregulin (HRG) is a pluripotent growth factor that can
stimulate the growth of some human mammary tumor cells and the
differentiation of others. Two members of the epidermal growth factor
receptor family of receptor/tyrosine kinases, p180 and p180
, serve as receptors for
the HRG ligand. While HRG appears to be capable of stimulating the
autophosphorylation activity of p180
, the
co-expression of p185
with
p180
is necessary for the HRG-stimulated
tyrosine phosphorylation of both of these receptors. On the basis of
the sequences surrounding their putative tyrosine phosphorylation
sites, we predict that the different HRG-responsive receptors couple to
different intracellular SH2 domain-containing proteins. Hence, the
different receptors may mediate different cellular responses to the HRG
ligand. In the present study we show that HRG
1 is mitogenic for erbB3-transfected DHFR/G8 cells, an NIH3T3 mouse fibroblast
derivative that overexpresses
p185
. HRG stimulated the
incorporation of [
H]thymidine into the DNA of
these cells with an EC
of 70 ± 7 pM. HRG
was not mitogenic for parental DHFR/G8 cells that do not express the
ErbB3 protein. Phosphatidylinositol (PI) 3-kinase, an enzyme believed
to be important in cellular growth regulation by growth factors and
oncogenes, is predicted to couple to tyrosine-phosphorylated ErbB3. We
observed that HRG stimulated the association of PI 3-kinase with both
p185
and ErbB3 in transfected
DHFR/G8 cells, but not in the parental cell line. We conclude that the
ErbB3 protein is capable of mediating a proliferative response of
fibroblasts to HRG, and that the activation of PI 3-kinase is an
integral part of the growth signaling mechanism.
The epidermal growth factor (EGF) ()receptor and its
relatives p185
,
p180
, and p180
interact with specific polypeptide growth factor ligands to
regulate cellular growth and differentiation(1) . In general,
growth factor binding to its target receptor stimulates the intrinsic
protein tyrosine kinase activity and autophosphorylation of that
receptor. Receptor autophosphorylation then mediates the recruitment of
specific intracellular proteins that possess src homology-2 (SH2)
domains to the activated receptor complex(2) . This in turn
triggers cascades of events that propagate the signal to the nucleus,
culminating in a biological response(3) .
Heregulin (HRG) is
a recently identified EGF-like growth factor that can regulate the
growth of some cultured human mammary tumor
cells(4, 5) , and may be involved in regulating the
growth and differentiation of neuronal tissues(6, 7) .
HRG was originally proposed to be a ligand for
p185 on the basis of its
ability to stimulate the tyrosine phosphorylation of that receptor, as
well as its ability to become covalently cross-linked to
p185
in various cancer cells.
However, HRG does not interact with
p185
in all cells that express
this receptor(8) .
We have recently demonstrated that
p180 is a receptor for
HRG(9, 10) . Interestingly, it appears that
p180
is incapable of mediating transmembrane
signaling in response to HRG, probably because it possesses an impaired
intrinsic tyrosine kinase activity(11) . We have observed that
the co-expression of p180
with
p185
is necessary for the
heregulin-stimulated phosphorylation of both receptors, and for the
creation of a high-affinity binding site for HRG (10) . On the
basis of these observations, we have proposed that a ligand-stimulated
receptor heterodimerization event is responsible for transmembrane
signaling(12) . HRG also appears to be a ligand for
p180
, although in this case a receptor
heterodimerization event is not required to mediate signal
transduction(13, 14) .
Previous studies have
demonstrated that HRG is capable of stimulating the growth of some
human mammary tumor cell lines (5) and the differentiation of
others(4, 15) . The diverse responses elicited by HRG
undoubtedly arise from differences in the cellular signaling pathways
emanating from activated receptors. Hence, interest has developed in
characterizing the intracellular proteins that might participate in
cellular responses to HRG. p180 is unique
among the EGF receptor family members in that it possesses several
putative phosphorylation sites that fit the consensus for binding
phosphatidylinositol (PI) 3-kinase, an enzyme implicated in the
regulation of cellular growth and transformation(16) . It has
been observed that the treatment of some cells with EGF results in the
tyrosine phosphorylation of p180
and the
recruitment of PI 3-kinase to this receptor(17, 18) .
These observations suggest that p180
may play
a role in coupling PI 3-kinase to the action of EGF-like growth
factors, in a manner analogous to that whereby insulin receptor
substrate-1 couples PI 3-kinase to insulin action (19) .
The purpose of the studies described in this report was to determine whether ErbB3 mediates HRG-dependent cell growth and whether PI 3-kinase associates with the ErbB3 protein in response to HRG stimulation.
DHFR/G8 cells were transfected with bovine erbB3 cDNA under the MMTV promoter, and stably transfected clones were
selected by G418 resistance. As an initial screen for ErbB3 expression,
clones were analyzed for their ability to specifically bind
[I]rHRG
1
, the
bacterially-expressed EGF-like domain of human
HRG(5, 9, 10) . Fig. 1shows the
binding of 0.1 nM [
I]rHRG
1
to
several of the selected cell lines, in the presence and absence of an
excess of unlabeled rHRG
1
. The DHFR/G8
parental cells had no detectable affinity for
[
I]rHRG
1
,
indicating that these cells do not express significant levels of either
ErbB3 or ErbB4. However, many of the selected cell lines were capable
of specifically binding the iodinated growth factor. Clone D33 was
selected for further analysis. Scatchard analysis revealed that these
cells bind the labeled growth factor with a K
of
20 pM, and express modest levels (2-3
10
receptors/cell) of HRG receptors. (
)
Figure 1:
[I]rHRG
1
binding by erbB3-transfected DHFR/G8 cells. DHFR/G8
cells and G418-resistant transfectants were incubated with 0.1 nM [
I]rHRG
1
in
the presence and absence of 50 nM unlabeled
rHRG
1
, as indicated, and the
cell-associated radioactivity determined.
Fig. 2A shows the covalent
cross-linking of
[I]rHRG
1
to the
surface of DHFR/G8 parental cells and D33 transfectants. We observed
that the 7-8 kDa iodinated growth factor cross-linked to three
protein species in D33 cells, producing radiolabeled bands at
175,
190, and >300 kDa (right lane). The appearance of
these bands could be blocked with 50 nM unlabeled
rHRG
1
(middle lane), indicating
that the labeled bands represent receptors for HRG. In addition, no
labeled bands were observed after cross-linking
[
I]rHRG
1
to the
parental DHFR/G8 cells (left lane), indicating that ErbB3
expression is required for
[
I]rHRG
1
binding
activity. All three bands could be immunoprecipitated from RIPA lysates
of D33 cells with anti-ErbB3 (not shown). These observations together
with our previous results (9, 10) suggest that the
175- and 190-kDa bands represent
[
I]rHRG
1
cross-linked to monomeric ErbB3 (see below), while the
>300-kDa band represents a ligand-bound heterodimer of the ErbB3
with p185
.
Figure 2:
Expression of ErbB3 in DHFR/G8 cells. A, cross-linking of
[I]rHRG
1
to
transfectants. DHFR/G8 (left lane) and D33 cells (right
two lanes) were treated with 0.1 nM [
I]rHRG
1
, in
the presence and absence of 50 nM unlabeled
rHRG
1
, as indicated, and cell surface
proteins were cross-linked with 1 mM BS
. Lysates
from cells were resolved by 7% SDS-PAGE and visualized by
autoradiography. B, DHFR/G8 and D33 cells were treated without
and with HRG
1
, and RIPA lysates were
immunoprecipitated with anti-ErbB3(3184). Precipitates were analyzed by
6% SDS-PAGE followed by immunoblotting with anti-ErbB3(3183). C, precipitates similar to those described for B were
resolved by 7% SDS-PAGE and immunoblotted with antiphosphotyrosine. The
data presented are representative of at least three
experiments.
To further demonstrate the
expression of ErbB3 in transfected cells, we analyzed anti-ErbB3
immunoprecipitates from DHFR/G8 and D33 cells by immunoblotting with
anti-ErbB3 antibodies. RIPA lysates were immunoprecipitated with
anti-ErbB3 antibody 3184, precipitated proteins were resolved by
SDS-PAGE, and blotted with anti-ErbB3 antibody 3183. As shown in Fig. 2B, a strong band of 170 kDa and a weaker
band of
185 kDa was observed, but only in the D33 transfectants.
Interestingly, treatment of D33 cells with HRG caused a shift in the
mobility of both bands, consistent with a stimulation of ErbB3
phosphorylation. Immunoblotting of similar anti-ErbB3(3184)
immunoprecipitates with antiphosphotyrosine revealed that the tyrosine
phosphorylation of both bands was markedly increased after treatment of
D33 cells with rHRG
1
(Fig. 2C).
Taken together, the cross-linking
and immunoblotting data suggest that ErbB3 is expressed in transfected
DHFR/G8 cells as a doublet. A similar doublet was observed when wild
type NIH3T3 cells were transfected with bovine erbB3(9) . However, previous studies indicate that
ErbB3 in human cancer cells migrates as a single 180-kDa species (22) , and when we expressed this same bovine erbB3 clone in COS cells, a single 180-kDa species was also
observed(10) . Since the presence of the 171-kDa band varied in
extent from one experiment to the next, we suspect that NIH3T3 cells
and their derivatives may partially proteolyze or only partially
glycosylate the ErbB3 protein. We have observed that both bands may be
immunoprecipitated and immunoblotted with an antibody made to a peptide
corresponding to the carboxyl-terminal 17 amino acids of ErbB3 (not
shown). This suggests that the faster migrating species is not deleted
in its tail region, and that all the putative tyrosine phosphorylation
sites are present in this form.
Figure 3:
Stimulation of
[H]thymidine uptake by HRG. A,
DHFR/G8 and D33 cells were treated without stimulus, or with 5 nM HRG, 10 ng/ml PDGF, or 10% calf serum, and the incorporation of
[
H]thymidine into the trichloroacetic
acid-insoluble cellular fraction was determined after 18 h. The data
were normalized to the extent of [
H]thymidine
incorporation observed in unstimulated cells. B, HRG
dose-response of [
H]thymidine uptake by DHFR/G8 (open squares) and D33 (closed squares) cells. Data
are plotted as the fraction of the response to serum. The curve through
the data points for the D33 cells represents the nonlinear least
squares fit to a single class of HRG binding sites. Error bars in each panel represent the standard error of triplicate samples,
and the data presented are representative of at least four
experiments.
To
test the involvement of PI 3-kinase in HRG signaling, we first examined
stringently washed antireceptor immunoprecipitates for lipid kinase
activity. In the experiment shown in Fig. 4A, DHFR/G8
and D33 cells were treated without and with
rHRG1
and then Nonidet P-40 lysates were
prepared. Antiphosphotyrosine,
anti-p185
, and anti-ErbB3
immunoprecipitates from lysates were assayed for PI 3-kinase activity,
and the stimulation of lipid kinase activity associated with the
various immunoprecipitates by rHRG
1
was
determined for the two cell lines. We observed that HRG stimulated the
association of PI 3-kinase with antiphosphotyrosine and anti-ErbB3
immunoprecipitates in the D33 transfectants by
5-fold, and its
association with anti-p185
immunoprecipitates by
2-fold. No stimulation was observed in
any of the immunoprecipitations from the DHFR/G8 parental cells.
Figure 4:
HRG stimulation of PI 3-kinase. DHFR/G8
and D33 cells were treated without and with HRG, and Nonidet P-40
lysates were immunoprecipitated with antiphosphotyrosine
(-pY), anti-p185
, or anti-ErbB3
antibodies. A, precipitates were assayed for PI 3-kinase
activity, and the fold stimulation observed with HRG treatment was
plotted for the two cell lines. Error bars represent the
standard error of at least three determinations for each
immunoprecipitating antibody. B, precipitates were analyzed by
immunoblotting with anti-p85. Data are representative of at least three
separate experiments.
The
stimulation of associated PI 3-kinase activity with immunoprecipitates
correlated with a stimulation of association with the 85-kDa regulatory
subunit of PI 3-kinase. In the experiment shown in Fig. 4B, antiphosphotyrosine,
anti-p185, and anti-ErbB3
immunoprecipitates from DHFR/G8 and D33 cells were analyzed for the
presence of p85 by immunoblotting. We observed an increased level of
p85 in the various immunoprecipitates only in the D33 cells that had
been treated with rHRG
1
. Since
immunoblotting with antireceptor antibodies revealed identical levels
of receptor in the anti-p185
and
anti-ErbB3 immunoprecipitates (not shown), these results indicate that
PI 3-kinase is recruited to both the ErbB3 and
p185
receptors upon activation with HRG
(see ``Discussion'').
To determine whether PI 3-kinase
might contribute to HRG-stimulated mitogenesis, we examined the effect
of wortmannin on [H]thymidine uptake in D33
cells. Wortmannin is a fungal metabolite that has been demonstrated to
covalently bind to the 110-kDa catalytic subunit of PI 3-kinase. At a
concentration of 100 nM wortmannin specifically targets PI
3-kinase, and inhibits >90% of its lipid kinase activity. (
)We treated D33 cells for 30 min without or with 100 nM wortmannin prior to the initiation of a thymidine uptake assay.
(The conditions of the assay were identical to those used in the
experiment illustrated in Fig. 3A.) We observed that
wortmannin only modestly inhibited the basal and serum-stimulated
[
H]thymidine uptake in D33 cells. However,
wortmannin reproducibly inhibited PDGF-stimulated thymidine uptake by
75% and rHRG
1
-stimulated uptake by
45% (Table 1). These results suggest that the activation of
PI 3-kinase activity by HRG significantly contributes to the mitogenic
response of erbB2- and erbB3-transfected fibroblasts
to this growth factor.
A number of reports suggest that members of the HRG family are mitogenic for a variety of cell types, including cultured Schwann cells (7) and some human mammary carcinoma cells such as the SKBR3 line(5) . In contrast, HRG appears to elicit a differentiative response in certain other breast cancer cells such as the AU547 and MDA-MB-453 lines(4, 15) . The observed differences in cellular responses to the HRGs must arise from differences in the signaling pathways engaged by the growth factor in the different cells. An obvious point of divergence concerns the receptors present at the surfaces of HRG-responsive cells.
Members
of the HRG family can bind to either p180 or
p180
(9, 13) . However, the mechanisms
of receptor activation by the growth factor appears to differ for the
two receptors (12) . HRG stimulation of p180
is
similar to that of the activation of EGF receptor by EGF; ligand
binding stimulates receptor autophosphorylation. On the other hand,
although HRG binds with relatively high affinity to
p180
, interaction of the ligand with this receptor
appears not to be sufficient for the stimulation of p180
tyrosine phosphorylation, probably because p180
has an impaired kinase activity(11) . It appears that the
presence of p185
is necessary for
p180
to respond to HRG(10) , probably the
outcome of a ligand-stimulated receptor heterodimerization
event(12) .
The purpose of the current study was to
determine whether the ErbB2-ErbB3 heterodimeric complex is capable of
mediating a proliferative response to HRG. To address this question, we
employed the DHFR/G8 cell line, an NIH3T3 mouse fibroblast derivative
that expresses high levels of
p185(21) . Although the
constitutive tyrosine phosphorylation of p185
is very high, these cells are not transformed and their growth
responses to serum and PDGF are quite similar to wild-type NIH3T3 cells
(not shown). These observations suggest that the abundance of
p185
and its basal phosphorylation do
not significantly affect the growth properties of the cells. Hence,
these cells provide an ideal system for examining cellular responses to
HRG after introduction of the ErbB3 receptor.
We observed that the
treatment of erbB3-transfected DHFR/G8 cells with HRG1
resulted in a marked stimulation of ErbB3 tyrosine phosphorylation.
However, because of its high degree of basal autophosphorylation it was
impossible to discern an effect of HRG treatment on the tyrosine
phosphorylation of p185
(not shown). Our
results indicate that ErbB3 mediates a mitogenic response of rodent
fibroblasts to HRG
1 by acting as a receptor for the HRG ligand.
Although ErbB3 expression in this system is necessary, it appears that
it is not sufficient to mediate the mitogenic response; the presence of
p185
also appears to be required for
growth signaling. We have observed that clones of erbB3-transfected NIH3T3 cells give a very weak and often
undetectable mitogenic response to HRG
1, (
)consistent
with the observation that there is a relatively low amount of
endogenous p185
in these cells.
Since
the intrinsic tyrosine kinase activity of the ErbB3 receptor appears to
be impaired relative to other members of the class I receptor
family(11) , and since p185 is
already heavily tyrosine phosphorylated in these cells, it is possible
that the mitogenic effect of HRG is due exclusively to the
cross-phosphorylation of ErbB3 by p185
(12) . On the basis of peptide selection experiments with
various SH2 domains(23, 24) , it appears that
p180
has the potential to interact with a different
subset of intracellular signaling proteins than the other three members
of the EGF receptor family(12) . Among the proteins predicted
to interact uniquely with the p180
receptor is p85, the
85-kDa regulatory subunit of PI 3-kinase(12, 17) .
We have observed that both p85 and PI 3-kinase activity associate
with ErbB3 in HRG-treated D33 cells. The observation that wortmannin
significantly inhibits HRG-stimulated
[H]thymidine uptake in D33 cells strongly
suggests that PI 3-kinase is a major contributor to the HRG/ErbB3
mitogenic pathway. Interestingly, PI 3-kinase also appears to associate
with p185
in these cells, even though no
p85 binding sites exist in this receptor. We propose that the observed
association of PI 3-kinase with p185
results from the co-immunoprecipitation of a HRG-stimulated
heterodimeric complex of ErbB3 with
p185
. This notion is supported by our
very recent observations that HRG promotes the co-immunoprecipitation
of p185
with anti-ErbB3 and vice versa. (
)
On the basis of the observations presented here, we
conclude that the ErbB2-ErbB3 receptor complex is capable of mediating
a mitogenic response in rodent fibroblasts, and that PI 3-kinase is
involved in this response. Future experiments will be aimed at
determining the role of the p180 receptor in mediating
cellular responses to HRG.