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-Shc Association and Shc Pathway Activation
Departments of Internal Medicine (R.X.-D.S., R.A.M., M.S., R.J.S.) and Biomolecular Research Facility (Y.B.), University of Virginia School of Medicine, Charlottesville, Virginia 22908; and Department of Molecular & Cellular Oncology (L.A., R.K.), University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Robert X. Song, Division of Hematology and Oncology, University of Virginia Health Science Center, Charlottesville, Virginia 22901. E-mail: rs5wf{at}virginia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and Src are upstream
regulators of Shc. A rapid physical association between ER and Shc
upon E2 stimulation further evidenced the role of ER on Shc
activation. Mutagenesis studies showed that the phosphotyrosine binding
and SH2 domains of Shc are required to interact with the activation
function 1, but not activation function 2, domain of
ER. Using a glutathione-S-transferanse-Shc
pull-down assay, we demonstrated that this ER-Shc association was
direct. Biological consequences of this pathway were further
investigated at the genomic and nongenomic levels. E2 stimulated
MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy
studies showed that E2 rapidly induced formation of membrane ruffles,
pseudopodia, and ER membrane translocation. The E2-induced
morphological changes were prevented by antiestrogen. Together
our results demonstrate that ER can mediate the rapid effects of E2
on Shc, MAPK, Elk-1, and morphological changes in breast cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß, which are mainly located
in the nucleus. As ligand-dependent transcriptional factors, the
hormone-bound ERs interact with estrogen response elements to
stimulate certain genes in E2-responsive tissues and to regulate gene
transactivation (2). The genomic actions of E2 are
characterized by delayed onset of action and sensitivity to inhibitors
of transcription and translation. Many of the E2-induced
transcriptional mechanisms involve ER, which can be functionally
regulated by posttranslational phosphorylation of serine residues at
the 104, 106, 118, 167, and 236 positions (3, 4, 5, 6). In
addition, tyrosine-537 of ER has been reported to be phosphorylated
in MCF-7 cells and in ER-expressing Sf9 insect cells
(7). The biological function of ER is also regulated by
association with a subset of nuclear proteins, such as coactivators,
corepressors, and integrator proteins (8, 9, 10, 11).
More recently, investigators have recognized rapid nongenomic actions
of E2 on several cellular processes, such as activation of Ras
(12), Raf-1 (13), PKC (14), PKA
(15), Maxi-K channels (16), increments in
intracellular calcium levels (17), and an increase of
nitric oxide (17). Elicited responses depend upon the cell
types studied and the conditions used. In contrast to its
transcriptional effects, the precise nongenomic signaling pathways of
E2, especially the involvement of ER in this process, are not well
understood and have not been extensively studied
(18, 19, 20).
Several recent reports demonstrated that E2 rapidly activates MAPK in a
number of model systems (13, 21, 22), but the mechanisms
responsible for this are still controversial (23, 24, 25, 26).
Many growth factor receptors on the cell membrane, such as the
receptors for epidermal growth factor (27), nerve growth
factor (28), platelet-derived growth factor
(27), and IGF (29), activate MAPK through a
Shc-mediated pathway. We hypothesized that E2 might co-opt a pathway
using Shc-Grb2-Sos (Src homology-growth factor receptor binding
protein 2-son of sevenless) to activate MAPK. The adapter protein Shc
has no intrinsic kinase domain and transduces signals dependent on the
association with membrane receptors. Three domains mediating
protein-protein interactions have been reported on Shc. Two of these,
the phosphotyrosine binding (PTB) domain in the amino-terminal region
and the Src homology 2 (SH2) domain in the carboxy-terminal region are
separated by a region rich in proline and glycine residues, called the
collagen homology (CH) domain (30). Shc binds rapidly to
the specific phosphotyrosine residues of receptors through its PTB or
SH2 domain and becomes phosphorylated itself on tyrosine residues of
the CH domain (27). The phosphorylated tyrosine residues
on the CH domain provide the docking sites for the binding of the SH2
domain of Grb2 and hence recruit Sos, a guanine nucleotide exchange
protein. This adapter complex formation allows Ras activation via the
Sos protein, leading to the activation of the MAPK pathway (27, 31, 32). Since ER has been reported to be associated with the
membrane of MCF-7 cells (20), we hypothesized that this
receptor might be involved in mediating E2 action on MAPK activation by
physical interaction with Shc in a manner analogous to that in growth
factor pathways.
To test our hypothesis, we used MCF-7 and its variant LTED cells. LTED
cells were developed from parental MCF-7 cells growing long-term in
estrogen-depleted medium (33). This maneuver causes a 5-
to 10-fold up-regulation of ER expression (33). In the
present study, we demonstrated that E2 rapidly stimulated both MAPK and
Shc phosphorylation and induced the association of Shc with ER.
Using selective inhibitors and a dominant mutant Shc, we further
demonstrated that Shc is upstream of MAPK and that the rapid actions of
E2 are ER-dependent and involve ER/Shc association. Furthermore,
a direct physical association of ER with Shc was evidenced by
protein pull-down assay. Our results show that the PTB and SH2 domains
of Shc interacted with the AF-1 region of ER. Tyrosine-537 of ER
was not involved in this interaction. Measurement of Elk-1 activation
and morphological changes by confocal microscopy provided further
evidence of the rapid, nongenomic role of E2 in these cells. Together,
our data suggest that the nongenomic effects of E2 on Shc and MAPK
activation are mediated by the classical ER receptor, that E2
can induce an association of ER with Shc, and that Shc appears to be
a crucial step for the activation of MAPK.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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shows that E2 increased both p42 and
p44 MAPK phosphorylation in a time-dependent manner with the peak
at 15-min treatment. We then questioned whether the effects of E2 on
MAPK involved ER, MEK (MAPK kinase), and Src tyrosine kinase. As
shown in Fig. 1B, the antiestrogen ICI, the MEK inhibitor
PD98059, and a pan Src inhibitor PP2 all blocked E2-induced MAPK
phosphorylation, implying that ER, MEK, and Src family members are
required for this step. PP2 alone abolished basal MAPK phosphorylation,
suggesting the role of Src in maintaining basal MAPK function (Fig. 1B).
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). Together, these results indicated that ER, Src, and Shc are all
upstream components regulating MAPK activation.
E2 Rapidly Induces Shc Pathway Activation
To further confirm the involvement of Shc in the E2 rapid
signaling pathway, we evaluated this step in both MCF-7 and LTED cells
that express a 10-fold elevation of ER levels. As shown in
the top panel of Fig. 2A, E2
at 10-10 M increased p52
kDa Shc phosphorylation in a time-dependent manner with the
maximum response at 3 min. When LTED cells were compared with MCF-7
cells, Shc was phosphorylated to a greater extent in LTED cells
(left in Fig. 2A) than parental MCF-7 cells
(right in Fig. 2A) under basal conditions and increased
proportionately in response to E2. To validate the results, the
polyvinylidene difluoride (PVDF) membranes were reprobed
with anti-Shc antibodies. The results show a similar amount of Shc
proteins of both p46 and p52 kDa in MCF-7 and LTED cells (Fig. 2A, bottom panels). E2-induced Shc phosphorylation was also
dose-dependent with the optimal dose at 10-10
M (data not shown).
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, Grb2/Shc
(left panel) and Grb2/Sos (middle and
right panels) complexes were faintly apparent in the
vehicle-treated group, but greatly increased by
10-10 M E2 treatment at 3
min. Together, these results suggest that E2 is capable of activating
the Shc-mediated signaling pathway in MCF-7 cells, which is regulated
by the level of ER expression.
Effects of Selective Signaling Pathway Inhibitors on E2-Induced Shc
Phosphorylation
Because Shc is responsible for the activation of MAPK by E2, we
wished to determine components responsible for Shc phosphorylation.
Additionally, we wished to demonstrate a role for ER in this process
and to exclude MAPK as the cause of Shc phosphorylation. Shc has been
reported to be a substrate of c-Src in HEK-293 cells (37).
Accordingly, we examined the effects of PP2, ICI, and PD98059 on
E2-induced Shc phosphorylation. In the presence of the inhibitors,
MCF-7 cells were stimulated with vehicle or
10-10 M E2 for 3 min, and the status
of Shc phosphorylation was examined. Figure 2C shows that both PP2 and
ICI effectively inhibited E2-induced Shc phosphorylation, indicating
that Src family kinases and ER are required for Shc activation. As
expected, PD98059 did not influence Shc phosphorylation status,
suggesting that MAPK functions downstream of Shc. No effects of
these inhibitors were apparent in the absence of E2 stimulation. These
results indicate that both ER and Src are upstream components of Shc
functionality, and their involvement is required for Shc
phosphorylation.
E2-Stimulated ER Association with Shc
We postulated that ER might mediate effects of E2 on Shc by
directly or indirectly associating with the Shc adapter protein. This
concept was based on the fact that activated Shc transduces signals by
associating with membrane receptors and our evidence that ICI
blocked E2-induced Shc phosphorylation in MCF-7 cells. To test this
hypothesis, we immunoprecipitated Shc protein and detected ER on
Western blots. As shown in Fig. 3, E2
rapidly induced the association of ER with Shc in both LTED and
MCF-7 cells (top panel). The induction was seen as early as
1 min of E2 treatment and reached a plateau from 3 to 10 min.
Compared with MCF-7 cells, ER/Shc complexes were present basally in
LTED cells, which express 5- to 10-fold higher levels of ER, and
greatly increased in response to E2 treatment. Consistency of protein
loading was confirmed by reprobing the membrane with anti-Shc antibody
(bottom panel). Thus, the association between ER and Shc
was observed not only in MCF-7 cells, but it was greatly enhanced in
ER up-regulated LTED cells. The results were also confirmed by the
immunoprecipitation of ER and the detection of Shc on immunoblot
(data not shown). Together, our data suggest that as an upstream signal
of Shc, the level of ER expression affects the level of Shc
phosphorylation and ER/Shc association.
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/Shc Interactions
and Shc, various GST-tagged Shc and ER mutants were employed (Fig. 4A). Furthermore, to
minimize factors specific to breast cancer cells, COS-1 cells that do
not have detectable endogenous ER were used and electroporated with
different ER and Shc expressing vectors (Fig. 4A). First, we
examined the Shc-interacting domain(s) on ER by electroporating
wild-type Shc (ShcWT) with either wild-type ER expressing vector
(HEGO) or a mutant ER expressing vector in which phenylalanine was
substituted for tyrosine in the 537-position (Y537F). As shown in Fig. 4B, the Y537F mutation did not alter the Shc/ER complex formation
compared with wild-type ER, suggesting that Y537 of ER is not the
site interacting with Shc. Coexpression of either the truncated
activation function 1 (AF-1) or activation function 2 (AF-2)
domains of ER with ShcWT in COS-1 cells confirmed that it is the
AF-1, but not AF-2 (contains Y537), domain of ER that interacts with
Shc (Fig. 4, C and D). E2 increased the association of AF-1
with Shc (Fig. 4C). The mechanism for this is not known and was
unexpected, since AF-1 does not contain a binding site for E2. We
postulate that E2 might exert effects on AF-1 or Shc binding proteins
independently of ER, leading to an increase of AF-1/Shc
association.
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-interacting site(s) on Shc. When ShcWT
was coexpressed with ER (HEGO) in COS-1 cells, we demonstrated an
association of GST-Shc protein with ER under basal conditions and
further enhancement of this association with 5 min of E2 treatment
(Fig. 4E, top left panel). We then used selective
domain-expressing Shc mutants to examine which domains of Shc were
necessary for this association. Both the PTB (ShcPTB)- and the SH2
(ShcSH2) domain-expressing proteins formed complexes with ER, which
further increased in intensity upon E2 stimulation (Fig. 4E, top
right panel). Proteins that only expressed the GST or CH domain of
Shc failed to interact with ER. After normalizing the GST-Shc
immunoprotein bands (Fig. 4E, top panels) for ER protein
loading (Fig. 4E, bottom panels), we show that the Shc/ER
association induced by E2 is 2- to 3-fold higher than the control in
cells expressing PTB or SH2 and is similar to that in cells containing
ShcWT.
Shc can be a substrate of c-Src that associates with ER in MCF-7
cells after E2 treatment (38). It would be of interest to
know whether c-Src is involved in this ER-Shc complex in our breast
cancer system. Using several different anti-c-Src (B-12, N-16, and
SRC2, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
anti-ER antibodies (D-12 and MC-20, Santa Cruz Biotechnology, Inc.), we did not detect c-Src in ER-Shc complexes of either
MCF-7 or LTED cells (data not shown). To further address the molecular
basis of Shc-ER interaction, we tested whether this interaction is
direct or mediated by third-party proteins using GST-pull-down assays.
Both GST-tagged SH2 and PTH+CH domain fusion proteins of Shc were
produced at the size of 37 kDa and 68 kDa (Fig. 4F, bottom
panel). The 62-kDa recombinant full-length ER was pulled down
by both GST-tagged Shc proteins (Fig. 4F, top panel). As
negative control, the beads and GST protein alone did not pull down the
ER protein. The specificity was confirmed by immunoprecipitation and
detection of ER with anti-ER antibodies, but not by the
nonspecific or anti-vitamin D receptor antibodies. Together, our
results demonstrate that the interaction between ER and Shc is
direct and does not require the presence of any other proteins.
Biological Effects of E2-Induced Nongenomic Pathway Activation
To further provide evidence that the ER-Shc-MAPK pathway is
biologically relevant, we used two approaches. First, we evaluated the
role of MAPK on the activation of Elk-1, which is a transcriptional
factor phosphorylated and activated by MAPK (39). Second,
we examined the rapid effects of E2 on morphological changes of MCF-7
cells.
To study Elk-1 activation by E2, a well characterized reporter system
utilizing both GAL4-Elk-1 and GAL4-luciferase vectors (40)
was cotransfected into MCF-7 cells. Figure 5 shows that E2 increased Elk-1
activation in a dose-dependent manner at 6 h as shown by
luciferase assay. The E2 doses higher than 10-12
M significantly stimulated Elk-1 activation, indicating the
involvement of MAPK in E2 action.
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/Shc association and Shc phosphorylation upon E2
treatment.
Under basal conditions (Fig. 6A), LTED
cells exhibited a low level of membrane ruffles (red),
indicating minimal actin polymerization sites. ER (green)
appeared to be predominantly in the nucleus with some also present in
the cytoplasm. Beginning at 10 min (data not shown) and increasing by
20 min, E2 induced dramatic morphological effects, including the
formation of additional membrane ruffles and cell shape alterations
(Fig. 6B). Even more dramatic was the formation of pseudopodia shown in
Fig. 6C as an arm-like extension of the cell membrane with a fist-like
structure at its terminus. To demonstrate that ER mediated these
morphological responses, the effects of E2 were abrogated by
coadministration of the pure antiestrogen ICI along with E2 (Fig. 6D).
ICI alone resulted in no morphological changes (Fig. 6E).
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location and alterations in response to E2.
Accordingly, we focused on the region contiguous to the cell membrane.
The insets in Fig. 6F show enlarged areas indicated by
arrows in panels A–E. Under basal conditions, minimal
green staining could be observed along the cell membrane of
the cells (Fig. 6A, inset a, right panel). In
marked contrast, E2 appeared to translocate ER into the region along
the membrane ruffles as indicated by the strong appearance of
green staining (Fig. 6B, inset b,right panel). As
shown by merging the red (actin) and green
(ER) views, the ER appeared as yellow (Fig. 6B, inset b, left panel), indicating colocalization with actin
in the membrane ruffles. Striking also was the translocation of
the ER into the fist-like region of the pseudopodia as shown by both
the green staining (Fig. 6C, inset c, right
panel) and yellow merged views (Fig. 6C, inset c,
left panel). ICI blocked E2-induced ER membrane
translocation with little effect by it alone (Fig. 6, D and E;
inset d, right and left panels; and inset
e, right and left panels). The immunofluorescence
staining for vinculin (blue) represents the focal adhesion
contacts of the cells that are not highly visible.
The morphological changes demonstrated in Fig. 6 represent examples of
consistent changes observed in many cells. To establish that these
changes were statistically significant, we used an integrated measure
of membrane changes called dynamic membrane formation (DM). This
integrated parameter includes both membrane ruffles and pseudopodia. As
shown in Fig. 7, the E2-induced increase
in DM was statistically significant and could be blocked by ICI.
Collectively, these results demonstrated that E2 rapidly induced
specific changes on cell morphology in addition to its effects on
membrane-mediated Shc-MAPK activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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with Shc, the phosphorylation of Shc, and the formation of Shc-Grb2-Sos
complexes. Shc is critical for E2-induced MAPK activation. Using a
mutagenesis approach, we demonstrated that the SH2 and PTB domains of
Shc are the sites interacting with ER and that the AF-1 region of
ER is required to interact with Shc.
Evidence of the biological relevance of this pathway is inferred from
the demonstration that Elk-1, one of the mediators of MAPK, was
increased by E2. More importantly, we obtained direct biological
evidence of the nongenomic effects of E2 by demonstrating morphological
changes with confocal microscopy. These studies showed that E2 rapidly
induced formation of membrane ruffles and pseudopodia, but also
translocation of ER to the membrane and into perimembrane regions of
pseudopodia. Finally our results demonstrated that
E2, mediated by ER, activated a classical
growth factor-signaling pathway involving Shc adapter proteins.
The adapter protein Shc mediates the effects of many growth factors to
activate MAPK. However, several recent studies demonstrated different
mechanisms whereby E2 can induce MAPK activation. McDonnell
and associates (34) showed that calcium is required for
E2-induced MAPK activation in MCF-7 cells, and this event is ER
dependent. E2-induced MAPK activation was also reported to be
independent of ER or ERß but required the existence of epidermal
growth factor receptor (EGFR) and GRP30, a G protein-coupled receptor
homolog in an ER-negative breast cancer cell line MDA-MB-231
(25). EGFR expression in MCF-7 cells is very low, and
Parsons and colleagues (42) have reported that EGFR
protein is not detectable in MCF-7 cells either by immunoprecipitation
or by immunoblotting methods. In our system, we do not detect EGFR in
either MCF-7 or its variant cells (data not shown).
In the present study, we demonstrated that ER, as a key mediator, is
required for E2 action on Shc/ER association and Shc and MAPK
phosphorylation. Approximately 0.5% of total ER was estimated to be
associated with Shc after E2 stimulation as shown by an ER-Shc
coimmunoprecipitation study (data not shown). The activation of MAPK
with E2 is less than that by growth factors, as reported by other
investigators (34). Our unpublished data also show that
MAPK phosphorylation induced by 10 ng/ml TGF was 35-fold higher than
untreated controls, compared with 2.5-fold induction induced by
10-10 M E2. ER-dependent MAPK
activation has been reported by many laboratories using MCF-7 and other
cell lines treated with E2 (39, 43). The
differences regarding ER involvement in MAPK activation under
varying conditions are unknown. Nevertheless, it is not surprising
that E2 can activate multiple rapid signaling pathways in different
cell lines due to the fact that it can exert nongenomic effects on
nitrous oxide, PKC, PKA, and MAPK. Detailed examination of E2 induction
through each of these mechanisms will be required before a full
understanding of the biological relevance of these various pathways
emerges.
A single time point of Shc phosphorylation induced by E2 was first
reported in MCF-7 cells (13). We extended these
observations by conducting detailed time studies and showed the effects
as early as 1 min after E2 treatment. ER is required for Shc
phosphorylation, and the association of ER with Shc appears to be
important for Shc activation. It has been suggested that PTB and SH2
are two distinct binding domains, which may link to phosphorylated
tyrosine residues in a different manner (44). Both PTB and
SH2 domains of Shc are required to associate with EGFR
(45), but Neuregulin-activated ErbB2 and ErbB3 bind only
to PTB domain of Shc (46). The functional roles of the PTB
or SH2 sites in their interaction with ER are now under
investigation.
The PTB and SH2 domains of Shc interact with phosphorylated tyrosine
residue(s) of other proteins. The exact tyrosine residue of the ER
responsible for binding to Shc is unknown. Since tyrosine-537 of human
ER may serve as a constitutively phosphorylated site
(7), we considered it a possible docking site for Shc.
However, our data show that the AF-1 domain of ER, but not the AF-2
or tyrosine-537 region, interacts with Shc, indicating that there might
be other tyrosine residues phosphorylated on the N-terminal ER. We
postulated that 1 of 10 potential tyrosine residues in the AF-1 region
(amino acids 1–179) might be involved in the association with Shc,
such as tyrosine 43, 52, 54, 60, 73, 80, 130/131, 139, and 150. Further
investigation of these sites is now being conducted in our
laboratory.
ER has been reported to associate functionally with many cell
membrane proteins, such as IRS-1, the p85 subunit of PI3K, and caveolin
(47, 48, 49). c-Src has been reported to physically associate
with ER of bovine uterus, but there is no direct evidence that
ER-associated c-Src is responsible for Shc phosphorylation
(13). The present study demonstrated that the Src
inhibitor PP2 blocked Shc phosphorylation induced by E2. This evidence
suggests that a Src protein family member is functionally involved in
E2-induced Shc phosphorylation, even though Src is not detectable in
the ER-Shc complex. It is possible that other Src family members are
involved in phosphorylating Shc or that the Src-ER complex is
unstable during immunoprecipitation.
The breast cancer models used in our studies involve parental MCF-7 and
its variant LTED cells. When compared with its parental cells, LTED
cells show an increased growth rate in E2-deprived media and respond to
very low concentrations of E2 when grown in nude mice
(50). ER, but not ERß, is detectable in our MCF-7 and
its variant cells (33, 50). The presence of increased
amounts of ER appears to amplify the components examined in the
ER-Shc signaling pathway. Whenever effects, such as ER/Shc
association and Shc phosphorylation, were compared in LTED and MCF-7
cells, the responses were qualitatively similar but quantitatively
greater in the variants. For this reason, we used this variant for
demonstrating certain effects such as Grb2/Sos interactions and
morphological changes by confocal microscopy.
Our studies demonstrated rapid, nongenomic effects of E2 by two
separate methods, biochemical assessment of the Shc-MAPK pathway and
morphological effects on cell shape, membrane ruffles, pseudopodia, and
ER translocation. Both effects could be blocked by the antiestrogen
ICI, indicating that Shc-MAPK activation and morphological changes were
all initiated by ER. Even though we do not yet have direct evidence
that the observed E2 effects on cell structure are mediated by
activation of the Shc and MAPK pathway, the role of Shc and MAPK on
cell adhesion and motility has been demonstrated in several cell lines
(51, 52). The interaction of Shc with membrane integrin in
MCF-7 cells was reported to regulate the locomotion and cell adhesion
of MCF-7 cells (53). We postulate that the E2-induced
membrane signaling pathway activation, especially ER-involved
activation of Shc, might be linked to cell morphology changes in our
cell models.
The suggestion that ER can reside in or near the cell membrane has
been controversial in the past (54). Recent transfection
studies provided additional support that ER can be present in the
cell membrane (55). Razandi et al.
(55) transfected ER into Chinese hamster ovary (CHO)
cells and demonstrated that 3% of expressed ER was associated with
cell membranes. Immunochemical studies with several anti-ER
antibodies on fixed cells including MCF-7 cells also suggested
the presence of membrane ER (20). Our observation using
confocal microscopy showed that E2 increased the amounts of ER in
the region of the cell membrane. Our findings provide the first
evidence that E2 can functionally influence the amount of ER
translocated into the perimembranous area. At the present time, we do
not have direct morphological evidence that Shc is associated with this
membrane-associated ER, but we plan to examine this issue in the
future.
In conclusion, we demonstrated that the signaling pathway mediating the
rapid action of E2 involved the interaction of Shc with ER, Shc
activation, and the phosphorylation of MAPK. The PTB and SH2 domains of
Shc as well as AF-1 domain of ER are the sites mediating the
interaction. Using selective pathway inhibitors and a dominant negative
Shc mutant, we confirmed that ER-mediated Shc activation resulted in
MAPK phosphorylation in MCF-7 cells. As evidence of functionality, E2
rapidly stimulated cell morphological changes and induced activation of
the MAPK substrate, Elk-1. Additional studies are now required to
provide further proof of the role of each of the signaling molecules in
the biological events observed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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recombinant protein was
purchased from Affinity BioReagents, Inc. (Golden, CO).
Protease inhibitors, leupeptin, pepstatin, and aprotinin, were
purchased from Sigma (St. Louis, MO).
Mammalian expression vectors encoding human 52-kDa forms or
partial regions of Shc with GST fusion proteins were gifts from Dr.
Ravichandran Kodi (University of Virginia, Charlottesville, VA) and
have been described previously (46). The amino acid
sequences encoded by the different Shc constructs are: full-length
(1–471), PTB (17–203), CH (233–377), and SH2 (377–471). The
full-length human ER expression vector (HEGO) was a gift from Dr.
Rosalie Uht (University of Virginia) and has been previously reported
(56). The ER expression vectors of AF-1, AF-2, and the
Y537F mutant in which tyrosine 537 was mutated into phenylalanine were
constructed in our laboratory by PCR and site-directed mutagenesis
(QuickChange, Stratagene, La Jolla, CA) as previously
described (57). The fusion gene GAL4-Elk-1 and its
reporter gene GAL4-E1B-Luc were generously provided by Dr. Michael J.
Weber (University of Virginia) and have been described previously
(40).
Cell Culture
The MCF-7 cells and COS-1 cells were maintained in 5%
FBS-IMEM and 10% FBS-DMEM, respectively. The LTED cells were developed
from MCF-7 cells by growing long term (i.e. 6 months to 2
yr) in phenol red-free IMEM supplemented with 5% Dextran-coated
charcoal-stripped FBS and have been extensively characterized with
respect to growth characteristics, responsiveness to E2, and ER content
(33, 50). As determined by ligand binding assays as well
as mRNA and Western blots, these cells contain 5- to 10-fold higher
levels of endogenous ER than the parental MCF-7 cells. The content
of ERß is negligible to absent as demonstrated by Western blot and
PCR (data not shown). All cell lines were routinely cultured in a
humidified 95% air-5% CO2 incubator at 37
C.
Cell Stimulation and Lysate Preparation
For rapid E2-induced signaling pathway studies, cells
cultured in IMEM containing 1% dextran-coated charcoal-stripped FBS
were treated at 37 C with vehicle or E2. Time and doses are indicated
in the figure legends. If inhibitors were used, cells were pretreated
with the inhibitors for 30 min and then challenged with E2. Cells were
washed once with ice-cold PBS containing 1 mM
Na2VO4 and extracted with
binding buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 25
mM NaF, 2 mM
Na2VO4, and 10 µg/ml of
each aprotinin, leupeptin, and pepstatin). Cell lysates were
centrifuged at 14,000 x g for 10 min at 4 C to pellet
insoluble material. The supernatant of cell extract was analyzed for
protein concentration by a DC protein assay kit based on the Lowry
method (Bio-Rad Laboratories, Inc.).
Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting were carried out as
described previously (58). Briefly, 1 mg of cell lysate
from each treatment was immunoprecipitated using one of the following
antibodies: 1 µg monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology, Inc.), 1 µg monoclonal anti-ER antibody
(D-12, Santa Cruz Biotechnology, Inc.), 1.2 µg
polyclonal anti-Grb2 antibody (C-23, Santa Cruz Biotechnology, Inc.), 1 µg polyclonal anti-GST antibody, and 1 µg
monoclonal anti-Sos antibody (Transduction Laboratories, Inc., Lexington, KY). Incubations proceeded for 4 h at room
temperature or overnight at 4 C in the presence of 35 µl of 50%
slurry protein-G or protein-A Sepharose beads (Life Technologies, Inc.). The beads were washed three times in cold binding
buffer. The proteins eluted from the beads were analyzed on 10%
SDS-polyacrylamide gels and transferred to PVDF membranes. The PVDF
membranes were probed with one of the following primary antibodies:
horseradish peroxidase-conjugated monoclonal antiphosphotyrosine
antibody (PY20, Transduction Laboratories, Inc.),
polyclonal anti-ER antibody (HC-20, Santa Cruz Biotechnology, Inc.), polyclonal anti-Shc (Transduction Laboratories, Inc.), polyclonal anti-GST antibodies (Z-5, Santa Cruz Biotechnology, Inc.), monoclonal anti-Grb2 (Transduction Laboratories, Inc.), monoclonal anti-GST, monoclonal anti-ER
N-terminal antibody (COIM2133, Fisher Scientific),
polyclonal anti-C-terminal ER antibodies (MC-20, Santa Cruz Biotechnology, Inc.), and polyclonal anti-Sos antibodies
(Transduction Laboratories). After washing the PVDF membrane, the
immunoblots were incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 h [donkey against rabbit IgG from
Pierce Chemical Co. (Rockford, IL) or sheep against mouse
IgG from Amersham Pharmacia Biotech (Piscataway, NJ)] and
further developed using the chemiluminescence detection system
(Pierce Chemical Co.). The reciprocal studies for all
protein-protein interactions were conducted and the same results were
confirmed. All experiments were done at least three times.
GST Pull-Down Assay
Prokaryotic vectors expressing both PTB+CH and SH2
domains of Shc were gifts from Dr. Ravichandran Kodi (University of
Virginia), expressed in Escherichia coli BL21 and purified
with a GST-glutathione affinity system (Amersham Pharmacia Biotech). GST, GST-ShcPTB+CH, and GST-ShcSH2 proteins were
induced, solubilized, and bound to Glutathione beads. To test the
protein interaction, 15 µl of the suspension were incubated in
binding buffer with 5 ng of human full-length ER expressed and
purified from Baculovirus. After incubation, the beads were washed
three times with the buffer. Bound proteins were eluted with 40 µl of
2x SDS-PAGE buffer, electrophoretically separated in 10% SDS gel, and
transferred on the PVDF membrane. The membrane was probed with
monoclonal anti-ER antibody, and the immunoprotein bands were
visualized as described above.
Electroporation and Transfection
COS-1 cells were electroporated exactly as described by Roy
et al. (59) with 5 µg each of Shc constructs
and 5 µg of each ER-expressing vectors. Cells were then incubated
in DMEM supplemented with 10% FBS for 48 h. The medium was
changed again with serum-free and phenol-free DMEM for 1 d. Four
hours before E2 treatment, the medium was changed again with fresh
serum-free and phenol-free medium. Cells were then treated with E2 for
5 min and extracted with binding buffer. At this step, the samples were
either stored at -80 C or prepared for protein-protein association
assay. To study MAPK-mediated Elk-1 activation, MCF-7 cells were
transiently transfected with both 2 ng of GAL4-Elk-1 and 0.1 µg of
GAL4-E1B-Luc reporter vectors encoding GAL-Elk fusion protein and
GAL-luciferase, respectively. Transfection was performed with Effectene
transfection reagent (QIAGEN, Valencia, CA)
according to company specifications; 24 h after transfection,
cells were treated with different doses of E2 for 6 h. Luciferase
activity was measured for each lysates, and samples were normalized by
ß-galactosidase cotransfected with above vectors. All transfections
were conducted at least three times and the data are presented as
mean ± SD.
Confocal Analysis of E2-Induced Cell Morphology Changes
Cells were fixed in paraformaldehyde followed by chilled acetone
and blocked in normal goat serum followed by incubation with monoclonal
antibody against vinculin (Sigma) and with polyclonal
antibodies against ER (Santa Cruz Biotechnology, Inc.).
For triple costaining of filamentous actin, ER and vinculin and 0.1
µM Alexa 546-conjugated phalloidin were included during
incubation with the Alexa 633-goat antimouse and Alexa 488 goat
antirabbit secondary antibodies. Slides were mounted, and analyzed by a
LSM inverted microscope (Carl Zeiss, Thornwood, NY)
(60). Each image represents Z sections at the same
cellular level and magnification. Confocal analysis was performed using
a Carl Zeiss laser scanning confocal microscope and
established methods, involving processing of the same section for each
detector and comparing pixel by pixel. Localization of ER with
ruffles is demonstrated by the development of yellow color
due to red and green overlapped pixels. The
effect of E2 on the changes of filamentous actin in terms of dynamic
membrane was measured based on the formation of ruffles and pseudopodia
from 12 cells in each treatment. Extensive application of identical
methods for analysis of effects of heregulin has been recently
published by Adam et al. (41).
|
ACKNOWLEDGMENTS |
|---|
-expressing
vector. |
FOOTNOTES |
|---|
Abbreviations: AF-1 and AF-2 domains, Activation function 1 and
2 domains; CH domain, collagen homology domain; EGFR, epidermal
growth factor receptor; Grb2, growth factor receptor binding protein 2;
GST, glutathione-S-transferase; HEGO, human ER
expression vector; ICI, ICI 182 780; IMEM, improved MEM; LTED cells,
MCF-7 cells growing long-term in estrogen-depleted medium; MEK, MAPK
kinase; PTB domain, phosphotyrosine binding domain; PVDF,
polyvinylidene difluoride; Shc, Src-homology and collagen homology;
ShcWT, wild-type Shc; Sos, Son of Sevenless; Src, Src family of
tyrosine kinases; c-Src, p60 of Src.
Received for publication February 20, 2001. Accepted for publication September 6, 2001.
|
REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
by protein kinase A
regulates dimerization. Mol Cell Biol 19:1002–1015 by S118
phosphorylation involves a ligand-dependent interaction with TFIIH and
participation of CDK7. Mol Cell 6:127–137[CrossRef][Medline]
transcriptional activation
through phosphorylation of serines 104 and 106 by the cyclin A-CDK2
complex. J Biol Chem 274:22296–22302. Steroids 64:5–13[CrossRef][Medline]
knock-out mice. J Neurosci 20:1694–1700-subunit promoter by
gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
-subunit gene requires ErbB interaction with Shc. J Neurochem 73:2358–2368[CrossRef][Medline]
(ER)
signaling: Caveolin-1 drives ligand-independent nuclear translocation
and activation of ER. J Biol Chem 274:33551–33556 rapidly activates the IGF-1
receptor pathway. J Biol Chem 275:18447–184535ß1 integrin interactions
regulate breast cancer cell adhesion and motility. Exp Cell Res 252:439–448[CrossRef][Medline]
and ERß expressed in Chinese hamster
ovary cells. Mol Endocrinol 13:307–319 and ß.
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|
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|
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|
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|
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|
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|
|
|
|
|
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|
|
|
|
|
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|
|
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|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
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|
|
|
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|
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|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
E Horner-Glister, M Maleki-Dizaji, C J Guerin, S M Johnson, J Styles, and I N H White Influence of oestradiol and tamoxifen on oestrogen receptors-{alpha} and -{beta} protein degradation and non-genomic signalling pathways in uterine and breast carcinoma cells J. Mol. Endocrinol., December 1, 2005; 35(3): 421 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S Zhang, X Li, R Burghardt, R Smith III, and S H Safe Role of estrogen receptor (ER) {alpha} in insulin-like growth factor (IGF)-I-induced responses in MCF-7 breast cancer cells J. Mol. Endocrinol., December 1, 2005; 35(3): 433 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L-A Martin, S Pancholi, C M W Chan, I Farmer, C Kimberley, M Dowsett, and S R D Johnston The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling Endocr. Relat. Cancer, December 1, 2005; 12(4): 1017 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Boerner, M. A. Gibson, E. M. Fox, E. D. Posner, S. J. Parsons, C. M. Silva, and M. A. Shupnik Estrogen Negatively Regulates Epidermal Growth Factor (EGF)-Mediated Signal Transducer and Activator of Transcription 5 Signaling in Human EGF Family Receptor-Overexpressing Breast Cancer Cells Mol. Endocrinol., November 1, 2005; 19(11): 2660 - 2670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Cui, R. Schiff, G. Arpino, C. K. Osborne, and A. V. Lee Biology of Progesterone Receptor Loss in Breast Cancer and Its Implications for Endocrine Therapy J. Clin. Oncol., October 20, 2005; 23(30): 7721 - 7735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. F. Zheng, R.-C. Wu, C. L. Smith, and B. W. O'Malley Rapid Estrogen-Induced Phosphorylation of the SRC-3 Coactivator Occurs in an Extranuclear Complex Containing Estrogen Receptor Mol. Cell. Biol., September 15, 2005; 25(18): 8273 - 8284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Rude, T.-A. S. Duhaney, G. M. Kuster, S. Judge, J. Heo, W. S. Colucci, D. A. Siwik, and F. Sam Aldosterone Stimulates Matrix Metalloproteinases and Reactive Oxygen Species in Adult Rat Ventricular Cardiomyocytes Hypertension, September 1, 2005; 46(3): 555 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Haas, S. N. White, L. B. Lutz, M. Rasar, and S. R. Hammes The Modulator of Nongenomic Actions of the Estrogen Receptor (MNAR) Regulates Transcription-Independent Androgen Receptor-Mediated Signaling: Evidence that MNAR Participates in G Protein-Regulated Meiosis in Xenopus laevis Oocytes Mol. Endocrinol., August 1, 2005; 19(8): 2035 - 2046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-B. Poirier, G. Hamann, M.-E. Domingue, M. Roy, T. Bardati, and M.-F. Langlois General Receptor for Phosphoinositides 1, a Novel Repressor of Thyroid Hormone Receptor Action that Prevents Deoxyribonucleic Acid Binding Mol. Endocrinol., August 1, 2005; 19(8): 1991 - 2005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boulay, J. Rudloff, J. Ye, S. Zumstein-Mecker, T. O'Reilly, D. B. Evans, S. Chen, and H. A. Lane Dual Inhibition of mTOR and Estrogen Receptor Signaling In vitro Induces Cell Death in Models of Breast Cancer Clin. Cancer Res., July 15, 2005; 11(14): 5319 - 5328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Grossmann, A. Benesic, A. W. Krug, R. Freudinger, S. Mildenberger, B. Gassner, and M. Gekle Human Mineralocorticoid Receptor Expression Renders Cells Responsive for Nongenotropic Aldosterone Actions Mol. Endocrinol., July 1, 2005; 19(7): 1697 - 1710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Arreguin-Arevalo and T. M. Nett A Nongenomic Action of 17{beta}-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone Biol Reprod, July 1, 2005; 73(1): 115 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R J Santen, R X Song, Z Zhang, R Kumar, M-H Jeng, A Masamura, J Lawrence Jr, L Berstein, and W Yue Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S61 - S73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Rai, A. Frolova, J. Frasor, A. E. Carpenter, and B. S. Katzenellenbogen Distinctive Actions of Membrane-Targeted Versus Nuclear Localized Estrogen Receptors in Breast Cancer Cells Mol. Endocrinol., June 1, 2005; 19(6): 1606 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Bjornstrom and M. Sjoberg Mechanisms of Estrogen Receptor Signaling: Convergence of Genomic and Nongenomic Actions on Target Genes Mol. Endocrinol., April 1, 2005; 19(4): 833 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Osborne and R. Schiff Estrogen-Receptor Biology: Continuing Progress and Therapeutic Implications J. Clin. Oncol., March 10, 2005; 23(8): 1616 - 1622. [Full Text] [PDF]
|
|
|
|
|
|
S. Curtis Hewitt, J. Collins, S. Grissom, B. Deroo, and K. S. Korach Global Uterine Genomics in Vivo: Microarray Evaluation of the Estrogen Receptor {alpha}-Growth Factor Cross-Talk Mechanism Mol. Endocrinol., March 1, 2005; 19(3): 657 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.J. Haisenleder, L.L. Burger, K.W. Aylor, A.C. Dalkin, H.E. Walsh, M.A. Shupnik, and J.C. Marshall Testosterone Stimulates Follicle-Stimulating Hormone {beta} Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells Biol Reprod, March 1, 2005; 72(3): 523 - 529. [Abstract] [Full Text] [PDF]
|
|
|
|
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J. S Lewis, T J Thomas, R. G Pestell, C. Albanese, M. A Gallo, and T. Thomas Differential effects of 16{alpha}-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells J. Mol. Endocrinol., February 1, 2005; 34(1): 91 - 105. [Abstract] [Full Text] [PDF]
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K. L. Chambliss, L. Simon, I. S. Yuhanna, C. Mineo, and P. W. Shaul Dissecting the Basis of Nongenomic Activation of Endothelial Nitric Oxide Synthase by Estradiol: Role of ER{alpha} Domains with Known Nuclear Functions Mol. Endocrinol., February 1, 2005; 19(2): 277 - 289. [Abstract] [Full Text] [PDF]
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P. Thomas, Y. Pang, E. J. Filardo, and J. Dong Identity of an Estrogen Membrane Receptor Coupled to a G Protein in Human Breast Cancer Cells Endocrinology, February 1, 2005; 146(2): 624 - 632. [Abstract] [Full Text] [PDF]
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C. K. Osborne, J. Shou, S. Massarweh, and R. Schiff Crosstalk between Estrogen Receptor and Growth Factor Receptor Pathways as a Cause for Endocrine Therapy Resistance in Breast Cancer Clin. Cancer Res., January 15, 2005; 11(2): 865s - 870s. [Abstract] [Full Text] [PDF]
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 F. Acconcia, P. Ascenzi, A. Bocedi, E. Spisni, V. Tomasi, A. Trentalance, P. Visca, and M. Marino Palmitoylation-dependent Estrogen Receptor {alpha} Membrane Localization: Regulation by 17{beta}-Estradiol Mol. Biol. Cell, January 1, 2005; 16(1): 231 - 237. [Abstract] [Full Text] [PDF]
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 L. Moro, S. Reineri, D. Piranda, D. Pietrapiana, P. Lova, A. Bertoni, A. Graziani, P. Defilippi, I. Canobbio, M. Torti, et al. Nongenomic effects of 17{beta}-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor {beta} and Src kinase Blood, January 1, 2005; 105(1): 115 - 121. [Abstract] [Full Text] [PDF]
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R I Nicholson, C Staka, F Boyns, I R Hutcheson, and J M W Gee Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy Endocr. Relat. Cancer, December 1, 2004; 11(4): 623 - 641. [Abstract] [Full Text] [PDF]
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M. Razandi, A. Pedram, I. Merchenthaler, G. L. Greene, and E. R. Levin Plasma Membrane Estrogen Receptors Exist and Functions as Dimers Mol. Endocrinol., December 1, 2004; 18(12): 2854 - 2865. [Abstract] [Full Text] [PDF]
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J. A. Huhtakangas, C. J. Olivera, J. E. Bishop, L. P. Zanello, and A. W. Norman The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1{alpha},25(OH)2-Vitamin D3 in Vivo and in Vitro Mol. Endocrinol., November 1, 2004; 18(11): 2660 - 2671. [Abstract] [Full Text] [PDF]
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I. M. Abraham, M. G. Todman, K. S. Korach, and A. E. Herbison Critical in Vivo Roles for Classical Estrogen Receptors in Rapid Estrogen Actions on Intracellular Signaling in Mouse Brain Endocrinology, July 1, 2004; 145(7): 3055 - 3061. [Abstract] [Full Text] [PDF]
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R. I. Fernando and J. Wimalasena Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and Akt Mol. Biol. Cell, July 1, 2004; 15(7): 3266 - 3284. [Abstract] [Full Text] [PDF]
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Z. Yang, C. J. Barnes, and R. Kumar Human Epidermal Growth Factor Receptor 2 Status Modulates Subcellular Localization of and Interaction with Estrogen Receptor {alpha} in Breast Cancer Cells Clin. Cancer Res., June 1, 2004; 10(11): 3621 - 3628. [Abstract] [Full Text] [PDF]
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 S. Cabodi, L. Moro, G. Baj, M. Smeriglio, P. Di Stefano, S. Gippone, N. Surico, L. Silengo, E. Turco, G. Tarone, et al. p130Cas interacts with estrogen receptor {alpha} and modulates non-genomic estrogen signaling in breast cancer cells J. Cell Sci., March 15, 2004; 117(8): 1603 - 1611. [Abstract] [Full Text] [PDF]
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R. X. Song, C. J. Barnes, Z. Zhang, Y. Bao, R. Kumar, and R. J. Santen The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor {alpha} to the plasma membrane PNAS, February 17, 2004; 101(7): 2076 - 2081. [Abstract] [Full Text] [PDF]
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 A. M. FOWLER, N. SOLODIN, M. T. PREISLER-MASHEK, P. ZHANG, A. V. LEE, and E. T. ALARID Increases in estrogen receptor-{alpha} concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen FASEB J, January 1, 2004; 18(1): 81 - 93. [Abstract] [Full Text] [PDF]
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R. Schiff, S. A. Massarweh, J. Shou, L. Bharwani, S. K. Mohsin, and C. K. Osborne Cross-Talk between Estrogen Receptor and Growth Factor Pathways as a Molecular Target for Overcoming Endocrine Resistance Clin. Cancer Res., January 1, 2004; 10(1): 331S - 336S. [Abstract] [Full Text] [PDF]
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R. J. Santen, R. X. Song, Z. Zhang, W. Yue, and R. Kumar Adaptive Hypersensitivity to Estrogen: Mechanism for Sequential Responses to Hormonal Therapy in Breast Cancer Clin. Cancer Res., January 1, 2004; 10(1): 337S - 345S. [Abstract] [Full Text] [PDF]
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 C. S. Watson and B. Gametchu Proteins of Multiple Classes May Participate in Nongenomic Steroid Actions Experimental Biology and Medicine, December 1, 2003; 228(11): 1272 - 1281. [Abstract] [Full Text] [PDF]
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Y. Kinoshita and S. Chen Induction of Aromatase (CYP19) Expression in Breast Cancer Cells through a Nongenomic Action of Estrogen Receptor {alpha} Cancer Res., July 1, 2003; 63(13): 3546 - 3555. [Abstract] [Full Text] [PDF]
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L. B. Lutz, M. Jamnongjit, W.-H. Yang, D. Jahani, A. Gill, and S. R. Hammes Selective Modulation of Genomic and Nongenomic Androgen Responses by Androgen Receptor Ligands Mol. Endocrinol., June 1, 2003; 17(6): 1106 - 1116. [Abstract] [Full Text] [PDF]
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M. Marino, F. Acconcia, and A. Trentalance Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells Mol. Biol. Cell, June 1, 2003; 14(6): 2583 - 2591. [Abstract] [Full Text] [PDF]
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 G. Castoria, M. Lombardi, M. V. Barone, A. Bilancio, M. Di Domenico, D. Bottero, F. Vitale, A. Migliaccio, and F. Auricchio Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action J. Cell Biol., May 12, 2003; 161(3): 547 - 556. [Abstract] [Full Text] [PDF]
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G. E. Stoica, T. F. Franke, A. Wellstein, F. Czubayko, H.-J. List, R. Reiter, E. Morgan, M. B. Martin, and A. Stoica Estradiol Rapidly Activates Akt via the ErbB2 Signaling Pathway Mol. Endocrinol., May 1, 2003; 17(5): 818 - 830. [Abstract] [Full Text] [PDF]
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E. R. Levin Bidirectional Signaling between the Estrogen Receptor and the Epidermal Growth Factor Receptor Mol. Endocrinol., March 1, 2003; 17(3): 309 - 317. [Abstract] [Full Text] [PDF]
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 D. P. Edwards and V. Boonyaratanakornkit Rapid Extranuclear Signaling by the Estrogen Receptor (ER): MNAR Couples ER and Src to the MAP Kinase Signaling Pathway Mol. Interv., February 1, 2003; 3(1): 12 - 15. [Abstract] [Full Text] [PDF]
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M. Razandi, A. Pedram, S. T. Park, and E. R. Levin Proximal Events in Signaling by Plasma Membrane Estrogen Receptors J. Biol. Chem., January 17, 2003; 278(4): 2701 - 2712. [Abstract] [Full Text] [PDF]
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D. W. Singleton, Y. Feng, C. J. Burd, and S. A. Khan Nongenomic Activity and Subsequent c-fos Induction by Estrogen Receptor Ligands Are Not Sufficient to Promote Deoxyribonucleic Acid Synthesis in Human Endometrial Adenocarcinoma Cells Endocrinology, January 1, 2003; 144(1): 121 - 128. [Abstract] [Full Text] [PDF]
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 K. J. Ho and J. K. Liao Nonnuclear Actions of Estrogen Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1952 - 1961. [Abstract] [Full Text] [PDF]
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C.-W. Wong, C. McNally, E. Nickbarg, B. S. Komm, and B. J. Cheskis Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade PNAS, November 12, 2002; 99(23): 14783 - 14788. [Abstract] [Full Text] [PDF]
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 D. M. Brownson, N. G. Azios, B. K. Fuqua, S. F. Dharmawardhane, and T. J. Mabry Flavonoid Effects Relevant to Cancer J. Nutr., November 1, 2002; 132(11): 3482S - 3489. [Abstract] [Full Text] [PDF]
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M. Marino, F. Acconcia, F. Bresciani, A. Weisz, and A. Trentalance Distinct Nongenomic Signal Transduction Pathways Controlled by 17beta -Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells Mol. Biol. Cell, October 1, 2002; 13(10): 3720 - 3729. [Abstract] [Full Text] [PDF]
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W. Yue, J.-P. Wang, M. Conaway, S. Masamura, Y. Li, and R. J. Santen Activation of the MAPK Pathway Enhances Sensitivity of MCF-7 Breast Cancer Cells to the Mitogenic Effect of Estradiol Endocrinology, September 1, 2002; 143(9): 3221 - 3229. [Abstract] [Full Text] [PDF]
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K. J. Ho and J. K. Liao Non-nuclear Actions of Estrogen: New Targets for Prevention and Treatment of Cardiovascular Disease Mol. Interv., July 1, 2002; 2(4): 219 - 228. [Abstract] [Full Text] [PDF]
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 A. C. B. Cato, A. Nestl, and S. Mink Rapid Actions of Steroid Receptors in Cellular Signaling Pathways Sci. Signal., June 25, 2002; 2002(138): re9 - re9. [Abstract] [Full Text] [PDF]
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V. G. Keshamouni, R. R. Mattingly, and K. B. Reddy Mechanism of 17-beta -Estradiol-induced Erk1/2 Activation in Breast Cancer Cells. A ROLE FOR HER2 AND PKC-delta J. Biol. Chem., June 14, 2002; 277(25): 22558 - 22565. [Abstract] [Full Text] [PDF]
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