Role of receptor complexes in the extranuclear actions of estrogen receptor α in breast cancer

in Endocrine-Related Cancer
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  • Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA

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Our recent studies have examined the role of various receptor complexes in the mediation of rapid, extranuclear effects of estradiol. This review describes 17β-estradiol (E2)-initiated extranuclear signaling pathways, which involve the insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) and result in the activation of several kinase cascades. The biologic results of these effects are the enhancement of cell proliferation and diminution of programmed cell death (apoptosis). Until recently, most studies assigned priority to the nuclear transcriptional actions of estrogen receptor α (ERα). Present investigative emphasis focuses on the additional importance of ERα residing in or near the plasma membrane. A small fraction of ERα is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as IGF-1R and EGFR, ERα has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming protein/protein complexes with many signaling molecules. Our recent studies demonstrate that the IGF-1R is involved in tethering ERα to the plasma membrane, in activating the EGFR, and in the initiation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling. The formation of a multi-protein complex containing these receptors as well as adaptor proteins is a critical step in this process. A full understanding of the mechanisms underlying these relationships with the ultimate aim of abrogating specific steps, should lead to more targeted strategies for treatment of hormone-dependent breast cancer.

Abstract

Our recent studies have examined the role of various receptor complexes in the mediation of rapid, extranuclear effects of estradiol. This review describes 17β-estradiol (E2)-initiated extranuclear signaling pathways, which involve the insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) and result in the activation of several kinase cascades. The biologic results of these effects are the enhancement of cell proliferation and diminution of programmed cell death (apoptosis). Until recently, most studies assigned priority to the nuclear transcriptional actions of estrogen receptor α (ERα). Present investigative emphasis focuses on the additional importance of ERα residing in or near the plasma membrane. A small fraction of ERα is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as IGF-1R and EGFR, ERα has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming protein/protein complexes with many signaling molecules. Our recent studies demonstrate that the IGF-1R is involved in tethering ERα to the plasma membrane, in activating the EGFR, and in the initiation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling. The formation of a multi-protein complex containing these receptors as well as adaptor proteins is a critical step in this process. A full understanding of the mechanisms underlying these relationships with the ultimate aim of abrogating specific steps, should lead to more targeted strategies for treatment of hormone-dependent breast cancer.

Introduction

In the normal mammary gland, estrogen receptor (ER)-positive cells are largely quiescent. 17β-Estradiol (E2) stimulates these cells to produce growth factors that cause the neighboring ER-negative cells to proliferate through paracrine effects (Cunha et al. 2004). As a result of this process, the ER-positive cells in the breast are Ki67 negative, whereas adjoining ER-negative cells represent the Ki67 positive proliferating pool. As an early step in the neoplastic transformation of the breast, the regulation of proliferation evolves from paracrine to autocrine mechanisms (Streuli & Haslam 1998). In hormone-dependent breast cancer cells, E2 stimulates growth and prevents apoptosis largely through auto-crine mechanisms. Notably, ER-positive breast cancer cells are directly stimulated to grow by E2 as evidenced by the fact that the ER-positive cells have now become Ki67 positive (Anderson & Clarke 2004).

Autocrine mechanisms mediated by the ER can take place in the nucleus of the cell or via extranuclear events. For the past two decades, major investigative emphasis in breast cancer research has focused upon transcriptional events resulting from the binding of E2 to ERs (ERα and ERβ) in the nucleus of the cell. Specifically, the liganded receptor interacts with canonical or non-canonical estrogen response elements in the promoter region of E2 responsive genes, leading to gene transactivation. Other nuclear actions of E2 involve the tethering of the ERα to nuclear transcription factors, such as NF-Y and Sp-1, which also lead to gene transactivation (Wang et al. 1999). These nuclear events usually require hours or days for maximal gene activation. More recently, multiple studies demonstrated very rapid ERα-mediated actions, which occur in the region of the cell membrane and mitochondria. This evolution in understanding of the ERα function has led to a new terminology, which distinguishes the nuclear (genomic) and extranuclear (non-genomic) actions of E2. An alternative but parallel nomenclature describes nuclear-initiated steroid signaling and membrane-initiated steroid signaling (Nemere et al. 2003).

A series of E2-induced extranuclear events has been described in benign as well as in malignant cells of various origins. These effects occur from seconds to minutes after administration of E2 and involve rapid activation of many signaling molecules (Cheskis 2004, Shupnik 2004, Levin 2005), such as (i) the insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR); (ii) p21ras and Raf; (iii) mitogen-activated protein kinase (MAPK) and Akt; (iv) protein kinase C; (v) intracellular calcium transients; (vi) nitric oxide and prolactin secretion; and (vii) Maxi-K channels. While the majority of studies describing extranuclear events focus on biochemical changes, convincing biologic evidence from many systems has accumulated. Within 2 min, vasodilatation in association with nitrous oxide formation occurs in response to physiologic concentrations of E2 in isolated femoral arterial wall (Chambliss et al. 2005, Guo et al. 2005). These effects can be blocked by inhibitors of MAPK and by anti-estrogens. E2 also rapidly stimulates the secretion of prolactin in isolated pituitary cells (Watters et al. 2000). In cancer cells, E2 causes a rapid increase in dynamic membrane structures that can be abrogated with an anti-estrogen (Song et al. 2002).

A recent focus of ERα research has been precisely to define the mechanisms underlying extranuclear actions. Substantial attention has been directed toward proving the hypothesis that kinases activated in the membrane are involved in later nuclear transcriptional events. According to the recent model of O’Malley (2005) ERα on the membrane initially activates cytoplasmic kinases, which in turn phosphorylate and activate coactivator proteins in the cytoplasm. These coactivators then travel to the nucleus and modulate ER-mediated transcriptional events. This integrated view of extranuclear ERα signaling suggests that events occurring initially at the level of the plasma membrane later modulate more classical transcriptional events by crosstalk mechanisms.

Our recent studies have examined the protein–protein complexes, which occur when E2 binds to its receptor in breast cancer cells. Special emphasis is given to the formation of large protein complexes that activate various kinases and mediate downstream effects. We hypothesize that the formation of this ERα-centered large protein complex plays a critical role in initiation of E2 rapid action in breast cancer cells. Activation of many signaling pathways by E2 is regulated by the formation of such complexes and could lead to the activation of MAPK and Akt and translocation of ERα to the region of the plasma membrane. In terms of ERα-centered protein complex formation, c-Src tyrosine kinase, Shc, PELP-1, modulator of nongenomic activity of estrogen receptor (MNAR), phosphoinositide 3-kinase (PI3K), caveolins, and G-proteins have all been reported to serve as components of large complexes of interacting proteins (Song et al. 2005). Through the mediation of these molecules, E2 activates the Shc/MAPK and PI3K/Akt pathways, which are likely to be the major effectors of cell proliferation and cell survival.

Identity of membrane E2-binding proteins

Several possible candidates have been suggested to mediate binding of E2 in the plasma membrane. These include: (i) full length ERα (Razandi et al. 1999); (ii) a truncated form of the ERα with a molecular weight of 46 kDa (Li et al. 2003); (iii) a unique estrogen membrane protein called ER-X, whose ligand affinity differs from that of the classical ER but is recognized by antibodies directed against ERα ligand-binding domain (Toran-Allerand et al. 2002); (iv) sex steroid-binding protein acting in concert with a membrane protein megalin (Catalano et al. 1997, Hammes et al. 2005); (v) G-protein-coupled receptor 30 (Thomas et al. 2005); and (vi) an unknown protein present in non-ERα, non-ERβ expressing, chinese hamster ovary (CHO) and COS-7 cells (Nethrapalli et al. 2005). While these various E2-binding proteins exist in specific systems, accumulating evidence supports the classical full length ERα as the membrane estradiol-binding protein. This has been demonstrated in several types of cells, including breast epithelial cells, osteoblasts, endothelial cells, and vascular smooth muscle cells. Using multiple antibodies against ERα, Watson & Gametchu (1999) demonstrated that the classical ERα is expressed near the region of the cell membrane in MCF-7 cells. Using confocal microscopic methods, our group demonstrated translocation of full length ERα into or near the plasma membrane in response to E2. A proof of principle experiment demonstrated that transfection of ERα-negative breast cancer cells with ERα resulted in 5% of ERα located in the plasma membrane and the remainder predominantly in the nucleus (Razandi et al. 1999).

Biochemical studies also demonstrated full length ERα protein in isolated plasma membranes as detected by mass spectrometry analysis (Pedram et al. 2006). Functional studies demonstrated that the rapid biochemical and biological effects of E2 seen in wild-type mice were abolished in ERKO (ERα knockout) and BERKO (ERβ knockout) animals and that these animals have no demonstrable ERs in isolated plasma membrane preparations (Razandi et al. 2004). Additional evidence supporting the classical ERα in the membrane was obtained from studies in which this receptor was knocked down by a selective siRNA (Song et al. 2004). In this study, E2 activated MAPK in a matter of minutes but abrogation of ERα with a selective siRNA abolished this effect in human breast cancer MCF-7 cells.

Additional studies demonstrated that E2 stimulates MAPK activation only in ERα transfected cells but not in those non-transfected (Razandi et al. 2004). Studies with ERα transfected COS-1 cells demonstrated that only the membrane localized receptor could activate MAPK (Zhang et al. 2002). Harrington et al. demonstrated rapid activation of MAPK using membrane impermeable dendrimers (Harrington et al. 2005). Taken together, these data provide strong evidence that ERα can localize to the region of the cell membrane and initiate kinase-mediated events. However, a critical overview of this field would suggest that truncated ERα, G-protein-coupled receptors, or other proteins may also be involved in rapid estrogen-initiated effects in some cell types and under certain circumstances (Filardo et al. 2000, Chambliss & Shaul 2002, Li et al. 2003).

Mechanism of membrane localization of ERα

Even though ERα has no transmembrane domain and is not intrinsically a membrane protein, several laboratories have demonstrated that extranuclear actions of E2 require ERα translocation in or near the plasma membrane (Lu et al. 2004, Song et al. 2004). Whether ERα membrane translocation and its activation on the cell membrane are sequential or an independent event is not presently known. We also do not understand the biological role of cytosol ERα in E2 rapid action, although effects on the mitochondria have been reported (Chen & Yager 2004).

Our working model suggests that translocation of the ERα to the membrane involves Shc as a transporter. In response to E2, Shc is phosphorylated and binds to ERα. At the same time, the Shc binding sites on the IGF-1R are phosphorylated, allowing Shc to bind to the IGF-1R. This is analogous to Shc acting as a bus, picking up ERα and then delivering ERα to the Shc binding site on IGF-1R. ERα can then be tethered to the membrane through Shc interaction with IGF-1R (Razandi et al. 2002, Song et al. 2004). Substantial proof for our working model has emanated from immunoprecipitation/western blot experiments, from the use of siRNA and dominant negative constructs, from confocal microscopy, and from biochemical studies of kinase activation (Song et al. 2002, 2004). We demonstrated that E2 induces formation of a ternary complex consisting of ERα, Shc, and IGF-1R in the cell membrane of MCF-7 cells. We also showed that E2 causes phosphorylation of the IGF-1R and of Shc. Elimination of Shc either by siRNA expression or by dominant negative constructs abrogates estrogen-induced MAPK activation (Fig. 1). More importantly, knockdown of Shc with a specific siRNA prevents E2-induced ERα translocation to the plasma membrane (Song et al. 2004). However, this knockdown strategy does not eliminate estrogen-induced membrane ruffling, a morphologic change of the plasma membrane, which is associated with cell mobility. Our results suggest that pathways not involving Shc might mediate E2-induced cell mobility changes. Recently, the adapter protein p130Cas was reported to associate with ERα and c-Src in T47D breast cancer cells (Cabodi et al. 2004). As p130Cas is known to interact with focal adhesion kinase, it is conceivable that estrogen-induced cell morphologic changes might be mediated by p130Cas independently of Shc and IGF-1R.

Another protein, striatin was also reported to play a role in translocating ERα into the plasma membrane of EAhy926 human aortic endothelial hybridoma cells, by interacting with amino acid 185–253 of N-terminal ERα (Lu et al. 2004). The protein complex of striatin and ERαleads to the targeting of ERαon the cell membrane. Notably, our preliminary data suggest that E2 also increased the binding of the ERα to the EGFR (Song et al. 2003). Furthermore, palmitoylation of ERα has been reported to enable the association of a truncated ERα with the plasma membrane (Acconcia et al. 2005). Therefore, it is possible that Shc transports ERα to the membrane and that ERα palmitoylation facilitates the persistent residence of this receptor in the membrane. Data from Levin et al. (Razandi et al. 2002) suggest that caveolin is also involved in the reversible shuttling of ERα from the cytosol to the membrane.

Role of the IGF-1R and EGFR in E2 rapid action

IGF-1R is a key receptor involved in the growth of breast cancer cells and its expression is always positively correlated with ERα expression in breast cancer. Estrogens appear to favor synergistic interactions with the IGF-1 system, leading to the expression of several components, such as IGF-1R, IRS1, and IGF-1 (Mauro et al. 2001). These upregulated proteins act locally in autocrine loops to mediate the biologic effects of estrogen. A close relationship between ERα and IGF-1R has been confirmed by the fact that uterine cells do not respond to estrogen if the IGF-IR pathway is blocked and that IGF-1 also loses its effect on gene transactivation and cell proliferation in ERα knockout cells (Klotz et al. 2002). A direct interaction between two pathways also occurs at the receptor level whereby estrogen can induce IGF-1R phosphorylation in uterine epithelia cells, ERα transfected COS-7 cells, and MCF-7 breast cancer cells (Richards et al. 1996, Kahlert et al. 2000). These data indicate that the IGF-1R-mediated signaling pathway is important in estrogen action. Interestingly, the expression of EGFR correlates inversely with the expression of ERα. However, a variety of evidence shows that both receptors are functional in the regulation of breast cancer growth regardless of which one is dominantly expressed (Klijn et al. 1992, van Agthoven et al. 1994). For example, EGFR is functionally involved in estrogen-induced MAPK activation in MCF-7 cells, a cell line with high expression of IGF-1R and ERα but low EGFR.

Both EGF and E2 also crosstalk at other levels. For example, administration of the anti-estrogen ICI 182780 reduces the response to EGF in the mammary gland (Ankrapp et al. 1998). Studies in ERα-knockout mice demonstrated that EGF could not induce DNA synthesis in the uterus even in the presence of wild-type levels of EGF and EGFR (Curtis et al. 1996, Ankrapp et al. 1998). These results indicate that there is a true requirement for the presence of ERα for EGF-mediated biological functions, at least in some cell types. These and other data suggest that ERα serves as a nodal point, which allows interactions between the ER and growth factor pathways.

Mechanism of ERα-mediated activation of the MAPK and Akt pathways

Both MAPK and Akt play important roles in estrogen-induced cancer cell growth. Using a fusion protein consisting of an oncogenic form of human Raf and the ERα HBD domain, ΔRaf:ERα, Samuels et al. in 1994 observed that quiescent 3T3 cells transfected with ΔRaf:ERα responded to estrogen and showed rapid, sustained activation of MAPK (Samuels & McMahon 1994). Migliaccio et al.(1996) then first demonstrated, in endogenous ERα expressing MCF-7 breast cancer cells that estrogen triggers rapid activation of MAPK. These findings were further confirmed in many other cell types, including osteoblasts (Endoh et al. 1997), rat brain cells (Bi et al. 2000) and human colon carcinoma-derived Caco-2 cells (Di Domenico et al. 1996). E2 also rapidly induces the activation of the PI3K/Akt pathway in endothelium cells (Koga et al. 2004), cardiomyocytes (Patten et al. 2004), rat PC-12 neuronal cells (Alexaki et al. 2004) and MCF-7 cells (Ahmad et al. 1999). In the presence of E2, ERα interacts with the regulatory subunit of PI3K, p85α, thus triggering activation of the catalytic subunit p110 of PI3K, leading to the downstream kinase Akt/PKB activation (Castoria et al. 2001). Activation of Akt mediates many of the downstream cellular effects of PI3K, including phosphorylation and inactivation of Bad to prevent Bad-mediated cell apoptosis as well as rapid activation of the endothelial isoform of eNOS (Chambliss & Shaul 2002).

Our studies demonstrated in MCF-7 breast cancer cells that E2 stimulation of MAPK occurs within 5 min. Later, MAPK phosphorylates and activates the transcription factor, Elk-1. The mechanism for E2-induced MAPK and Akt activation in breast cancer cells is now focused on the involvement of classical growth factor receptors, such as IGF-1R and EGFR (Kahlert et al. 2000, Levin 2003). E2 co-opts both IGF-1R and EGFR signaling pathways and utilizes their downstream common adapter proteins to transduce signals, leading to the activation of MAPK and Akt (Fig. 2). These steps occurred within the time frame that E2 stimulated the activation of MAPK and Akt. Knockdown of Shc blocked E2-induced MAPK activation, which was also confirmed by the expression of a dominant negative Shc in MCF-7 cells (Song et al. 2002). E2-induced activation of Akt could be attenuated by administration of the IGF-1R tyrosine kinase inhibitor AG1024. These experiments demonstrated a mechanistic link between the membrane growth factor receptors and their downstream kinase cascades in rapid E2 action.

ERα interacting molecules

Prior to MAPK and Akt activation, ERα needs to interact with many signaling molecules to form a protein complex. It is now known that c-Src activation in the ERα-centered complex is a key step in initiating many downstream signaling pathways in E2 action (Fig. 3). However, many other signaling molecules in the complex also play an important role in initiating and stabilizing the complex.

Role of c-Src

ERα has no intrinsic kinase domain and therefore is not capable of phosphorylating other proteins. Accordingly, E2 must stimulate the activity of a kinase, which serves this function. A key kinase candidate is c-Src tyrosine kinase, which has previously been identified to physically interact with ERα. Our working model (Fig. 3) suggests that E2 activates c-Src, which in turn phosphorylates Shc as well as the IGF-1R (Peterson et al. 1996). The c-Src then would serve as a crucial molecule to facilitate other protein/protein interactions involved in estrogen rapid action.

We have introduced the term, ‘activating particle’ for this multi-protein complex containing ERα, c-Src, and other proteins and suggested that this ‘activating particle’ is a key component involved in estrogen rapid action. This name implies that a complex of proteins is necessary to activate c-Src and that this complex is a discrete molecular entity, which is required to initiate membrane receptor-mediated growth factor-induced signals. The involvement of the above proteins with ERα and c-Src raises many questions regarding estrogen-induced signal initiation mechanisms which now require investigation. While our working model (Fig. 3) suggests that the EGFR and IGF-1R act downstream of c-Src activation, this remains to be precisely established (Song et al. 2004).

Role of p85α of PI3K

The PI3K molecule is composed of an 85Kd regulatory domain (p85α) and a p110 catalytic domain. p85α is an adapter protein known to be involved in estrogen rapid action by forming a protein complex with ERα and c-Src (Castoria et al. 2001). PI3K plays an important role in cell survival and in prevention of cell death (Vanhaesebroeck & Waterfield 1999). Activation of growth factor receptors, such as IGF-1R and EGFR on the cell membrane will recruit p85α to the receptors, leading to the activation of the p110 catalytic subunit (Yamamoto et al. 1992, Altschuler et al. 1994). The p85α as a substrate of IGF-1R and EGFR not only transduces IGF-1R signals to activate Akt, but also forms a protein complex with ERα and c-Src (Fig. 3), leading to estrogen-induced c-Src activation. In this complex, c-Src SH2 interacts with pY-537 of ERα and c-Src SH3 with a polyproline sequence in the linker region of p85α (Renzoni et al. 1996, Barletta et al. 2004).

Role of Shc, G-protein, GPCR, and caveolins

The adapter protein Shc, the G-proteins (Gαs and Gαi), the G-protein-coupled receptor 30 (GPR30), and caveolin-1 have all been involved in estrogen-induced MAPK activation by association with ERα (Migliaccio et al. 1996, Kousteni et al. 2001, Song et al. 2002, Razandi et al. 2003, Revankar et al. 2005). Among these proteins, Shc was demonstrated to directly interact with ERα in MCF-7 cells (Fig. 3), in which the SH2 domain of Shc physically interacts with ERα A/B region (Song et al. 2002). Shc has no intrinsic kinase domain. It transduces signals based on protein–protein interactions through three functional domains: a PTB domain, a region rich in proline, and glycine residues called the collagen-homology domain, and a carboxy-terminal SH2 domain (Pelicci et al. 1996).

Both G-proteins and caveolin-1 might also be potential candidates in the formation of the ‘activating particle’ mediating the rapid effects of E2 since they have been reported to be in the complex with ERα (Wyckoff et al. 2001, Razandi et al. 2002). However, so far there is no evidence showing that they directly interact with ERα in a cell-free system. Presently the physiological significance of this protein–protein interaction is unclear. GPR30, a G-protein-coupled receptor (GPCR), was recently reported to specifically localize to the endoplasmic reticulum and bind to estrogen (Revankar et al. 2005).

ERα-initiated sequential interactions among receptor components

Recent studies in non-breast cancer models have suggested a linear pathway whereby the IGF-R signals through the EGFR to activate MAPK. This process involves the sequential phosphorylation of the IGF-1R, activation of the matrix metalloproteinases (MMPs) 2 and 9, shedding of the extracellular domains of HB-EGF, binding of HB-EGF to the EGFR, and enhanced EGFR tyrosine kinase activity (Fig. 2). Meantime another report demonstrated that IGF-1R-induced EGFR phosphorylation is c-SRC dependent in IGF-II action (Knowlden et al. 2005). IGF-1R is not the only receptor to act upstream of the EGFR in this way. The receptors for angiotensin I, thrombin, α and β adrenergic and cholinergic ligands, human growth hormone, and prolactin can all act in a similar fashion (Hackel et al. 1999, Luttrell et al. 1999, Zwick et al. 1999). Accordingly, the EGFR has been considered a nodal point upon which converges many cytokine- and hormone-induced signals, which can lead to MAPK activation. Our present studies examine the hypothesis that the E2/ERα complex can similarly engage the IGF-1R pathway and sequentially active metaloproteinases, and the EGFR. The biologic consequences of this putative pathway could involve the mediation of E2-induced cell growth and protection from cell death.

We have examined the effect of E2 on the phosphorylation of the IGF-1R, EGFR, and MAPK. Our preliminary data were presented in Endo2006, Boston (Song & Santen 2006) and show the following evidence. AG1024, a specific IGF-1R phosphorylation inhibitor blocked E2-induced MAPK phosphorylation as did AG1478, an EGFR phosphorylation inhibitor. In order to focus on the upstream/downstream relationships of IGF-1R and EGFR on E2-induced MAPK activation in MCF-7 cells, we utilized a strategy, which bypassed the ER and stimulated both receptors directly with their cognate ligands. Our preliminary data show that IGF-1, but not EGF, increased IGF-1R phosphorylation in a dose-dependent manner. Unlike IGF-1R, the phosphorylation status of EGFR was increased not only by EGFR, but also by IGF-1, indicating that IGF-1 is an upstream molecule that mediates EGFR tyrosine kinase activation. Matrix metaloproteinases are involved in this process since a MMP2/9 inhibitor blocked the effect of IGF-1 to activate the EGFR.

We then focused on the ability of IGF-1 and EGF to activate MAPK. IGF-1R and EGFR were specifically knocked down with a selective siRNA and the resultant effects on MAPK examined. Using a non-specific siRNA as control, IGF-1 and EGF produced in a 9- and 12-fold increase of MAPK phosphorylation respectively. The siRNA against IGF-1R significantly blocked IGF-1-stimulated, but not EGF-stimulated, MAPK phosphorylation. Together, the results indicate that EGFR not only plays a role in maintaining the basal level of MAPK activation, but also one of the molecules is affected by the upstream actions of IGF-1R in MCF-7 cells.

After biochemical demonstration of the involvement of EGFR in IGF-1-induced MAPK activation, we wished to assess the biological consequences of both receptors on E2 action in MCF-7 cell and examined both cell proliferation and the apoptosis in response to E2. For this purpose, both siRNA and tyrosine kinase inhibitor strategies were employed. First, we examined proliferation. Knockdown of either IGF-1R or EGFR significantly decreased the numbers of cells growing in the absence of E2. Compared with vehicle treatment, knockdown of IGF-1R significantly blocked E2 or IGF-1, but not EGF, induced stimulation of cell number, suggesting that ligand-induced EGFR activation is a downstream event in E2 or IGF-1 action. At the same time, knockdown of EGFR with siRNA significantly retarded the stimulatory effect induced by E2, IGF-1, and EGF supporting that EGFR is a common converging point mediating E2 and IGF-1R action. Together, these results reinforce the hypothesis that EGFR activation is a downstream signal involved in E2 and IGF-1 action.

Next, we examined apoptosis. We determined whether blockade of IGF-1R or EGFR could reverse the effects of E2 or growth factors on protection against cell death. Approximately a twofold increase of apoptosis was observed in both AG1024- and AG1478-treated groups. Treatment of cells with E2, IGF-1, and EGF not only significantly decreased cell death rate compared with vehicle-treated control, but also in general protected cell apoptosis induced by both AG1024 and AG1478. Together, the data indicate that both IGF-1R and EGFR play a role in the effect of E2 in protecting against cell death and that IGF-1R might exert a stronger anti-apoptotic effect than on proliferation.

ER involved non-genomic action in anti-hormonal therapy

Exposure to hormonal therapy can cause breast cancer cells to adapt and change their properties. We postulate that long-term exposure to tamoxifen might enhance the utilization of the EGFR pathways. Our experimental model was to expose MCF-7 cells to tamoxifen for a period of 3 months to 2 years until they became resistant to tamoxifen (TAM-R). Our published data show that TAM-R cells were more sensitive to the stimulatory effect of EGF (Fan et al. in press). Wild-type control MCF-7 cells did not respond to 0.1 ng/ml EGF with MAPK phosphorylation, whereas the TAM-R cells responded robustly. In addition, the rapid, extranuclear, MAPK response to E2 was also increased in TAM-R cells, which is in agreement with enhanced sensitivity to EGF-stimulated MAPK activation. TAM-R cells became more sensitive to growth inhibition by small molecule inhibitors that block kinase activity of EGFR or MEK. These results suggest that MCF-7 cells become more dependent on the EGFR/MAPK pathway for growth after long-term exposure to tamoxifen.

As a possible explanation for our findings, we questioned whether or not ERα bound more effectively to the EGFR in the TAM-R cells. These studies demonstrated an enhanced interaction between ERα and the EGFR in the TAM-R cells. However, the levels of ERα and EGFR were not increased in TAM-R cells. This result suggested that an enhanced interaction between EGFR and ERα might result from redistribution of ERα. In support of this possibility, confocal microscopy studies demonstrated a marked enhancement of cytoplasmic ERα content in the TAM-R cells. To confirm that long-term tamoxifen treatment increased the pool of cytoplasmic ERα, fractions of the plasma membrane, nucleus, and cytoplasm were prepared from TAM-R and control cells. In the control cells, ERα was recovered from both cytoplasmic and nuclear fractions with higher levels in the nucleus. In TAM-R cells, ERα was detectable in the membrane fraction. The level of ERα in cytoplasm was increased, whereas the level of nuclear ERα was reduced compared with the control cells.

Summary and conclusions

Breast cancer cells utilize both the nuclear and extranuclear mechanisms of ERα action to enhance cell proliferation and diminish apoptosis. The formation of large protein/protein complexes, which involve the IGF-1R, the EGFR, c-Src, Shc, and ERα are required for extranuclear ERα actions. Depending upon the cell type and context, additional proteins in this complex can include PELP-1 (MNAR), striatin, caveolin, P130Cas, and certain G-protein-coupled receptors. Complex interactions occur between the extranuclear signaling pathways and the nuclear transcriptional events. Our studies suggest that E2, when bound to ERα, can co-opt classical growth factor signaling pathways to activate MAPK and PI3K. Our working model emphasizes a sequential process involving several steps. Initially E2 binds to ERα and induces the phosphorylation of Shc and subsequent binding to ERα. Shc acts as a ‘bus’ to transport ERα to Shc binding sites on the IGF-1R, which in turn is phosphorylated in response to E2. After formation of this complex, metaloproteinases 2 and 9 are activated, which release HB-EGF from its membrane tethering. HB-EGF serves as a ligand to activate the EGFR, which in turn enhances the activity of the MAPK and PI3K pathways. With exposure to anti-hormonal therapies, such as tamoxifen, breast cancer cells can adapt by upregulation of growth factor pathways. Our data demonstrate that long-term tamoxifen exposure results in an increase in the relative amounts of ERα in extranuclear sites of the cell, a process that can be abrogated by the c-Src inhibitor PP2. With more ERα outside the nucleus, the processes that utilize the IGF-1R and EGFR are enhanced. These adaptive mechanisms provide specific targets, such as blockade of the IGF-1R, EGFR, c-Src, and Shc, which could potentially abrogate development of resistance to anti-hormonal therapy (Knowlden et al. 2005).

Funding

The authors declare that there is no conflict of interest that would prejudice the impartiality of this research.

Figure 1
Figure 1

Downregulation of Shc blocked E2-induced MAPK phosphorylation. Cells were transfected with a smart pool of siRNA against either a non-specific target or Shc using the siPORT lipid transfection reagent. At day 2, cells were treated with vehicle or 0.1 nM E2 for 15 min and the MAPK activation was assayed with antiphosphotyrosine MAPK antibody to both isoforms. The graphed data are from three experiments combined. All values are mean±s.e.m. *P < 0.05 comparison of E2-treated cells with the vehicle-treated control in each siRNA transfected group. The figure is a reproduction from reference Song et al.(2004), Fig. 5A.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01322

Figure 2
Figure 2

Proposed model of both IGF-1R and EGFR involvement in E2 rapid action on MAPK and Akt activation. We propose here that E2-induced IGF-1R activation not only recruits Shc and p85α, leading to the activation of MAPK and Akt pathways, but also initiates a linear activation pathway involving matrix metalloproteinase, heparin-binding EGF (HB-EGF). HB-EGF as a ligand causes EGFR-dependent MAPK activation. Activation of both MAPK and Akt pathways by E2 eventually contributes to E2-mediated cell proliferation and anti-apoptosis.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01322

Figure 3
Figure 3

Proposed model of E2-induced activating particle formation and its downstream signaling pathway activation. In this model, there are two phases for ERα-mediated rapid E2 action, the interaction and stimulation phases. At the basal condition, ERα and c-Src form an inactive protein complex due to basal level of Y537 phosphorylation on ERα. In phase I, E2 induces the interaction of other proteins, such as Shc and PELP1, with the ERα/c-Src complex, leading to the formation of ‘activating particle’. In phase II, the activated c-Src in multiple protein complexes preferentially activates the downstream signaling pathways.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01322

References

  • Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, Visca P & Marino M 2005 Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Molecular Biology of the Cell 16 231–237.

    • Search Google Scholar
    • Export Citation
  • Ahmad S, Singh N & Glazer RI 1999 Role of AKT1 in 17beta-estradiol- and insulin-like growth factor I (IGF-I)-dependent proliferation and prevention of apoptosis in MCF-7 breast carcinoma cells. Biochemical Pharmacology 58 425–430.

    • Search Google Scholar
    • Export Citation
  • Alexaki VI, Charalampopoulos I, Kampa M, Vassalou H, Theodoropoulos P, Stathopoulos EN, Hatzoglou A, Gravanis A & Castanas E 2004 Estrogen exerts neuro-protective effects via membrane estrogen receptors and rapid Akt/NOS activation. FASEB Journal 18 1594–1596.

    • Search Google Scholar
    • Export Citation
  • Altschuler D, Yamamoto K & Lapetina EG 1994 Insulin-like growth factor-1-mediated association of p85 phospha-tidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction. Molecular Endocrinology 8 1139–1146.

    • Search Google Scholar
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  • Anderson E & Clarke RB 2004 Steroid receptors and cell cycle in normal mammary epithelium. Journal of Mammary Gland Biology and Neoplasia 9 3–13.

    • Search Google Scholar
    • Export Citation
  • Ankrapp DP, Bennett JM & Haslam SZ 1998 Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland. Journal of Cellular Physiology 174 251–260.

    • Search Google Scholar
    • Export Citation
  • Barletta F, Wong CW, McNally C, Komm BS, Katzenellenbogen B & Cheskis BJ 2004 Characterization of the interactions of estrogen receptor and MNAR in the activation of cSrc. Molecular Endocrinology 18 1096–1108.

    • Search Google Scholar
    • Export Citation
  • Bi R, Broutman G, Foy MR, Thompson RF & Baudry M 2000 The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus. PNAS 97 3602–3607.

    • Search Google Scholar
    • Export Citation
  • Cabodi S, Moro L, Baj G, Smeriglio M, Di Stefano P, Gippone S, Surico N, Silengo L, Turco E, Tarone G et al.2004 p130Cas interacts with estrogen receptor α and modulates non-genomic estrogen signaling in breast cancer cells. Journal of Cell Science 117 1603–1611.

    • Search Google Scholar
    • Export Citation
  • Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, Fiorentino R, Varricchio L, Barone MV & Auricchio F 2001 PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO Journal 20 6050–6059.

    • Search Google Scholar
    • Export Citation
  • Catalano MG, Comba A, Fazzari A, Benedusi-Pagliano E, Sberveglieri M, Revelli A, Massobrio M, Frairia R & Fortunati N 1997 Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer. Breast Cancer Research and Treatment 42 227–234.

    • Search Google Scholar
    • Export Citation
  • Chambliss KL & Shaul PW 2002 Estrogen modulation of endothelial nitric oxide synthase. Endocrine Reviews 23 665–686.

  • Chambliss KL, Simon L, Yuhanna IS, Mineo C & Shaul PW 2005 Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERα domains with known nuclear functions. Molecular Endocrinology 19 277–289.

    • Search Google Scholar
    • Export Citation
  • Chen JQ & Yager JD 2004 Estrogen’s effects on mitochondrial gene expression: mechanisms and potential contributions to estrogen carcinogenesis. Annals of the New York Academy of Sciences 1028 258–272.

    • Search Google Scholar
    • Export Citation
  • Cheskis BJ 2004 Regulation of cell signalling cascades by steroid hormones. Journal of Cellular Biochemistry 93 20–27.

  • Cunha GR, Cooke PS & Kurita T 2004 Role of stromal-epithelial interactions in hormonal responses. Archives of Histology and Cytology 67 417–434.

    • Search Google Scholar
    • Export Citation
  • Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF & Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. PNAS 93 12626–12630.

    • Search Google Scholar
    • Export Citation
  • Di Domenico M, Castoria G, Bilancio A, Migliaccio A & Auricchio F 1996 Estradiol activation of human colon carcinoma-derived Caco-2 cell growth. Cancer Research 56 4516–4521.

    • Search Google Scholar
    • Export Citation
  • Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu T, Kato S & Kawashima H 1997 Rapid activation of MAP kinase by estrogen in the bone cell line. Biochemical and Biophysical Research Communications 235 99–102.

    • Search Google Scholar
    • Export Citation
  • Fan P, Wang J, Santen RJ & Yue W 2006 Long term treatment with tamoxifen facilitates translocation of ERα out of the nucleus and enhances its interaction with epidermal growth factor receptor in MCF-7 breast cancer cells. Cancer Research (In Press).

    • Search Google Scholar
    • Export Citation
  • Filardo EJ, Quinn JA, Bland KI & Frackelton AR Jr 2000 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. Molecular Endocrinology 14 1649–1660.

    • Search Google Scholar
    • Export Citation
  • Guo X, Razandi M, Pedram A, Kassab G & Levin ER 2005 Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta. Journal of Biological Chemistry 280 19704–19710.

    • Search Google Scholar
    • Export Citation
  • Hackel PO, Zwick E, Prenzel N & Ullrich A 1999 Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Current Opinion in Cell Biology 11 184–189.

    • Search Google Scholar
    • Export Citation
  • Hammes A, Andreassen TK, Spoelgen R, Raila J, Hubner N, Schulz H, Metzger J, Schweigert FJ, Luppa PB, Nykjaer A et al.2005 Role of endocytosis in cellular uptake of sex steroids. Cell 122 751–762.

    • Search Google Scholar
    • Export Citation
  • Harrington WR, Kim SH, Funk CC, Madak-Erdogan Z, Schiff R, Katzenellenbogen JA & Katzenellenbogen BS 2005 Estrogen dendrimer conjugates that preferentially activate extranuclear, non-genomic versus genomic pathways of estrogen action. Molecular Endocrinology 20 491–502.

    • Search Google Scholar
    • Export Citation
  • Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R & Grohe C 2000 Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. Journal of Biological Chemistry 275 18447–18453.

    • Search Google Scholar
    • Export Citation
  • Klijn JG, Berns PM, Schmitz PI & Foekens JA 1992 The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocrine Reviews 13 3–17.

    • Search Google Scholar
    • Export Citation
  • Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP & Korach KS 2002 Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. Journal of Biological Chemistry 277 8531–8537.

    • Search Google Scholar
    • Export Citation
  • Knowlden JM, Hutcheson IR, Barrow D, Gee JM & Nicholson RI 2005 Insulin-like growth factor-I receptor signaling in tamoxifen-resistant breast cancer: a supporting role to the epidermal growth factor receptor. Endocrinology 146 4609–4618.

    • Search Google Scholar
    • Export Citation
  • Koga M, Hirano K, Hirano M, Nishimura J, Nakano H & Kanaide H 2004 Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells. Biochemical and Biophysical Research Communications 324 321–325.

    • Search Google Scholar
    • Export Citation
  • Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS et al.2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104 719–730.

    • Search Google Scholar
    • Export Citation
  • Levin ER 2003 Bidirectional signaling between the estrogen receptor and the epidermal growth factor receptor. Molecular Endocrinology 17 309–317.

    • Search Google Scholar
    • Export Citation
  • Levin ER 2005 Integration of the extra-nuclear and nuclear actions of estrogen. Molecular Endocrinology 19 1951–1959.

  • Li L, Haynes MP & Bender JR 2003 Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. PNAS 100 4807–4812.

    • Search Google Scholar
    • Export Citation
  • Lu Q, Pallas DC, Surks HK, Baur WE, Mendelsohn ME & Karas RH 2004 Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha. PNAS 101 17126–17131.

    • Search Google Scholar
    • Export Citation
  • Luttrell LM, Daaka Y & Lefkowitz RJ 1999 Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Current Opinion in Cell Biology 11 177–183.

    • Search Google Scholar
    • Export Citation
  • Mauro L, Salerno M, Panno ML, Bellizzi D, Sisci D, Miglietta A, Surmacz E & Ando S 2001 Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells. Biochemical and Biophysical Research Communications 288 685–689.

    • Search Google Scholar
    • Export Citation
  • Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E & Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol- receptor complex in MCF-7 cells. EMBO Journal 15 1292–1300.

    • Search Google Scholar
    • Export Citation
  • Nemere I, Pietras RJ & Blackmore PF 2003 Membrane receptors for steroid hormones: signal transduction and physiological significance. Journal of Cellular Biochemistry 88 438–445.

    • Search Google Scholar
    • Export Citation
  • Nethrapalli IS, Tinnikov AA, Krishnan V, Lei CD & Toran-Allerand CD 2005 Estrogen activates mitogen-activated protein kinase in native, nontransfected CHO-K1, COS-7, and RAT2 fibroblast cell lines. Endocrinology 146 56–63.

    • Search Google Scholar
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  • O’Malley BW 2005 A life-long search for the molecular pathways of steroid hormone action. Molecular Endocrinology 19 1402–1411.

  • Patten RD, Pourati I, Aronovitz MJ, Baur J, Celestin F, Chen X, Michael A, Haq S, Nuedling S, Grohe C et al.2004 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circulation Research 95 692–699.

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  • Pedram A, Razandi M & Levin ER 2006 Nature of functional estrogen receptors at the plasma membrane. Molecular Endocrinology 20 1996–2009.

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  • Pelicci G, Dente L, De Giuseppe A, Verducci-Galletti B, Giuli S, Mele S, Vetriani C, Giorgio M, Pandolfi PP, Cesareni G et al.1996 A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene 13 633–641.

    • Search Google Scholar
    • Export Citation
  • Peterson JE, Kulik G, Jelinek T, Reuter CWM, Shannon JA & Weber MJ 1996 Src phosphorylates the insulin-like growth factor type I receptor on the autophosphorylation sites requirement for transformation by src. Journal of Biological Chemistry 271 31562–31571.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Pedram A, Greene GL & Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Molecular Endocrinology 13 307–319.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Oh P, Pedram A, Schnitzer J & Levin ER 2002 ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Molecular Endocrinology 16 100–115.

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    • Export Citation
  • Razandi M, Pedram A, Park ST & Levin ER 2003 Proximal events in signaling by plasma membrane estrogen receptors. Journal of Biological Chemistry 278 2701–2712.

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    • Export Citation
  • Razandi M, Pedram A, Merchenthaler I, Greene GL & Levin ER 2004 Plasma membrane estrogen receptors exist and functions as dimers. Molecular Endocrinology 18 2854–2865.

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    • Export Citation
  • Renzoni DA, Pugh DJ, Siligardi G, Das P, Morton CJ, Rossi C, Waterfield MD, Campbell ID & Ladbury JE 1996 Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase. Biochemistry 35 15646–15653.

    • Search Google Scholar
    • Export Citation
  • Revankar CM, Cimino DF, Sklar LA, Arterburn JB & Prossnitz ER 2005 A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307 1625–1630.

    • Search Google Scholar
    • Export Citation
  • Richards RG, DiAugustine RP, Petrusz P, Clark GC & Sebastian J 1996 Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus. PNAS 93 12002–12007.

    • Search Google Scholar
    • Export Citation
  • Samuels ML & McMahon M 1994 Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1. Molecular and Cellular Biology 14 7855–7866.

    • Search Google Scholar
    • Export Citation
  • Shupnik MA 2004 Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 23 7979–7989.

    • Search Google Scholar
    • Export Citation
  • Song RX, Zhang Z & Santen RJ 2006 Estradiol signals via a novel, linear, extra-nuclear pathway involving IGF-1R, metaloproteinase and EGF-R to activate MAPK in MCF-7 breast cancer cells. 88th Annual Meeting of the Endocrine Society, Boston, USA. Abstract, P-2 393, pg 494.

  • Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R & Santen RJ 2002 Linkage of rapid estrogen action to MAPK activation by ERalpha-Shc association and Shc pathway activation. Molecular Endocrinology 16 116–127.

    • Search Google Scholar
    • Export Citation
  • Song RX, Barnes CJ, Zhang Z, Bao Y, Kumar R & Santen RJ 2004 The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. PNAS 101 2076–2081.

    • Search Google Scholar
    • Export Citation
  • Song RX, Zhang Z & Santen RJ 2004 A novel role of EGF and Shc in ER alpha membrane translocation and activation of MAPK in breast cancer cells. 86th Annual Meeting of the Endocrine Society, New Orleans, USA. Abstract, OR-4, pg 74.

  • Song RX, Zhang Z & Santen RJ 2005 Estrogen rapid action via protein complex formation involving ERalpha and Src. Trends in Endocrinology and Metabolism 16 347–353.

    • Search Google Scholar
    • Export Citation
  • Streuli CH & Haslam SZ 1998 Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. Journal of Mammary Gland Biology and Neoplasia 3 107–108.

    • Search Google Scholar
    • Export Citation
  • Thomas P, Pang Y, Filardo EJ & Dong J 2005 Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146 624–632.

    • Search Google Scholar
    • Export Citation
  • Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ES Jr, Nethrapalli IS & Tinnikov AA 2002 ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. Journal of Neuroscience 22 8391–8401.

    • Search Google Scholar
    • Export Citation
  • van Agthoven T, Timmermans M, Foekens JA, Dorssers LC & Henzen-Logmans SC 1994 Differential expression of estrogen, progesterone, and epidermal growth factor receptors in normal, benign, and malignant human breast tissues using dual staining immunohistochemistry. American Journal of Pathology 144 1238–1246.

    • Search Google Scholar
    • Export Citation
  • Vanhaesebroeck B & Waterfield MD 1999 Signaling by distinct classes of phosphoinositide 3-kinases. Experimental Cell Research 253 239–254.

    • Search Google Scholar
    • Export Citation
  • Wang W, Dong L, Saville B & Safe S 1999 Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Molecular Endocrinology 13 1373–1387.

    • Search Google Scholar
    • Export Citation
  • Watson CS & Gametchu B 1999 Membrane-initiated steroid actions and the proteins that mediate them. Proceedings of the Society for Experimental Biology and Medicine 220 9–19.

    • Search Google Scholar
    • Export Citation
  • Watters JJ, Chun TY, Kim YN, Bertics PJ & Gorski J 2000 Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Molecular Endocrinology 14 1872–1881.

    • Search Google Scholar
    • Export Citation
  • Wyckoff MH, Chambliss KL, Mineo C, Yuhanna IS, Mendelsohn ME, Mumby SM & Shaul PW 2001 Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i). Journal of Biological Chemistry 276 27071–27076.

    • Search Google Scholar
    • Export Citation
  • Yamamoto K, Altschuler D, Wood E, Horlick K, Jacobs S & Lapetina EG 1992 Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85. Journal of Biological Chemistry 267 11337–11343.

    • Search Google Scholar
    • Export Citation
  • Zhang Z, Maier B, Santen RJ & Song RX 2002 Membrane association of estrogen receptor alpha mediates estrogen effect on MAPK activation. Biochemical and Biophysical Research Communications 294 926–933.

    • Search Google Scholar
    • Export Citation
  • Zwick E, Hackel PO, Prenzel N & Ullrich A 1999 The EGF receptor as central transducer of heterologous signalling systems. Trends in Pharmacological Sciences 20 408–412.

    • Search Google Scholar
    • Export Citation

 

Society for Endocrinology

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    Downregulation of Shc blocked E2-induced MAPK phosphorylation. Cells were transfected with a smart pool of siRNA against either a non-specific target or Shc using the siPORT lipid transfection reagent. At day 2, cells were treated with vehicle or 0.1 nM E2 for 15 min and the MAPK activation was assayed with antiphosphotyrosine MAPK antibody to both isoforms. The graphed data are from three experiments combined. All values are mean±s.e.m. *P < 0.05 comparison of E2-treated cells with the vehicle-treated control in each siRNA transfected group. The figure is a reproduction from reference Song et al.(2004), Fig. 5A.

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    Proposed model of both IGF-1R and EGFR involvement in E2 rapid action on MAPK and Akt activation. We propose here that E2-induced IGF-1R activation not only recruits Shc and p85α, leading to the activation of MAPK and Akt pathways, but also initiates a linear activation pathway involving matrix metalloproteinase, heparin-binding EGF (HB-EGF). HB-EGF as a ligand causes EGFR-dependent MAPK activation. Activation of both MAPK and Akt pathways by E2 eventually contributes to E2-mediated cell proliferation and anti-apoptosis.

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    Proposed model of E2-induced activating particle formation and its downstream signaling pathway activation. In this model, there are two phases for ERα-mediated rapid E2 action, the interaction and stimulation phases. At the basal condition, ERα and c-Src form an inactive protein complex due to basal level of Y537 phosphorylation on ERα. In phase I, E2 induces the interaction of other proteins, such as Shc and PELP1, with the ERα/c-Src complex, leading to the formation of ‘activating particle’. In phase II, the activated c-Src in multiple protein complexes preferentially activates the downstream signaling pathways.

  • Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, Visca P & Marino M 2005 Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Molecular Biology of the Cell 16 231–237.

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  • Ahmad S, Singh N & Glazer RI 1999 Role of AKT1 in 17beta-estradiol- and insulin-like growth factor I (IGF-I)-dependent proliferation and prevention of apoptosis in MCF-7 breast carcinoma cells. Biochemical Pharmacology 58 425–430.

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    • Export Citation
  • Alexaki VI, Charalampopoulos I, Kampa M, Vassalou H, Theodoropoulos P, Stathopoulos EN, Hatzoglou A, Gravanis A & Castanas E 2004 Estrogen exerts neuro-protective effects via membrane estrogen receptors and rapid Akt/NOS activation. FASEB Journal 18 1594–1596.

    • Search Google Scholar
    • Export Citation
  • Altschuler D, Yamamoto K & Lapetina EG 1994 Insulin-like growth factor-1-mediated association of p85 phospha-tidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction. Molecular Endocrinology 8 1139–1146.

    • Search Google Scholar
    • Export Citation
  • Anderson E & Clarke RB 2004 Steroid receptors and cell cycle in normal mammary epithelium. Journal of Mammary Gland Biology and Neoplasia 9 3–13.

    • Search Google Scholar
    • Export Citation
  • Ankrapp DP, Bennett JM & Haslam SZ 1998 Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland. Journal of Cellular Physiology 174 251–260.

    • Search Google Scholar
    • Export Citation
  • Barletta F, Wong CW, McNally C, Komm BS, Katzenellenbogen B & Cheskis BJ 2004 Characterization of the interactions of estrogen receptor and MNAR in the activation of cSrc. Molecular Endocrinology 18 1096–1108.

    • Search Google Scholar
    • Export Citation
  • Bi R, Broutman G, Foy MR, Thompson RF & Baudry M 2000 The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus. PNAS 97 3602–3607.

    • Search Google Scholar
    • Export Citation
  • Cabodi S, Moro L, Baj G, Smeriglio M, Di Stefano P, Gippone S, Surico N, Silengo L, Turco E, Tarone G et al.2004 p130Cas interacts with estrogen receptor α and modulates non-genomic estrogen signaling in breast cancer cells. Journal of Cell Science 117 1603–1611.

    • Search Google Scholar
    • Export Citation
  • Castoria G, Migliaccio A, Bilancio A, Di Domenico M, de Falco A, Lombardi M, Fiorentino R, Varricchio L, Barone MV & Auricchio F 2001 PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO Journal 20 6050–6059.

    • Search Google Scholar
    • Export Citation
  • Catalano MG, Comba A, Fazzari A, Benedusi-Pagliano E, Sberveglieri M, Revelli A, Massobrio M, Frairia R & Fortunati N 1997 Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer. Breast Cancer Research and Treatment 42 227–234.

    • Search Google Scholar
    • Export Citation
  • Chambliss KL & Shaul PW 2002 Estrogen modulation of endothelial nitric oxide synthase. Endocrine Reviews 23 665–686.

  • Chambliss KL, Simon L, Yuhanna IS, Mineo C & Shaul PW 2005 Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERα domains with known nuclear functions. Molecular Endocrinology 19 277–289.

    • Search Google Scholar
    • Export Citation
  • Chen JQ & Yager JD 2004 Estrogen’s effects on mitochondrial gene expression: mechanisms and potential contributions to estrogen carcinogenesis. Annals of the New York Academy of Sciences 1028 258–272.

    • Search Google Scholar
    • Export Citation
  • Cheskis BJ 2004 Regulation of cell signalling cascades by steroid hormones. Journal of Cellular Biochemistry 93 20–27.

  • Cunha GR, Cooke PS & Kurita T 2004 Role of stromal-epithelial interactions in hormonal responses. Archives of Histology and Cytology 67 417–434.

    • Search Google Scholar
    • Export Citation
  • Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF & Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. PNAS 93 12626–12630.

    • Search Google Scholar
    • Export Citation
  • Di Domenico M, Castoria G, Bilancio A, Migliaccio A & Auricchio F 1996 Estradiol activation of human colon carcinoma-derived Caco-2 cell growth. Cancer Research 56 4516–4521.

    • Search Google Scholar
    • Export Citation
  • Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu T, Kato S & Kawashima H 1997 Rapid activation of MAP kinase by estrogen in the bone cell line. Biochemical and Biophysical Research Communications 235 99–102.

    • Search Google Scholar
    • Export Citation
  • Fan P, Wang J, Santen RJ & Yue W 2006 Long term treatment with tamoxifen facilitates translocation of ERα out of the nucleus and enhances its interaction with epidermal growth factor receptor in MCF-7 breast cancer cells. Cancer Research (In Press).

    • Search Google Scholar
    • Export Citation
  • Filardo EJ, Quinn JA, Bland KI & Frackelton AR Jr 2000 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. Molecular Endocrinology 14 1649–1660.

    • Search Google Scholar
    • Export Citation
  • Guo X, Razandi M, Pedram A, Kassab G & Levin ER 2005 Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta. Journal of Biological Chemistry 280 19704–19710.

    • Search Google Scholar
    • Export Citation
  • Hackel PO, Zwick E, Prenzel N & Ullrich A 1999 Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Current Opinion in Cell Biology 11 184–189.

    • Search Google Scholar
    • Export Citation
  • Hammes A, Andreassen TK, Spoelgen R, Raila J, Hubner N, Schulz H, Metzger J, Schweigert FJ, Luppa PB, Nykjaer A et al.2005 Role of endocytosis in cellular uptake of sex steroids. Cell 122 751–762.

    • Search Google Scholar
    • Export Citation
  • Harrington WR, Kim SH, Funk CC, Madak-Erdogan Z, Schiff R, Katzenellenbogen JA & Katzenellenbogen BS 2005 Estrogen dendrimer conjugates that preferentially activate extranuclear, non-genomic versus genomic pathways of estrogen action. Molecular Endocrinology 20 491–502.

    • Search Google Scholar
    • Export Citation
  • Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R & Grohe C 2000 Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. Journal of Biological Chemistry 275 18447–18453.

    • Search Google Scholar
    • Export Citation
  • Klijn JG, Berns PM, Schmitz PI & Foekens JA 1992 The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocrine Reviews 13 3–17.

    • Search Google Scholar
    • Export Citation
  • Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP & Korach KS 2002 Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. Journal of Biological Chemistry 277 8531–8537.

    • Search Google Scholar
    • Export Citation
  • Knowlden JM, Hutcheson IR, Barrow D, Gee JM & Nicholson RI 2005 Insulin-like growth factor-I receptor signaling in tamoxifen-resistant breast cancer: a supporting role to the epidermal growth factor receptor. Endocrinology 146 4609–4618.

    • Search Google Scholar
    • Export Citation
  • Koga M, Hirano K, Hirano M, Nishimura J, Nakano H & Kanaide H 2004 Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells. Biochemical and Biophysical Research Communications 324 321–325.

    • Search Google Scholar
    • Export Citation
  • Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS et al.2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104 719–730.

    • Search Google Scholar
    • Export Citation
  • Levin ER 2003 Bidirectional signaling between the estrogen receptor and the epidermal growth factor receptor. Molecular Endocrinology 17 309–317.

    • Search Google Scholar
    • Export Citation
  • Levin ER 2005 Integration of the extra-nuclear and nuclear actions of estrogen. Molecular Endocrinology 19 1951–1959.

  • Li L, Haynes MP & Bender JR 2003 Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. PNAS 100 4807–4812.

    • Search Google Scholar
    • Export Citation
  • Lu Q, Pallas DC, Surks HK, Baur WE, Mendelsohn ME & Karas RH 2004 Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha. PNAS 101 17126–17131.

    • Search Google Scholar
    • Export Citation
  • Luttrell LM, Daaka Y & Lefkowitz RJ 1999 Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Current Opinion in Cell Biology 11 177–183.

    • Search Google Scholar
    • Export Citation
  • Mauro L, Salerno M, Panno ML, Bellizzi D, Sisci D, Miglietta A, Surmacz E & Ando S 2001 Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells. Biochemical and Biophysical Research Communications 288 685–689.

    • Search Google Scholar
    • Export Citation
  • Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E & Auricchio F 1996 Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol- receptor complex in MCF-7 cells. EMBO Journal 15 1292–1300.

    • Search Google Scholar
    • Export Citation
  • Nemere I, Pietras RJ & Blackmore PF 2003 Membrane receptors for steroid hormones: signal transduction and physiological significance. Journal of Cellular Biochemistry 88 438–445.

    • Search Google Scholar
    • Export Citation
  • Nethrapalli IS, Tinnikov AA, Krishnan V, Lei CD & Toran-Allerand CD 2005 Estrogen activates mitogen-activated protein kinase in native, nontransfected CHO-K1, COS-7, and RAT2 fibroblast cell lines. Endocrinology 146 56–63.

    • Search Google Scholar
    • Export Citation
  • O’Malley BW 2005 A life-long search for the molecular pathways of steroid hormone action. Molecular Endocrinology 19 1402–1411.

  • Patten RD, Pourati I, Aronovitz MJ, Baur J, Celestin F, Chen X, Michael A, Haq S, Nuedling S, Grohe C et al.2004 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circulation Research 95 692–699.

    • Search Google Scholar
    • Export Citation
  • Pedram A, Razandi M & Levin ER 2006 Nature of functional estrogen receptors at the plasma membrane. Molecular Endocrinology 20 1996–2009.

    • Search Google Scholar
    • Export Citation
  • Pelicci G, Dente L, De Giuseppe A, Verducci-Galletti B, Giuli S, Mele S, Vetriani C, Giorgio M, Pandolfi PP, Cesareni G et al.1996 A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene 13 633–641.

    • Search Google Scholar
    • Export Citation
  • Peterson JE, Kulik G, Jelinek T, Reuter CWM, Shannon JA & Weber MJ 1996 Src phosphorylates the insulin-like growth factor type I receptor on the autophosphorylation sites requirement for transformation by src. Journal of Biological Chemistry 271 31562–31571.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Pedram A, Greene GL & Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Molecular Endocrinology 13 307–319.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Oh P, Pedram A, Schnitzer J & Levin ER 2002 ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Molecular Endocrinology 16 100–115.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Pedram A, Park ST & Levin ER 2003 Proximal events in signaling by plasma membrane estrogen receptors. Journal of Biological Chemistry 278 2701–2712.

    • Search Google Scholar
    • Export Citation
  • Razandi M, Pedram A, Merchenthaler I, Greene GL & Levin ER 2004 Plasma membrane estrogen receptors exist and functions as dimers. Molecular Endocrinology 18 2854–2865.

    • Search Google Scholar
    • Export Citation
  • Renzoni DA, Pugh DJ, Siligardi G, Das P, Morton CJ, Rossi C, Waterfield MD, Campbell ID & Ladbury JE 1996 Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase. Biochemistry 35 15646–15653.

    • Search Google Scholar
    • Export Citation
  • Revankar CM, Cimino DF, Sklar LA, Arterburn JB & Prossnitz ER 2005 A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307 1625–1630.

    • Search Google Scholar
    • Export Citation
  • Richards RG, DiAugustine RP, Petrusz P, Clark GC & Sebastian J 1996 Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus. PNAS 93 12002–12007.

    • Search Google Scholar
    • Export Citation
  • Samuels ML & McMahon M 1994 Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1. Molecular and Cellular Biology 14 7855–7866.

    • Search Google Scholar
    • Export Citation
  • Shupnik MA 2004 Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 23 7979–7989.

    • Search Google Scholar
    • Export Citation
  • Song RX, Zhang Z & Santen RJ 2006 Estradiol signals via a novel, linear, extra-nuclear pathway involving IGF-1R, metaloproteinase and EGF-R to activate MAPK in MCF-7 breast cancer cells. 88th Annual Meeting of the Endocrine Society, Boston, USA. Abstract, P-2 393, pg 494.

  • Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R & Santen RJ 2002 Linkage of rapid estrogen action to MAPK activation by ERalpha-Shc association and Shc pathway activation. Molecular Endocrinology 16 116–127.

    • Search Google Scholar
    • Export Citation
  • Song RX, Barnes CJ, Zhang Z, Bao Y, Kumar R & Santen RJ 2004 The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. PNAS 101 2076–2081.

    • Search Google Scholar
    • Export Citation
  • Song RX, Zhang Z & Santen RJ 2004 A novel role of EGF and Shc in ER alpha membrane translocation and activation of MAPK in breast cancer cells. 86th Annual Meeting of the Endocrine Society, New Orleans, USA. Abstract, OR-4, pg 74.

  • Song RX, Zhang Z & Santen RJ 2005 Estrogen rapid action via protein complex formation involving ERalpha and Src. Trends in Endocrinology and Metabolism 16 347–353.

    • Search Google Scholar
    • Export Citation
  • Streuli CH & Haslam SZ 1998 Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. Journal of Mammary Gland Biology and Neoplasia 3 107–108.

    • Search Google Scholar
    • Export Citation
  • Thomas P, Pang Y, Filardo EJ & Dong J 2005 Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146 624–632.

    • Search Google Scholar
    • Export Citation
  • Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ES Jr, Nethrapalli IS & Tinnikov AA 2002 ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. Journal of Neuroscience 22 8391–8401.

    • Search Google Scholar
    • Export Citation
  • van Agthoven T, Timmermans M, Foekens JA, Dorssers LC & Henzen-Logmans SC 1994 Differential expression of estrogen, progesterone, and epidermal growth factor receptors in normal, benign, and malignant human breast tissues using dual staining immunohistochemistry. American Journal of Pathology 144 1238–1246.

    • Search Google Scholar
    • Export Citation
  • Vanhaesebroeck B & Waterfield MD 1999 Signaling by distinct classes of phosphoinositide 3-kinases. Experimental Cell Research 253 239–254.

    • Search Google Scholar
    • Export Citation
  • Wang W, Dong L, Saville B & Safe S 1999 Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Molecular Endocrinology 13 1373–1387.

    • Search Google Scholar
    • Export Citation
  • Watson CS & Gametchu B 1999 Membrane-initiated steroid actions and the proteins that mediate them. Proceedings of the Society for Experimental Biology and Medicine 220 9–19.

    • Search Google Scholar
    • Export Citation
  • Watters JJ, Chun TY, Kim YN, Bertics PJ & Gorski J 2000 Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Molecular Endocrinology 14 1872–1881.

    • Search Google Scholar
    • Export Citation
  • Wyckoff MH, Chambliss KL, Mineo C, Yuhanna IS, Mendelsohn ME, Mumby SM & Shaul PW 2001 Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i). Journal of Biological Chemistry 276 27071–27076.

    • Search Google Scholar
    • Export Citation
  • Yamamoto K, Altschuler D, Wood E, Horlick K, Jacobs S & Lapetina EG 1992 Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85. Journal of Biological Chemistry 267 11337–11343.

    • Search Google Scholar
    • Export Citation
  • Zhang Z, Maier B, Santen RJ & Song RX 2002 Membrane association of estrogen receptor alpha mediates estrogen effect on MAPK activation. Biochemical and Biophysical Research Communications 294 926–933.

    • Search Google Scholar
    • Export Citation
  • Zwick E, Hackel PO, Prenzel N & Ullrich A 1999 The EGF receptor as central transducer of heterologous signalling systems. Trends in Pharmacological Sciences 20 408–412.

    • Search Google Scholar
    • Export Citation