Estrogen-Induced Activation of Erk-1 and Erk-2 Requires the G Protein-Coupled Receptor Homolog, GPR30, and Occurs via Trans-Activation of the Epidermal Growth Factor Receptor through Release of HB-EGF
Edward J. Filardo,
Jeffrey A. Quinn,
Kirby I. Bland1 and
A. Raymond Frackelton, Jr.
Department of Medicine and Surgery (E.J.F., J.A.Q., K.I.B.)
Rhode Island Hospital and Brown University
Department of
Medicine (A.R.F.) Roger Williams Hospital and Departments of
Medicine and Pathology and Laboratory Medicine Brown University
Providence, Rhode Island 02903
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ABSTRACT
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Estrogen rapidly activates the
mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet
unknown mechanism. Here, evidence is provided that estrogen-induced
Erk-1/-2 activation occurs independently of known estrogen receptors,
but requires the expression of the G protein-coupled receptor homolog,
GPR30. We show that 17ß-estradiol activates Erk-1/-2 not only in
MCF-7 cells, which express both estrogen receptor
(ER
) and
ERß, but also in SKBR3 breast cancer cells, which fail to express
either receptor. Immunoblot analysis using GPR30 peptide antibodies
showed that this estrogen response was associated with the presence of
GPR30 protein in these cells. MDA-MB-231 breast cancer cells
(ER
-, ERß+) are
GPR30 deficient and insensitive to Erk-1/-2 activation by
17ß-estradiol. Transfection of MDA-MB-231 cells with a GPR30
complementary DNA resulted in overexpression of GPR30 protein and
conversion to an estrogen-responsive phenotype. In addition,
GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists,
including ICI 182,780, yet not by 17
-estradiol or progesterone.
Consistent with acting through a G protein-coupled receptor, estradiol
signaling to Erk-1/-2 occurred via a Gß
-dependent, pertussis
toxin-sensitive pathway that required Src-related tyrosine kinase
activity and tyrosine phosphorylation of tyrosine 317 of the Shc
adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2
was dependent upon trans-activation of the epidermal growth
factor (EGF) receptor via release of heparan-bound EGF (HB-EGF).
Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting
EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with
antibodies, or 3) down-modulating HB-EGF from the cell surface with the
diphtheria toxin mutant, CRM-197. Our data imply that ER-negative
breast tumors that continue to express GPR30 may use estrogen to drive
growth factor-dependent cellular responses.
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INTRODUCTION
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Estrogen exerts multiple biological effects on a diverse array of
target tissues. Its actions are required for the development and
maintenance of reproductive tissues, and in some instances are
essential for the growth and survival of tumors that arise from these
tissues. In addition to its impact on the reproductive system, estrogen
regulates bone structure (1), cardiovascular function (2), and the
central nervous system (3). At present, it is unclear whether these
diverse estrogen-mediated biological effects are entirely manifested
via the known estrogen receptors, ER
and ERß. These ERs belong to
the steroid hormone receptor superfamily (reviewed in Ref. 4) and
function as ligand-activated transcription factors. Upon interaction
with estrogen, they undergo conformational changes that result in their
ability to bind DNA and promote gene transcription. In this sense,
estrogen appears to bypass second messenger signaling and directly
promote the transcription of genes required for estrogen-dependent
proliferation. However, in addition to its ability to promote
ER-dependent gene transcription, estrogen rapidly triggers a variety of
second messenger signaling events, including mobilization of
intracellular calcium (5), production of cAMP (6), generation of
inositol phosphate (7), and activation of the mitogen-activated protein
(MAP) kinases, Erk-1 and Erk-2 (8, 9, 10). Although the mechanism by which
these rapid signaling events occur is unknown, due to the rapidity
(within 5 min) by which they are activated it is presumed that they are
initiated at the plasma membrane and do not involve ER-mediated gene
transcription.
Several studies have suggested that ER
and ERß facilitate
this rapid estrogen-stimulated signaling and activation of Erk-1/-2 (5, 11, 12). However, ER
and ERß proteins lack known functional motifs
that would allow for nongenomic mechanisms of estrogen action (13).
Further questions regarding the roles of ER
and ERß in rapid
estrogen signaling are suggested by the effect of pure ER antagonists,
such as ICI 182,780 and ICI 164,384, on these second messenger
signaling pathways. It has been reported that ICI 182,780 prevents
estrogen-induced activation of Erk-1 and Erk-2 (5, 8). In contrast, it
has also been noted that this antiestrogen activates MAP kinases
(MAPKs) and releases intracellular calcium stores (5). Similarly, ICI
164,384 has been shown to potentiate activation of adenylyl cyclase
(6). These observations parallel other studies that have shown that
several steroid hormones and their antihormones may act through
membrane receptors to facilitate rapid nongenomic signaling (14, 15, 16, 17).
Because rapid activation of diverse second messenger signaling pathways
by a single ligand is often mediated by G protein-coupled receptors
(GPCRs), many have speculated that rapid steroid hormone signaling
events may use GPCR signaling mechanisms. This idea is consistent with
data that have implicated G proteins in second messenger signaling by
androgens (18) or progesterone (19).
Recently, Weigel and colleagues isolated a complementary DNA
(cDNA) encoding an orphan member of the G protein-coupled receptor
superfamily, termed GPR30, whose expression is elevated in some
ER-positive vs. ER-negative human breast tumors and cell
lines (20). Here, we test the hypothesis that GPR30 may promote rapid
estrogen-induced activation of Erk-1 and Erk-2. We provide several
lines of evidence that, independent of ER
or ERß, estrogen
activates the MAPK pathway via rapid, GPR30-dependent activation of an
HB-EGF autocrine loop.
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RESULTS
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Estrogen-Induced Erk-1/-2 Activation Is ER Independent and Requires
Expression of the GPCR Homolog, GPR30
Estrogen promotes rapid activation of the MAPKs, Erk-1 and -2 (5, 8, 9, 10). At present, it is unclear whether ER
or ERß is required
for this mechanism of estrogen action. To determine whether
estrogen-induced Erk activation is associated with expression of ER
or ERß, human breast cancer cell lines exhibiting different ER
expression profiles were tested for their ability to activate Erk-1/-2
after exposure to estrogen. Detergent lysates were prepared from
quiescent cells that were either untreated or exposed to estrogens or
EGF for various lengths of time. Erk activity and expression in these
cellular lysates were measured by immunoblotting using phosphorylation
state-dependent and -independent antibodies. In agreement with
observations by others (5, 8), 1 nM 17ß-estradiol induced
a rapid, 5- to 10-fold increase in the phosphorylation state of Erk-1
and Erk-2 in MCF-7 cells, which express both ER
and ERß protein
(21) (Fig. 1
). Surprisingly, however,
17ß-estradiol induced a similar rapid increase in Erk-1/-2 in SKBR3
cells (Fig. 1
) that express neither ER
nor ERß messenger RNA (22).
Erk-1 and -2 activation in these cell types could also be achieved
using 1 µM of the pure anti-estrogen ICI 182,780, a
concentration that blunts both ER
and ERß (23). Although, in
general, the activation kinetics for Erk-1/-2 phosphorylation by
estradiol were similar in each of these cell lines, minor differences
in the onset of Erk phosphorylation were observed. These differences
appeared to be associated with the level of baseline phosphorylated
Erk-1/-2 expressed before estrogen stimulation. In contrast,
17ß-estradiol and ICI 182,780 each failed to activate Erk-1/-2 in
MDA-MB-231 cells, which express only ERß (21). Yet, suggesting no
global defect in signaling to MAPKs in these cells, EGF strongly
activated Erk-1/-2 (Fig. 1
). Collectively, these results support the
hypothesis that estrogen-induced Erk-1/-2 activation occurs via a
non-ER-dependent mechanism.

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Figure 1. Activation of Erk-1/-2 by Estrogens or
Antiestrogens Does Not Correlate with ER Expression
Human MCF-7, SKBR3, or MDA-MB-231 breast carcinoma cells, untreated or
exposed to 1 nM 17ß-estradiol, 1 µM ICI
182,780, or 1 ng/ml EGF for the lengths of time indicated (minutes),
were lysed in ice-cold RIPA detergent. Cellular proteins (50 µg) were
resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and
probed with antibodies specific for phosphorylated Erk-1 and -2. The
nitrocellulose membrane was then stripped and reprobed with antibodies
that recognize total (phosphorylation state-independent) Erk-2 protein.
The relative positions of phosphorylated Erk-1 and -2 and total Erk-2
proteins are designated at the right. The ER expression
profile of each cell line is indicated. The data shown
above are representative of at least three independent
experiments. Below, Band intensities from these
individual experiments were quantified using NIH Image software.
Results were normalized to total Erk-2 expression in each sample and
are plotted with SEM. *, Erk-1/-2 activation significantly
(P < 0.05, by Students t test)
greater than that in unstimulated cells.
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Because a diverse array of extracellular ligands (reviewed in Refs. 24, 25) signal through GPCRs to activate the MAPKs, Erk-1 and Erk-2, we
questioned whether activation of these MAPKs by estrogen may also occur
through a GPCR mechanism. Although there are many orphan receptors that
could be considered candidates for promoting estrogen-induced
activation of Erk-1/-2, we speculated that GPR30, an orphan GPCR whose
expression is elevated in MCF-7 cells relative to MDA-MB-231 cells
(20), may promote estrogen-induced MAPK activation. Therefore, to test
this possibility we asked whether MDA-MB-231 cells, when forced to
overexpress GPR30 protein, would acquire the capacity to activate
Erk-1/-2 in response to estrogen stimulation.
To facilitate the study of GPR30 expression, antibodies were raised in
rabbits to a C-terminal peptide derived from the deduced amino acid
sequence of GPR30. These peptide antibodies identified a single 38-kDa
band that was abundant in MCF-7 and SKBR3 cells and in MDA-MB-231 cells
that had been transfected with a GPR30 expression vector, but was
barely detected in vector-transfected MDA-MB-231 cells (Fig. 2A
). The apparent molecular mass of the
38-kDa band closely approximates the predicted molecular mass (39,815
Da) of the mature 351-amino acid GPR30 polypeptide. Although ER
was
readily detectable in MCF-7 cells, it was not detected in MDA-MB-231 or
SKBR3 cells and was not reacquired in MDA-MB-231 cells upon
transfection with GPR30 cDNA (Fig. 2A
). A small amount of ERß protein
was detected in lysates from MCF-7 and MDA-MB-231 cells; however, no
detectable ERß protein was present in lysates from SKBR3 cells,
consistent with a recent report (22).

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Figure 2. Estrogen-Induced Activation of Erk-1/2 Requires
Cellular Expression of the G Protein Receptor Homolog, GPR30
A, Expression of ER , ERß, and GPR30 in MCF-7, SKBR3, or
MDA-MB-231 cells stably expressing GPR30 or transfected with control
vector was assessed by immunoblotting with antibodies specific for
ER , ERß, or GPR30. B and C, Using phosphorylation state-dependent
or -independent antibodies, phospho-Erk or Erk expression was
determined from whole cell lysates of MDA-MB-231 (GPR30) cells that
were untreated, stimulated with EGF (1 ng/ml; 10 min), or exposed to
17ß-estradiol (1 nM) or ICI 182,780 (1 µM),
or to 17 -estradiol (1 nM), 4-hydroxytamoxifen (1
µM), or progesterone (1 nM) for the indicated
times (minutes). The positions of phosphorylated Erk-1/-2 and total
Erk- 2 proteins are indicated at the right. The data
shown are representative of at least three independent experiments. As
in Fig. 1 , band intensities from independent experiments have been
quantified by densitometry and are plotted with the SEMs.
*, Erk-1/-2 activation significantly (P < 0.05, by
Students t test) greater than unstimulated cells.
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GPR30-transfected MDA-MB-231 cells supported rapid activation of
Erk-1/-2 in response to either 17ß-estradiol or ICI 182,780 (Fig. 2B
). These hormones did not promote Erk-1/-2 activation in MDA-MB-231
cells transfected with the empty vector (data not shown). Activation of
Erk-1/-2 in MDA-MB-231 (GPR30) cells was also induced by 1
µM tamoxifen (partial agonist), but not by the inactive
estrogen isomer, 17
-estradiol, or other sex steroid hormones, such
as progesterone (Fig. 2C
). No differences were observed in total Erk-2
protein expression under any of these conditions (Fig. 2
, B and C).
Thus, these data suggest that when forced to express levels of GPR30
protein comparable to those expressed in MCF-7 or SKBR3 cells,
MDA-MB-231 cells become estrogen responsive with respect to their
ability to activate Erk-1/-2.
Estrogen-Mediated GPR30-Dependent Erk-1/-2 Activation Occurs via a
Gß
, Pertussis Toxin-Sensitive Pathway That Requires Src-Related
Tyrosine Kinase Activity and Shc
Activation of Erk-1/-2 by GPCRs is mediated through the action of
receptor-associated heterotrimeric G proteins (reviewed in Ref. 24).
After ligand-receptor interaction, the G
-subunit protein dissociates
from the heterotrimeric G
ß
complex. Both free G
and the
remaining Gß
complex have been shown to participate in signaling
pathways that may promote Erk-1/-2 activation (reviewed in Ref. 24).
These signaling mechanisms are commonly discriminated based on their
sensitivity to pertussis toxin, tyrosine kinase inhibitors, and
dominant negative effector proteins. Therefore, to further test the
role of GPR30 in promoting estrogen-induced Erk-1/-2 activation, we
examined whether estrogen-induced Erk-1/-2 activation was sensitive to
inhibitors known to disrupt G protein-mediated signaling.
Erk activity and expression were assessed in GPR30-transfected
MDA-MB-231 cells that were untreated or pretreated with either
pertussis toxin or the Src family tyrosine kinase inhibitor, PP2
(26), before stimulation with either 17ß-estradiol or EGF. As shown
in Fig. 3A
, pertussis toxin completely
abrogated the ability of estradiol to activate Erk-1/-2, yet had no
impact on EGF-mediated Erk activation. Likewise, pertussis toxin
inhibited estradiol-induced Erk activation in MCF-7 cells (data not
shown). Similarly, PP2 completely blocked estradiol-induced Erk
activation, indicating a requirement for a Src-related tyrosine kinase
(Fig. 3A
). In contrast, PP2 had no discernible effect on EGF-stimulated
Erk phosphorylation, consistent with a recent report that Src does not
lie on the pathway from the EGF receptor to MAPKs (27). Because MAPK
activation via pertussis toxin-sensitive, Src-dependent, G protein
signaling commonly occurs via a Gß
-subunit protein-mediated
pathway that uses the Shc adaptor protein (28, 29, 30), we next tested
whether estrogen-induced Erk activation could be inhibited by either a
Gß
-sequestrant peptide (31) or a dominant negative Shc protein. To
accomplish this aim, MDA-MB-231 (GPR30) cells were transfected with a
minigene encoding the carboxyl-terminus of the ß-adrenergic
receptor kinase (ßark), dominant negative Shc (shcY317F) or control
vector, pcDNA3.1Zeo. Phospho-Erk and total Erk-2 protein expression
were assessed in MDA-MB-231 (GPR30/ßark), MDA-MB-231 (GPR30/dnshc),
or MDA-MB-231 (GPR30/Zeo) transfectants that had been stimulated with
estradiol or EGF. As observed in Fig. 3B
, cells expressing ßark or
dominant negative Shc failed to phosphorylate Erk-1/-2 in response to
estradiol stimulation, but remained fully competent to activate Erk in
response to EGF stimulation. Zeo-transfected MDA-MB-231 (GPR30) cells
maintained their estrogen responsiveness to Erk activation.

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Figure 3. Estrogen-Mediated, GPR30-Dependent Erk-1/-2
Activation Occurs via a Gß -Subunit Protein-Src-Shc Pathway and
Results in Shc-EGFR Association
A and B, MDA-MB-231 (GPR30) cells untreated or pretreated with
pertussis toxin (PT; 100 ng/ml; 16 h) or the Src family tyrosine
kinase inhibitor, PP2 (50 µM; 15 min), were stimulated
with 17ß-estradiol (1 nM; 5 min) or EGF (1 ng/ml; 15
min). Subsequently, phospho-Erk and Erk expression was determined by
immunoblotting with phosphorylation state-dependent or -independent
Erk-1/2 antibodies. C, MDA-MB-231 (GPR30) cells
transfected with Gß sequestrant peptide, ßark; dominant negative
Shc, GSTShc317Y/F; or control vector, pcDNA3.1Zeo were assessed for
their ability to phosphorylate Erk-1 or -2 after stimulation with
17ß-estradiol or EGF as described in A. D, p66Shc, p52Shc, and p46Shc
proteins were immunoprecipitated using pan-Shc specific antibodies from
1 mg total cellular protein extracted in modified RIPA buffer from
MDA-MB-231, MDA-MB-231 (GPR30), or MDA-MB-231 (GPR30ßark) cells,
untreated or stimulated for 5 min with 17ß-estradiol (1
nM) or EGF (10 ng/ml). Tyrosine-phosphorylated proteins
associated with the Shc immunoprecipitates were detected by
immunoblotting with the phosphotyrosine-specific monoclonal antibody,
4G10. Recovery of Shc protein in the immunoprecipitates was assessed by
stripping the membrane and reprobing with antibodies to Shc. The
170-kDa tyrosine-phosphorylated protein was identified as the EGFR by
reprobing the same membrane with ErbB1-specific antibodies. The
positions of p66Shc, p52Shc, p46Shc, and p170EGFR are indicated at the
right. Mol wt standards are indicated at the
left.
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Agonist stimulation of GPCRs results in rapid tyrosine phosphorylation
of Shc and EGF-related receptors and the formation of Shc-EGF-related
receptor complexes (32). To determine whether 17ß-estradiol might
similarly promote GPR30-dependent tyrosine phosphorylation of Shc and
the formation of Shc-EGF receptor complexes, lysates were prepared from
vector-, GPR30-, or GPR30/ßark-transfected MDA-MB-231 cells that had
been either untreated or treated with 17ß-estradiol or EGF,
immunoprecipitated with pan-Shc antibodies, and then assayed for
phosphotyrosyl proteins (Fig. 3C
). Estradiol stimulated tyrosine
phosphorylation of p66, p52, and p46 Shc isoforms as well as a
Shc-associated 170-kDa protein present in GPR30 expressors, but not in
control-transfected cells or in GPR30 cells coexpressing ßark. This
same pattern of Shc-associated tyrosine-phosphorylated proteins was
observed in each of these cell types when stimulated by EGF. The
170-kDa tyrosine-phosphorylated protein was identified as the EGF
receptor by reblotting with EGF receptor (EGFR)-specific antibody.
Reprobing this membrane with a pan-Shc antibody confirmed that there
was little difference in total Shc protein recovery in these Shc
immunoprecipitates. Taken together, these data suggest that GPR30
signaling occurs through a pertussis toxin-sensitive, Gß
-signaling
mechanism that requires Src family tyrosine kinase activity and
tyrosine phosphorylation of Shc on tyrosine residue 317.
GPR30-Mediated Erk-1/-2 Activation Requires EGF Receptor Tyrosine
Kinase Activity and Occurs through the Release of Cell
Surface-Associated HB-EGF
To further explore the mechanism by which GPR30 promotes EGFR
tyrosine phosphorylation, we examined the effect of specific tyrosine
kinase inhibitors on estrogen-induced activation of Erk-1/-2 and the
EGFR. MDA-MB-231 (GPR30) cells were treated with the EGF receptor
kinase inhibitor, tyrphostin AG-1478; the Her-2/Neu kinase inhibitor,
tyrphostin AG-879; or the Src family tyrosine kinase inhibitor, PP2,
before stimulation with 17ß-estradiol, the pure antiestrogen ICI
182,780, or EGF. Immunoblot analysis showed (Fig. 4A
) that tyrphostin AG-1478 blocked
EGFinduced as well as 17ß-estradiol-induced EGFR tyrosine
phosphorylation, and activation of Erk-1/-2. AG-1478 also similarly
inhibited ICI 182,780-induced activation of Erk-1/-2 (Fig. 4A
). In
contrast, tyrphostin AG-879 did not influence either Erk-1/-2
activation or EGFR tyrosine phosphorylation by estrogen, antiestrogen,
or EGF (Fig. 4A
). The Src family kinase inhibitor, PP2, completely
inhibited 17ß-estradiol-induced EGFR tyrosine phosphorylation (Fig. 4A
) and Erk-1/-2 activation (Figs. 3B
and 4A
). As observed previously,
PP2 pretreatment did not effect EGF-induced Erk-1/-2 activation.
However, PP2 did increase the mobility of the EGFR (Fig. 4A
), probably
due to less extensive EGFR tyrosine phosphorylation on residues 845 and
1101 (33). ICI 182,780-induced tyrosine phosphorylation of the EGF
receptor does not occur in vector-transfected MDA-MB-231 cells that
lack GPR30 (Fig. 4B
), but this antiestrogen does promote EGFR tyrosine
phosphorylation in SKBR3 cells that express elevated levels of GPR30
protein (Fig. 4B
). 17ß-Estradiol did not stimulate EGFR tyrosine
phosphorylation in MDA-MB-231 cells (data not shown), but acted
similarly to ICI 182,780 in SKBR3 cells promoting EGFR activation (Fig. 4B
). Considered together, these data imply that EGFR tyrosine kinase
activity is required for GPR30-dependent, estrogen-induced Erk-1/-2
activation.

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Figure 4. EGF Receptor Kinase Activity Is Required for
GPR30-Mediated Erk-1/-2 Activation
A, MDA-MB-231 (GPR30) cells were treated in the absence or
presence (15 min) of tyrphostins AG-1478 or AG-879 (50
µM) or the Src kinase inhibitor, PP2 (50
µM), before stimulation for 5 min with 17ß-estradiol (1
nM), ICI 182,780 (1 µM), or EGF (10 ng/ml).
Phospho-Erk and Erk-2 expressions in these samples were determined as
previously described. After immunoprecipitation with the ErbB1-specific
antibody, Ab-1, tyrosine-phosphorylated EGFR was detected by
immunoblotting with the phosphotyrosine-specific monoclonal antibody,
PY20. EGFR recovery was assessed by stripping these membranes and
reprobing with ErbB1-specific antibodies. B, MDA-MB-231 or SKBR3
cells were treated with 17ß-estradiol (1 nM), ICI 182,780
(1 µM), or EGF (10 ng/ml) for the indicated times
(minutes) and lysed in ice-cold RIPA buffer. EGFR tyrosine
phosphorylation and recovery were measured as described in A.
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Recent evidence from Ullrich and colleagues suggests that GPCRs
mediate EGFR trans-activation and downstream signaling
through the release of surface-associated heparan-binding EGF (HB-EGF)
precursor protein (34). Therefore, to determine whether
estrogen-induced activation of the EGFR and Erk may occur through a
similar mechanism, we measured EGFR tyrosine phosphorylation and
Erk-1/-2 phosphorylation in MDA-MB-231 (GPR30) cells that had been
treated with HB-EGF-neutralizing antibodies or control rabbit
antibodies before stimulation with ICI 182,780. Pretreatment with
anti-HB-EGF antibodies specifically inhibited ICI 182,780-induced EGFR
tyrosine phosphorylation and Erk-1/-2 phosphorylation, but had no
effect on the ability of exogenous EGF to activate EGFR or Erk-1/-2
(Fig. 5A
).

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Figure 5. GPR30-Mediated EGFR Trans-Activation
by Estrogen Requires HB-EGF
MDA-MB-231 (GPR30) cells were preincubated with rabbit anti-HB-EGF or
control antibodies (36 ng/ml; A) or pretreated with the diphtheria
toxin mutant, CRM-197 (200 ng/ml for 1 h; B) before stimulation
for 5 min with ICI 182,780 (1 µM) or EGF (10 ng/ml). EGFR
tyrosine phosphorylation, EGFR recovery, and phospho-Erk and Erk-2
expression were determined as previously described.
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In addition to its role as a growth factor precursor, pro-HB-EGF is
known to serve as the primary attachment site for diphtheria toxin
(35). The diphtheria toxin mutant, CRM-197, inhibits the mitogenic
activity of HB-EGF (36), and this is related to its ability to
sequester or down-modulate surface-expressed pro-HB-EGF (37).
Therefore, to test further the hypothesis that estrogen acts through
GPR30 to mediate HB-EGFdependent activation of the EGFR and
downstream activation of Erk-1/-2, we measured EGFR and Erk-1/-2
phosphorylation in MDA-MB-231 (GPR30) cells that had been pretreated
with CRM-197 before stimulation with ICI 182,780 or exogenous EGF.
CRM-197 markedly reduced EGFR and Erk-1/-2 activation promoted by ICI
182,780, but showed no effect on the ability of exogenous EGF to
activate either EGFR or MAPK (Fig. 5B
). Similarly, pretreatment with
CRM-197 specifically abrogated estradiol-mediated activation of the
EGFR and MAPK in both MDA-MB-231 cells (GPR30) and MCF-7 cells (data
not shown).
Thus, these data support the model that estrogen-mediated MAPK
activation requires GPR30 and is mediated via the activation of a
Gß
-subunit-Src-Shc pathway that results in
trans-activation of the EGFR and downstream signaling to the
MAPKs, Erk-1/-2, through the release of pro-HB-EGF.
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DISCUSSION
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Other investigators have concluded that estrogeninduced MAPK
activation is promoted by ER
or ERß (5, 8, 11, 12). Their studies
have suggested that in addition to functioning as ligand-activated
transcription factors, these ERs may promote nongenomic signaling
events by estrogen. Although this conclusion is possible, several
issues regarding the capacity of ERs to mediate nongenomic signaling
exist. The structure of the ER, a member of the steroid hormone
receptor superfamily, is well studied, and there are no known
functional motifs within its structure that promote second messenger
signaling (13). Moreover, studies investigating rapid MAPK activation
by estrogen have employed cell lines derived from tissues known to be
estrogen responsive, including MCF-7 breast cancer cells (5, 8),
osteosarcoma cells (9), and neuroblastoma cells (10), but these studies
have not directly addressed the roles of ER
and ERß in promoting
estrogen-induced Erk activation. To test this hypothesis, we examined
estrogen-induced Erk activation in breast cancer cell lines that have
various patterns of ER expression. We found no correlation between the
expression of either ER
or ERß and the ability of estrogen to
activate Erk-1/-2 in these cells (Fig. 1
). In fact, we demonstrate that
either estrogen or the pure antiestrogen ICI 182,780 activates Erk-1/-2
in human SKBR3 breast carcinoma cells, which we and others (22) have
demonstrated lack ER
and ERß, protein, and messenger RNA, strongly
suggesting that the ER is not involved.
Experiments conducted in other cell types have led to suggestions
that membrane-associated ER-like receptors and G proteins may be
responsible for nongenomic estrogen signaling (7, 11, 38). Strongly
supporting such an idea, our evidence indicate that cellular expression
of the orphan receptor, GPR30, is sufficient for estrogen-induced
activation of Erk-1/-2. Employing GPR30 peptide antibodies raised in
our laboratory, we found that human MCF-7 and SKBR3 breast cancer cell
lines that expressed elevated GPR30 protein were capable of activating
Erk-1/-2 in response to estrogen. Moreover, GPR30-deficient MDA-MB-231
breast cancer cells, which are normally nonresponsive to
estrogen-induced Erk-1/-2 activation, can be converted to a responsive
phenotype by overexpression of GPR30 protein (Fig. 2
). Based on our
results with breast cancer cell lines, it is tempting to speculate that
GPR30 may in part define the sensitivity of other tissues to estrogen.
Studies by others (20, 39) indicate that GPR30 has a restricted
expression pattern, with abundant levels in placenta, bone, and brain,
tissues that are considered to be estrogen responsive. Although our
data strongly suggest that GPR30 participates in rapid estrogen
signaling to Erk-1/-2, whether GPR30 acts alone or functions as a
subunit of a receptor complex remains to be determined.
Consistent with GPR30 promoting G protein-dependent activation of
Erk-1/-2, estrogen-induced Erk-1/-2 activation is inhibited by agents
that block G protein signaling. For example, Erk-1/-2 activity induced
by estrogen in GPR30-expressing breast cancer cells is blunted by
pertussis toxin as well as the Src family-specific tyrosine kinase
inhibitor, PP2 (Fig. 3
). In addition, cellular expression of the
carboxyl-terminus of the ß-adrenergic receptor kinase, ßark-1,
which is known to function as a Gß
-subunit protein sequestrant
peptide (31), specifically blocks estrogen-dependent Erk activation in
these cells. A similar inhibitory effect on estrogen-mediated Erk-1/-2
activity was observed upon transfection of a dominant negative Shc
protein (Fig. 3
). Thus, our results indicate that estrogen-induced
activation of Erk-1/-2 occurs via a Gß
-subunit protein
complex-dependent signaling mechanism that requires both Src and Shc.
This mechanism of Erk-1/-2 activation is used by a number of other
GPCRs and is typically Ras dependent (24, 25). Although we did not test
the role of Ras in estrogen-induced Erk-1/-2 signaling, increases in
GTP-bound Ras have been reported after exposure of MCF-7 cells to
estrogen (8). However, estrogen stimulation of these cells by others
did not result in phosphorylation of Raf-1 protein (5). These results
may indicate that estrogen-induced activation of Ras does not require
Raf-1. Alternatively, different G protein-dependent signaling pathways
leading to Erk-1/-2 activation may be used depending on the activation
state of MCF-7 cells before estrogen stimulation. This later
explanation is supported by the finding that intracellular signals have
been shown to determine the coupling of distinct G
ß
heterotrimers with the same GPCR (40).
Although it has been known for some time that Gß
complexes use Src
family nonreceptor tyrosine kinases and Shc to promote intracellular
activation of receptor tyrosine kinases, it has been demonstrated only
recently that many GPCRs activate metalloproteinases that release
pro-HB-EGF from the cell surface. The cleaved HB-EGF, in turn,
activates EGFR signaling pathways (34). Similarly, our data suggest
that estrogen activates Erk-1/-2 by pro-HB-EGFdependent
trans-activation of the EGFR (Figs. 4
and 5
). In this
regard, our findings support prior observations that estrogen
administration to rodents increases levels of local EGF (41) and
stimulates EGFR kinase activity in uterine membranes (42). Moreover,
estrogen-dependent trans-activation of the EGFR underscores
the potential significance of the EGFR in the growth and survival of
female reproductive tissues and breast tumors and is consistent with
studies that have shown high concentrations of EGF-related proteins
(43, 44) and EGFR in these tissues and tumors.
Breast tumors that fail to express ER normally do not respond favorably
to antiestrogen therapy (45). These tumors are referred to as estrogen
independent and are presumed to use growth factor-dependent signaling
mechanisms for their growth and survival. This biological distinction
is furthered by the observation that ER-negative breast tumors commonly
overexpress EGFR-related proteins (46) and that simultaneous expression
of elevated ER and EGFR are rarely observed in cultured breast lines
(47). Consistent with this, it is interesting to note that transfection
of the EGFR cDNA into ER-positive MCF-7 cells results in transient
expression of EGFR that is unstable in the presence of estrogen (48).
In light of our findings, ER-negative breast tumors that express GPR30
may remain estrogen responsive through their ability to promote growth
factor-dependent signals. To the extent that this is true, antagonism
of the EGFR may be beneficial for patients with estrogen-independent or
estrogen-dependent breast tumors. Further studies regarding the
expression of this GPCR in breast tumor specimens will be required to
test this hypothesis.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
Human MCF-7 (ER
+,
ERß+), SKBR3 (ER
-,
ERß-), and MDA-MB-231
(ER
-, ERß+) breast
carcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in phenol red-free
DMEM/Hams F-12 medium (1:1) containing 10% FBS and 100 µg/ml of
gentamicin. MDA-MB-231 transfectants were generated as described below
and were maintained in the same medium supplemented with 500 µg/ml
geneticin (Sigma, St. Louis, MO), 200 µg/ml Zeocin
(Invitrogen, La Jolla, CA), or both.
cDNAs and Dominant Negative Constructs
GPR-BR is a cDNA encoding the full-length human GPR30 protein
subcloned into the pBK-CMV expression vector (20) and was provided by
Ronald Weigel (Stanford University, Palo Alto, CA). The
carboxyl-terminus of ßark-1 has previously been shown to function as
a Gß
sequestrant peptide and was a gift from Robert Lefkowitz
(Duke University, Durham, NC) in the RK-5 vector (31). A molecular
clone encoding glutathione-S-transferase fused to mutant
mouse Shc protein containing a tyrosine to phenylalanine substitution
at residue 317, GSTShcY317F, has been demonstrated to block Shc
signaling and was a gift from Dr. Kodimengalam Ravichandran (49). To
generate constructs suitable for generating stable cell lines
expressing either Gß
sequestrant peptide or the dominant negative
Shc, the respective EcoRI inserts of these clones were
excised and subcloned into the EcoRI site of the
pcDNA3.1Zeo(+) expression vector.
Transfections and Selection of Stable Cell Lines Expressing
Dominant Negative Constructs
MDA-MB-231 cells were transfected with either pBK-CMV vector or
GPR-BR plasmid DNA using Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturers
suggestions. Three days after transfection, 500 µg/ml of geneticin
(Sigma) were added to the growth medium. The resulting
uncloned population of geneticinresistant cells was propagated to
generate cell lines used for further study. MDA-MB-231 (GPR30)
geneticin-resistant cells were retransfected with pcDNA3.1Zeo(+)
constructs expressing either Gß
sequestrant peptide (ßark) or
dominant negative Shc Y317F and were selected for dual resistance in
medium containing (500 µg/ml) geneticin and (200 µg/ml) Zeocin as
described above.
Growth Factors, Estrogens, and Inhibitors
Recombinant human EGF was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Water-soluble
17ß-estradiol; its inactive isomer, 17
-estradiol; progesterone;
and 4-hydroxytamoxifen were purchased from Sigma. The pure
ER antagonist, ICI 182,780, was obtained from Tocris Chemicals
(Ballwin, MO). The diphtheria toxin mutant, CRM 197, was purchased from
Berna Products (Coral Gables, FL). Tyrphostins AG-879 and AG-1478 were
purchased from BIOMOL Research Laboratories, Inc.
(Plymouth Meeting, PA). The former has been shown to preferentially
inhibit Her-2/neu autophosphorylation (50), and the latter has been
demonstrated to be a selective inhibitor of ErbB1 (EGFR) activity (51).
The Src family tyrosine kinase inhibitor PP2 (26) was purchased from
Calbiochem (La Jolla, CA).
Antibodies
Phospho-specific antibodies that recognize phosphorylated
Erk-1 and Erk-2 (phospho-erk) were purchased from New England Biolabs, Inc. (Beverly, MA). The Erk-2 antibodies were also
purchased from the same vendor and are also known to cross-react with
Erk-1. ER
-specific antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ERß-specific antibodies
raised against a synthetic peptide representing amino acids 4663 of
human ERß were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). GPR30-specific antibodies were generated
against a synthetic peptide, CAVIPDSTEQSDVRFSSAV (Multiple Peptide
Systems, San Diego, CA), derived from the carboxyl-terminus of the
deduced amino acid sequence of human GPR30 polypeptide. The GPR30
peptide was covalently coupled to keyhole limpet hemocyanin using the
bifunctional cross-linker,
m-maleimidobenzoyl-Nhydroxysuccinimide
ester, and injected intradermally into New Zealand White rabbits. The
IgG antibody fraction of the immune serum was enriched by affinity
chromatography using protein G-agarose columns. The pan-Shc antibody,
which detects all Shc isoforms, and sheep EGFR antibody, which detects
all ErbB family members, were purchased from Upstate Biotechnology, Inc.. The EGFR (ErbB1) monoclonal antibody (clone
Ab-1) purchased from Calbiochem, recognizes an epitope
within the extracellular domain of the p170 EGFR and does not react
with ErbB2 (Her-2/Neu), ErbB3, or ErbB4. Phosphotyrosine-specific
monoclonal antibodies, 4G10 and PY20, were purchased from Upstate Biotechnology, Inc. and Transduction Laboratories, Inc. (Lexington, KY), respectively. HB-EGF neutralizing
antibodies were purchased from R and D Systems (Minneapolis, MN).
Conditions for Cellular Stimulation and Detergent Lysates
One million cells were seeded onto 90-mm Falcon tissue culture
dishes in phenol red-free DMEM/F-12 medium containing 10% FCS. The
following day, the cell monolayers were washed twice in PBS and placed
in fresh phenol red-free, serum-free medium. Cells were maintained in
phenol red-free medium for an additional 3 days, an interval of time
that we have determined to be necessary to minimize basal levels of
Erk-1/-2 activity. Stimulations of quiescent cells were carried out at
37 C in serum-free medium as described in the figure legends.
Concentrations of 17ß-estradiol (1 nM) and the
anti-estrogen, ICI 182,780 (1 µM) were chosen from
preliminary experiments to provide more than half-maximum
17ß-estradiol activation of Erk-1/-2, in agreement with values
determined by others (8, 9, 10). After stimulation, monolayers were lysed
with ice-cold RIPA buffer consisting of 150 mM NaCl, 100
mM Tris (pH 7.5), 1% deoxycholate, 0.1% SDS, 1% Triton
X-100, 3.5 mM
Na3VO4, 2 mM
phenylmethylsulfonylfluoride, 50 mM NaF, 100 mM
sodium pyrophosphate, plus a protease inhibitor cocktail (Complete,
Roche Molecular Biochemicals, Indianapolis, IN). Crude
lysates were clarified by centrifugation, and protein concentrations
were determined by the bicinchoninic acid method according to the
manufacturers suggestions (Pierce Chemical Co.,
Rockford, IL). Detergent lysates were stored at -70 C until use.
Western Blotting
Total cellular protein (50 µg) was boiled in standard Laemmli
buffer with reducing reagents and resolved by SDS-PAGE. Proteins were
electrotransferred onto nitrocellulose membranes (0.45 µm pore size;
Schleicher and Schuell, Keene, NH) using a semidry transfer cell (CBS,
Del Mar, CA) at 1 mA/cm2 for 4 h.
Phospho-Erk was detected by probing membranes, which were blocked
overnight in Tris -buffered saline containing 0.1% Tween-20 and 2%
BSA (TBST-BSA), with phospho-Erk-specific rabbit antibodies diluted
1:1000 in TBST-BSA for 1 h at room temperature. Rabbit
antibody-antigen complexes were detected with horseradish
peroxidase-coupled goat antibodies to rabbit anti-IgG diluted 1:5000 in
TBST-BSA and visualized by enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech, Arlington Heights, IL).
Relative levels of total Erk-2 protein in each sample were determined
by stripping the phospho-specific rabbit antibodies from the
nitrocellulose membrane and reprobing with antibodies to Erk-2.
ER
and GPR30 proteins were detected on nitrocellulose membranes in
the same manner, except that filters to be probed with GPR30 peptide
antibodies were blocked in TBST containing 5% nonfat dry milk. ERß
was detected using ERß-specific peptide antibodies purchased from
Upstate Biotechnology, Inc., following specifications
provided by the manufacturer. In brief, membranes were blocked for 30
min in PBS containing 3% nonfat dry milk (PBS-MLK), and incubated with
1 µg/ml ERßspecific peptide antibodies diluted in fresh
(PBS-MLK). After an overnight incubation at 4 C, membranes were washed
in water, and immobilized rabbit antibodies were incubated with
horseradish peroxidase-coupled goat antibodies to rabbit anti-IgG
diluted 1:5000 in PBS-MLK for 1.5 h at room temperature. The
membrane was then rinsed in water and washed in PBS containing 0.05%
Tween-20 before visualizing ERß antibody-goat IgG horseradish
peroxidase complexes by ECL (Amersham Pharmacia Biotech).
Apparent mol wts were determined from Rainbow mol wt standards
(Amersham Pharmacia Biotech).
Detection of Tyrosine-Phosphorylated EGFR and Shc-Associated
Tyrosine-Phosphorylated Proteins
Tyrosine phosphorylation of the EGFR was assessed by
immunoblotting EGFR immunoprecipitates with phosphotyrosine-specific
antibodies. EGFR was immunoprecipitated from 500 µg total cellular
protein, extracted in RIPA buffer using 2 µg/sample Ab-1, a
monoclonal antibody to ErbB1. Similarly, Shc-associated
tyrosine-phosphorylated proteins were immunopurified from 1 mg total
cellular protein, prepared in RIPA buffer, and diluted 5-fold in 1%
Nonidet P-40, using 2 µg/sample pan-Shc antibodies. In either case,
antigen-antibody complexes were immunoprecipitated with 50 µl of a
50% slurry of protein G-agarose (Pierce Chemical Co.).
EGFR immunoprecipitates were washed, resuspended in standard Laemmli
buffer containing 875 mM ß-mercaptoethanol, and subjected
to SDS-PAGE. Immunoprecipitated proteins were electrotransferred to
nitrocellulose, blocked with TBS-BSA, and then immunoblotted with the
phosphotyrosine-specific monoclonal antibodies, PY20 or 4G10, diluted
1:1,000 or 1:10,000 in TBS-BSA. Immobilized mouse antibody-antigen
complexes were detected with horseradish peroxidase-coupled sheep
antibodies to mouse IgG diluted 1:5,000 in TBS-BSA and visualized by
ECL. Recovery of EGFR or Shc in each of these immunoprecipitates was
measured by stripping the phosphotyrosine antibodies from the membrane
and reprobing with EGFR or Shc antibodies, respectively.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ronald J. Weigel (Stanford University, Palo Alto,
CA) who supplied us with the GPR30 cDNA. We acknowledge Eva Paradis for
providing excellent secretarial support, and Dr. Timothy W. Baba for
carefully reviewing this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Edward J. Filardo, Department of Surgery, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: edward_filardo{at}brown.edu
This work was supported by Brown University Institutional Research
Training Grant IN-4538 from the American Cancer Society and the T. J.
Martell Foundation (to E.J.F.) and NCI Grants CA-7428501A1 and
A670818 (to K.I.B.).
1 Present address: Department of Surgery, University of Alabama,
Birmingham, Alabama 35294. 
Received for publication May 12, 2000.
Revision received June 26, 2000.
Accepted for publication June 28, 2000.
 |
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