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Membrane Initiated Estrogen Signaling in Breast Cancer¹

Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903

	ABSTRACT

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
Recent research has focused on effects of the estrogen receptor acting at the level of the cell membrane in breast cancer. In this review we describe 17beta-estradiol (E2)-initiated membrane signaling pathways involving the activation of several kinases that contribute to the regulation of cell proliferation and prevention of apoptosis. Although classical concepts had assigned priority to the nuclear actions of estrogen receptor, recent studies document the additional importance of estrogen receptor residing in or near the plasma membrane. A small fraction of estrogen receptor is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), estrogen receptor has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming a protein complex with many signaling molecules. The formation of the protein complex is a critical step, leading to the activation of the MAPK1/3 (also known as MAP kinase) and AKT1 (also known as Akt) pathways. 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.

estradiol, estradiol receptor, insulin-like growth factor receptor, mammary glands, signal transduction, CSK, IGF1R, PELP1, PIK3R1, and SHC1

	INTRODUCTION

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
The molecule 17beta-estradiol (E2) stimulates growth and prevents apoptosis in estrogen-responsive breast cancer cells. In contrast, in healthy mammary glands, estrogen receptor-positive cells are largely quiescent, and E2 stimulates these estrogen receptor-positive cells to produce growth factors that cause the growth of neighboring estrogen receptor-negative cells through paracrine effects [1]. As shown by Cunha et al. [1] in a series of experiments, both androgen-positive and estrogen receptor-positive cells can stimulate surrounding benign cells to proliferate through similar paracrine effects involving stromal-epithelial cell interactions. As an early step in the neoplastic transformation of the breast, the autocrine mechanism becomes predominant [2]. Estrogen receptor-positive breast cancer cells are themselves stimulated to grow by E2 through autocrine effects, and the estrogen receptor-positive cells are Ki67 positive [3].

For the past two decades major investigative emphasis in breast cancer research has focused upon genomic events resulting from the binding of E2 to estrogen receptor and ß (ESR1 and ESR2, also known as ER and ERß) in the nucleus of the cell. The liganded ESR further interacts with estrogen response elements in the promoter region of E2-responsive genes, leading to gene transactivation. Other nuclear actions of E2 involve the tethering of ESR to nuclear transcription factors, such as NFYB and SP1, which also lead to gene transactivation [4]. These nuclear events usually require hours or days for maximal gene activation. More recently, multiple studies demonstrated very rapid ESR1-mediated actions at the level of the plasma membrane. On the basis of the site of initiation of these events, a new terminology has been proposed. In this nomenclature, ESR-mediated nuclear events are referred to as "nuclear-initiated steroid signaling" (NISS), and membrane-initiated actions are termed "membrane-initiated steroid signaling" (MISS) [5].

A series of E2-induced MISS events has been described in benign cells and malignant cells of various origins. These effects occur from seconds to minutes after administration of E2 and involve rapid activation of many signaling molecules [6–8], such as 1) IGF1 and EGF receptors, 2) HRAS1 (also known as p21^ras) and RAF1, 3) MAPK1/3 and AKT1, 4) protein kinase C, 5) calcium channel, 6) nitric oxide and prolactin secretion, and 7) Maxi-K channels. These rapid effects are also called "nongenomic, extranuclear, or membrane-mediated effects," because they take place outside of the nucleus and are initiated predominantly in or near the plasma membrane.

The majority of studies describing MISS events focus on biochemical changes, such as those described above. Incontrovertible evidence of biologic actions mediated by MISS pathways is quite limited. The most convincing experimental support is the observation that vasodilatory responses to physiologic concentrations of E2 occur in isolated femoral arterial wall within 2 min in association with nitrous oxide formation [9, 10]. These effects can be blocked by inhibitors of MAPK1/3 and by antiestrogens. E2 also rapidly stimulates the secretion of prolactin in isolated pituitary cells [11]. In cancer cells E2 causes a rapid increase in dynamic membrane structures that can be abrogated with antiestrogens [12]. Less conclusive evidence of biologic effects are the observations that MISS events mediate the rapid morphologic effects of E2 on dendritic spines within the hippocampus in rodents [13] and on the rapid increase in water inhibition in uterus [14]. Others have suggested that the MISS events enhance cell proliferation and decrease apoptosis in many cell types and tissues, such as breast, bone, heart, endothelia, and brain, but supporting evidence is indirect [7, 15, 16].

A recent focus of ESR1 research has been to precisely define the mechanisms underlying membrane-initiated estrogen receptor signaling. 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 et al. [17], ESR1 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 ESR1-mediated transcriptional events. O'Malley's group has provided substantial evidence in support of this model [17]. This integrated view of MISS signaling suggests that events occurring initially at the level of the plasma membrane later modulate more-classic transcriptional events. Sequential interactions between MISS and NISS events broadly relate to the actions of multiple steroids. Progesterone [18], androgen [19], and 1,25(OH)₂D₃ [20] and their respective receptors also initiate events at the cell membrane.

With regard to MISS in breast cancer it is known that ESR1 has been a molecular target for breast cancer treatment, because it serves as a nodal point for mediation of several actions of E2. Current strategies in clinical use include blockade of ESR1 function through use of selective estrogen receptor modulators, such as tamoxifen, selective estrogen receptor downregulators, such as fulvestrant, and third-generation aromatase inhibitors to block E2 synthesis. A greater understanding of ESR1-mediated MISS and NISS events as well as the interactions of ESR1 with growth factor pathways should provide additional specific targets for development of new breast cancer treatment regimens.

The focus of this review is primarily to elucidate events occurring through the interaction of ESR1 with other proteins on the membrane in breast cancer cells. We will discuss the identity of membrane ESR1, the mechanism of ESR1 membrane association, and growth factor receptor involvement in rapid E2 action. Special emphasis is given to the formation of a large protein complex that activates various kinases and mediates downstream effects (Fig. 1). We hypothesize that the formation of this ESR1-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 large multiprotein complexes, leading to the activation of MAPK1/3 and AKT1. In terms of ESR1-centered protein complex formation, c-Src tyrosine kinase (CSK), SHC1, PELP1 (the modulator of nongenomic activity of estrogen receptor, also known as MNAR), PIK3R1 (also known as p85 of phosphoinositide-3-kinase), caveolins, and G proteins have all been reported to serve as components of large complexes of interacting proteins [21]. Through the mediation of these molecules, E2 activates the SHC1/MAPK1/3 and phosphoinositide-3-kinase/AKT1 pathways that are likely to be the major effectors of cell proliferation and cell survival.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Proposed model of E2-induced activating particle formation and its downstream signaling pathway activation. In this model, there are two phases for ESR1-mediated rapid E2 action, the interaction and stimulation phases. At the basal condition, ESR1 and CSK form an inactive protein complex due to basal level of Y537 phosphorylation on ESR1. In phase I, E2 induces the interaction of other proteins, such as SHC1 and PELP1, with the ESR1/CSK complex, leading to formation of activating particle. In phase II, the activated CSK in multiple protein complex preferentially activates the downstream signaling pathways.

	IDENTITY OF MEMBRANE E2 BINDING PROTEINS

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
Several possible candidates have been suggested to mediate binding of E2 in the plasma membrane. These include 1) full-length ESR1 [22]; 2) a truncated form of ESR1 with a molecular weight of 46 kDa [23]; 3) a unique estrogen membrane protein called ESR-X, whose ligand affinity differs from that of the classic ESR1 but is recognized by antibodies directed against ESR1 ligand binding domain [24]; 4) sex steroid binding protein acting in concert with a membrane protein megalin [25, 26]; 5) G protein-coupled receptor 30 (GPR30) [27]; and 6) an unknown protein present in non-ESR1-, non-ESR2-expressing CHO and COS-7 cells [28]. Although these various E2 binding proteins exist in specific systems, accumulating evidence supports the classic full-length ESR1 in or near the membrane functioning as the membrane estrogen receptor, which has been demonstrated in several types of cells, including breast epithelial cells, osteoblasts, endothelial cells, and vascular smooth muscle cells. Using multiple antibodies against ESR1, Watson et al. [12] demonstrated that the classical ESR1 is expressed near the region of the cell membrane in MCF-7 cells. Using confocal microscopic methods, our group demonstrated translocation of full-length ESR1 into or near the plasma membrane in response to E2. The antibodies we used were directed against only amino acids 2–184 in the N-terminus of the receptor (H-184, Santa Cruz). Accordingly, it would not recognize the 46-kDa truncated form described by Bender et al. [23] by using the F-10 anti-C-terminal ESR1 antibody (Santa Cruz). A proof-of-principle experiment demonstrated that transfection of ESR1-negative breast cancer cells with ESR1 resulted in 5% of ESR1 located in the plasma membrane and the remainder predominantly in the nucleus [22]. Biochemical studies also demonstrated full-length ESR1 protein in isolated plasma membranes, as detected by mass spectrometry analysis (Levin et al., unpublished results). Functional studies demonstrated that the rapid biochemical and biological effects of E2 seen in wild-type mice were abolished in ERKO (ESR1 knock out) and BERKO (ESR2 knock out) animals and that these animals have no demonstrable ESRs in isolated plasma membrane preparations [29]. Additional evidence supporting the classic ESR1 in the membrane was obtained from studies in which this receptor was knocked down by a selective siRNA [30]. In this study, E2 activated MAPK1/3 in a matter of minutes, but abrogation of ESR1 with a selective siRNA abolished this effect in human breast cancer MCF-7 cells.

Structurally, the classical ESR1 molecule contains 6 domains, named A to F (Fig. 2), which can be functionally divided into a hormone-independent activation function (AF-1) region, the DNA binding domain (DBD), a hinge region containing three nuclear localization sequences (NLS) that mediate the translocation of the receptor from cytosol to nucleus, and a hormone-binding domain (HBD) on the C-terminus of the receptor possessing dimerization and hormone-dependent activation function (AF-2). Binding of E2 to ESR1 leads to ESR1 dimer formation through the ligand binding domain and initiates a series of conformational changes in the protein structure of ESR1. The conformational changes uncover areas on the external surface of ESR1 that are responsible for the binding of coactivator molecules that facilitate gene transactivation in the nucleus. However, the necessity for the same conformational changes to initiate membrane signaling pathways, such as those involving MAPK1/3 and AKT1, has not yet been established. Posttranslational modifications of ESR1 through serine and tyrosine phosphorylations can also influence ESR1-mediated functions and presumably also induce conformational changes that could potentially modulate MISS events. One of these modifications involves phosphorylation of tyrosine-537, whereas others involve phosphorylation of serine 104, 116, and 167. Several other phosphorylations on ESR1 at other sites are possible but have not been extensively studied. The precise sites and conformational changes needed to allow ESR1 to localize in the plasma membrane are still poorly understood.

View larger version (3K): [in this window] [in a new window]   FIG. 2. Structure and functional domains of ESR1.

Additional studies demonstrated that it is the membrane ESR1 mediating E2 on MAPK1/3 activation [29]. For example, COS-1 cells expressing ESR1 only on the membrane responded to E2 on MAPK1/3 phosphorylation [31]. Katzenellenbogan et al. [32] demonstrated rapid activation of MAPK1/3 by use of E2 ligated to membrane impermeable dendrimers. Taken together, these data provide strong evidence that ESR1 can localize to the region of the cell membrane and initiate kinase-mediated events. A critical overview of this field would suggest, however, that truncated ESR1, G protein-coupled receptors, or other proteins may also be involved in rapid estrogen-initiated effects in some cell types and under certain circumstances [23, 33, 34].

	INTERACTIONS OF ESR1 WITH SIGNALING MOLECULES

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
ESR1 binds to many membrane and cytosol proteins as a means of initiating membrane signaling events. The regions of ESR1 involved in this process are not completely known. Results from various laboratories have often provided conflicting information, particularly regarding the role of the NLS domain of ESR1 in the mediation of MISS action. For example, NIH 3T3 cells transfected with a transcriptionally inactive ESR1 mutant lacking NLS domain responded to E2 by increasing CSK/HRAS1/MAPK1/3 activation and DNA synthesis [35]. Expression of this mutant in COS-1 cells also led to MAPK1/3 phosphorylation in response to E2 treatment [31]. In contrast, transfection of COS-7 cells with the same mutant was reported to prevent CSK and MAPK1/3 activation [10]. This mutant altered the ligand-induced interaction of phosphatidylinositide-3 kinase with ESR1 while inhibiting endothelial nitric oxide synthase NOS3 (also known as eNOS) phosphorylation [10]. These results leave open the question whether the NLS domain of ESR1 is needed for MISS events to occur. Other studies suggest that only the E domain of the ESR1 is needed for rapid activation of MAPK1/3. Expression of the E domain of ESR1 in CHO cells has been reported to mediate estrogen effects on MAPK1/3 activation and to prevent cells from undergoing apoptosis [36]. At face value, these results appear to be contradictory but may relate to differences in cell type or to the fact that all of these experiments were conducted in ESR1-negative cells transfected with ESR1.

	MECHANISM OF MEMBRANE LOCALIZATION OF ESR1

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
Even though ESR1 has no transmembrane domain and is not intrinsically a membrane protein, several laboratories have demonstrated that MISS actions of E2 require ESR1 translocation in or near the plasma membrane [30, 37]. Whether ESR1 membrane translocation and its activation on the cell membrane are sequential or an independent event is not currently known. We also do not understand the biological role of cytosol ESR1 in E2 rapid action, although effects on the mitochondria have been reported [38].

Our working model suggests that translocation of the ESR1 to the membrane involves SHC1 as a transporter. In response to E2, SHC1 is phosphorylated and binds to ESR1. At the same time, the SHC1 binding sites on the IGF1R are phosphorylated, allowing SHC1 to bind to the IGF1R. This is analogous to SHC1 acting as a bus, picking up ESR1 and then delivering ESR1 to the SHC1 binding site on IGF1R. ESR1 can then be tethered to the membrane through SHC1 interaction with IGF1R [30, 39]. Substantial proof for our working model has emanated from immunoprecipitation and Western blot experiments, from use of siRNA and dominant negative constructs, from confocal microscopy, and from biochemical studies of kinase activation [12, 30]. In these experiments, we demonstrated that E2 induces formation of a ternary complex consisting of ESR1, SHC1, and IGF1R in the cell membrane of MCF-7 cells. We also showed that E2 causes phosphorylation of the IGF1R and of SHC1. Elimination of SHC1 by either siRNA expression or by dominant negative constructs abrogates estrogen-induced MAPK1/3 activation (Fig. 3). More importantly, knock down of SHC1 with a specific siRNA prevents E2-induced ESR1 translocation to the plasma membrane, as shown by confocal microscopy studies (Fig. 4). 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 SHC1 might mediate E2-induced cell mobility changes. Recently, the adapter protein BCAR1 (also known as p130Cas) was reported to be associated with ESR1 and CSK in T47D breast cancer cells [40]. As BCAR1 is known to interact with focal adhesion kinase, it is conceivable that estrogen-induced cell morphologic changes might be mediated by BCAR1 independently of SHC1 and IGF1R.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Downregulation of SHC1-blocked E2-induced MAPK1/3 phosphorylation. Cells were transfected with a smart pool of siRNA against either a nonspecific target or SHC1 by means of the siPORT lipid transfection reagent. At day 2, cells were treated with vehicle or 0.1 nM E2 for 15 min, and the MAPK1/3 activation was assayed with antiphosphotyrosine MAPK1/3 antibody to both isoforms. The graphed data are from three experiments combined. All values are means ± SEM. *P < 0.05 for comparison of E2-treated cells with the vehicle-treated control in each siRNA transfected group. This figure is reproduced from Song et al. [30], Figure 5A, with permission of Proc Natl Acad Sci U S A.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Membrane colocalization of SHC1 and ESR1 in response to E2 with or without SHC1 siRNA transfection. The picture shows the merged images of confocal microscopy study. Under basal conditions (a) cells show fewer ruffles on the membrane (red color). Much of the SHC1 staining (blue color) was actually cytoplasmic rather than membranous. ESR1 (green color) was seen primarily in the nucleus but also was expressed in cytoplasm. E2 treatment stimulated SHC1 and ESR1 membrane region association, where they colocalized along the cell membrane, as indicated by the white color development due to the overlapping of the blue, green, and red colors (b). In many instances, ESR1 seemed to be collected specifically on the inner surface of extending filopodia rather than on the more basal extending edge. Expression of siRNA directed against SHC1 greatly reduced SHC1 protein expression, as indicated by decreased blue color (c), which also abolished ESR1 membrane association, but not morphologic changes, induced by E2. Original magnification x120; bar = 20 µm. This figure is reproduced from Song et al. [30], Figure 3B, with permission of Proc Natl Acad Sci U S A.

Another protein, striatin, was also reported to play a role in translocating ESR1 into the plasma membrane of EAhy926 human aortic endothelial hybridoma cells by interacting with amino acids 185–253 of N-terminal ESR1 [37]. The protein complex of striatin and ESR1 leads ESR1 targeting on the cell membrane. Notably, our preliminary data suggest that E2 also increased the binding of the ESR1 to EGFR. Furthermore, palmitoylation of ESR1 has been reported to enable the association of a truncated ESR1 with the plasma membrane [41]. Therefore, it is possible that SHC1 transports ESR1 to the membrane and that ESR1 palmitoylation facilitates the persistent residence of this receptor in the membrane. Data from Levin et al. [39] suggest that caveolin is also involved in the reversible shuttling of ESR1 from the cytosol to the membrane.

	ROLE OF THE IGF1R AND EGFR IN E2 RAPID ACTION

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
IGF1R is a key receptor involved in the growth of breast cancer cells, and its expression is always positively correlated with ESR1 expression in breast cancer. Estrogens appear to favor synergistic interactions with the IGF1 system, leading to the expression of several components, such as IGFIR, insulin receptor substrate 1, and IGF1 [42]. These upregulated proteins act locally in autocrine loops to mediate the biologic effects of estrogen. A close relationship between ESR1 and IGF1R has been confirmed by the fact that uterine cells do not respond to estrogen if the IGF-IR pathway is blocked and that IGF1 also loses its effect on gene transactivation and cell proliferation in ESR1-knockout cells [43]. A direct interaction between two pathways also occurs at the receptor level whereby estrogen can induce IGF1R phosphorylation in uterine epithelial cells, ESR1-transfected COS-7 cells, and MCF-7 breast cancer cells [44, 45]. These data indicate that the IGF1R-mediated signaling pathway is important in estrogen action.

Of interest, the expression of EGFR correlates inversely with the expression of ESR1. 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 [46, 47]. For example, EGFR is functionally involved in estrogen-induced MAPK1/3 activation in MCF-7 cells, a cell line with high expression of IGF1R and ESR1 but low EGFR. Interestingly, long-term blockade of ESR1 function with tamoxifen irreversibly causes cells to overexpress EGFR or HER2, another member of the EGFR family. Our preliminary data from immunoprecipitation and Western immunoblot studies suggest that E2 enhances the binding of ESR1 to the EGFR (unpublished results). In cells exposed to tamoxifen long term, increased binding of the EGFR to ESR1 occurs (unpublished results). These data support the hypothesis that the EGFR pathway can alter the function of tamoxifen and convert its estrogen antagonistic to agonistic activity.

Both EGF and E2 also cross talk at other levels. For example, administration of the antiestrogen ICI 182780 reduces the response to EGF in the mammary gland [48]. Studies in ESR1-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 [48, 49]. These results indicate that there is a true requirement for the presence of ESR1 for EGF-mediated biological functions, at least in some cell types. These and other data suggest that ESR1 serves as a nodal point that allows interactions between the ESR and growth factor pathways. On the basis of the observations presented above, studies involving nude mice demonstrated that the combination of Gefitinib (Irresa), a potent inhibitor of EGFR, and estrogen deprivation is more efficient at inhibiting ESR-positive breast cancer growth than either agent alone [50]. Clinical trials are now testing this concept in patients.

	MECHANISM OF ESR1-MEDIATED ACTIVATION OF THE MAPK1/3 AND AKT1 PATHWAYS

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
Both MAPK1/3 and AKT1 belong to the serine and threonine kinase family and play important roles in estrogen-induced cancer cell growth. Using a fusion protein consisting of an oncogenic form of human RAF1 and the ESR1 HBD domain (RAF1:ESR1), Samuels et al. [51] observed in 1994 that quiescent 3T3 cells transfected with RAF1:ESR1 responded to estrogen and showed rapid, sustained activation of MAPK1/3. Migliaccio et al. [52] then first demonstrated in studies of endogenous ESR1-expressing MCF-7 breast cancer cells that estrogen triggers rapid activation of MAPK1/3. These findings were further confirmed in many other cell types, including osteoblasts [53], rat brain cells [54], and human colon carcinoma-derived Caco-2 cells [55]. The observations that the pure antiestrogen ICI 182 780 blocks the membrane-initiated, rapid actions of E2 in many cell types, including breast cancer cells, provide further evidence that a membrane estrogen receptor is responsible for the rapid activation of many transduction pathways.

E2 also rapidly induces the activation of the phosphoinositide-3 kinase-AKT1 pathway in endothelium cells [56], cardiomyocytes [57], rat PC-12 neuronal cells [58], and MCF-7 cells [59]. In the presence of E2, ESR1 interacts with the regulatory subunit of phosphoinositide-3-kinase, PIK3R1, thus triggering activation of the catalytic subunit p110 of phosphoinositide-3-kinase, leading to the downstream kinase AKT1 activation [60]. Activation of AKT1 mediates many of the downstream cellular effects of phosphoinositide-3-kinase, including phosphorylation and inactivation of Bad to prevent Bad-mediated cell apoptosis, as well as rapid activation of the endothelial isoform of NOS3 [33].

Our studies in MCF-7 breast cancer cells demonstrated that E2 stimulation of MAPK1/3 occurs within 5 min and that MAPK1/3 later phosphorylates and activates the transcription factor Elk-1. Studies of the mechanism for E2-induced MAPK1/3 and AKT1 activation in breast cancer cells is now focused on the involvement of classical growth factor receptors, such as IGF1R and EGFR [45, 61]. E2 co-opts both IGF1R and EGFR signaling pathways and uses their downstream common adapter proteins to transduce signals, leading to the activation of MAPK1/3 and AKT1. These steps occurred within the time frame that E2 stimulated the activation of MAPK1/3 and AKT1. Knockdown of SHC1 blocked E2-induced MAPK1/3 activation, which was also confirmed by the expression of a dominant negative SHC1 in MCF-7 cells [12]. E2-induced activation of AKT1 could be attenuated by administration of the IGF1R 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.

	ESR1-INTERACTING MOLECULES

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
Before MAPK1/3 and AKT1 activation, ESR1 needs to interact with many signaling molecules to form a protein complex. It is now known that CSK activation in the ESR1-centered complex is a key step to initiate many downstream signaling pathways in E2 action. However, many other signaling molecules in the complex also play an important role in initiating and stabilizing the complex.

Role of CSK

ESR1 has no intrinsic kinase domain and therefore is not capable of phosphorylating other proteins. Accordingly, E2 must stimulate the activity of a kinase that serves this function. A key kinase candidate is CSK, which has previously been identified to physically interact with ESR1. Our working model suggests that E2 activates CSK, which in turn phosphorylates SHC1 and the IGF1R [62]. CSK then would serve as a crucial molecule to facilitate other protein-protein interactions involved in estrogen rapid action [6]. The precise mechanism of E2-induced CSK activation is not known. However, we now know that additional proteins are required for ESR1 interaction with CSK and CSK activation [19, 63]. This is supported by several recent observations, including the following: 1) in MCF-7 cells, Y-537 of ESR1 is basally tyrosine phosphorylated in vivo, even in the absence of ligand stimulation [64, 65]; 2) the pY-537 of ESR1 is the binding site for the SH2 domain of the CSK tyrosine kinase [19, 63]; and 3) the association of ESR1 with CSK is further increased after E2 treatment [19, 60, 63]. These data suggest that E2 induces a more stable protein complex between ESR1 and CSK, leading to CSK activation. The above observations support the fact that E2-induced CSK activation requires formation of a protein complex involving at least ESR1 and CSK.

CSK contains four domains: an SH3, an SH2, a tyrosine kinase domain, and a short carboxy-terminal tail (Fig. 5). In addition, CSK possesses two important regulatory tyrosine (Y) phosphorylation sites, Y-416 and Y-527. Under basal conditions, Y-416 in the activating loop of the kinase domain is unphosphorylated. Binding of CSK partner proteins to either the SH2 or SH3 domain of CSK can release CSK from its inhibited conformation into an unfolded position. Under these circumstances, pY-527 is dephosphorylated and Y-416 is phosphorylated, and changes in the phosphorylation status of Y-527 and Y-416 lead to CSK activation [66].

View larger version (18K): [in this window] [in a new window]   FIG. 5. Structure and functional domains of CSK, PELP1, PIK3R1, and SHC1.

At the present time, the detailed mechanisms by which ESR1 activates CSK via protein-protein interaction are still not clear. The Y-537 of ESR1 is phosphorylated under basal conditions, and this generates a binding site for the CSK SH2 domain [60, 63, 65]. On the basis of these findings, it is hypothesized that a transitional stage involving CSK and ESR1 association exists, which is not stable and cannot activate CSK to initiate downstream signaling events. Upon estrogen stimulation, the structure of ESR1 undergoes conformational changes, exposing both the N-terminal AF-1 and the C-terminal AF-2 domains which facilitate binding to other proteins. Of all the molecules that are potential partners with ESR1 and CSK in estrogen rapid action, PELP1 [63], PIK3R1 [67], SHC1 [12], G proteins [68], and caveolin-1 [39] are the most attractive candidates at this time.

We have introduced the term "activating particle" for this multiprotein complex containing ESR1, CSK, 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 CSK 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 ESR1 and CSK raises many questions regarding estrogen-induced signal initiation mechanisms that now require investigation. Although our working model suggests that EGFR and IGF1R act downstream of CSK activation, this remains to be precisely established [30].

Role of PELP1

PELP1 has been reported to interact directly with both ESR1 and CSK in the cytosol, stabilizing the protein complex and leading to CSK activation by unfolding CSK and inducing CSK Y416 phosphorylation [63]. PELP1 is widely expressed in a variety of estrogen-responsive tissues and developmentally regulated in mammary glands by interaction with many proteins, including nuclear transcriptional factors and the retinoblastoma protein [69, 70]. Owing to its involvement in estrogen-induced gene transactivation, PELP1 was initially recognized as a nuclear receptor-interacting protein. However, PELP1 has been recently demonstrated to act outside of the nucleus to interact with ESR1 and regulate the CSK activity of an ESR1/CSK complex [63]. PELP1 has 9 LXXLL motifs and 3 PXXP motifs (Fig. 5). X represents any amino acid. PELP1 and CSK interactions are mediated between a PELP1 PXXP motif and the CSK SH3 domain [63]. CSK and ESR1 interactions are mediated by ESR1 pY-537 and the CSK SH2 domain. The whole complex is further stabilized by the interaction between two N-terminal LXXLL motifs of PELP1 and AF-1 domain of ESR1 [63], although no specific motifs have been mapped on the E-domain of ESR1. The interaction of PELP1, ESR1, and CSK leads to CSK activation [63]. However, some questions regarding the role of PELP1 in regulating ESR1/CSK activation still remain. PELP1 has been reported to interact with most steroid family member receptors, such as the progesterone receptor (PR), and the androgen receptor (AR) [19, 71]. The PR alone, for example, is sufficient to activate CSK via direct interaction [71]. The AR also can interact and activate CSK in the presence of ESR1 but not PELP1 [19]. Further investigation of molecules besides PELP1 is still required to fully understand ESR1-induced CSK activation.

Role of PIK3R1

Phosphoinositide-3 kinase is composed of a regulatory domain, PIK3R1, and a p110 catalytic domain. PIK3R1 is an adapter protein and known to be involved in estrogen rapid action by forming a protein complex with ESR1 and CSK [60]. Phosphoinositide-3 kinase plays an important role in cell survival and in prevention of cell death [72]. Structurally, PIK3R1, as an adapter protein, is composed of an N-terminal SH3 domain, linker-containing polyproline residues, and N-terminal and C-terminal SH2 domains (Fig. 5) [73]. Activation of growth factor receptors, such as IGF1R and EGFR, on the cell membrane will recruit PIK3R1 to the receptors, leading to the activation of the p110 catalytic subunit of phosphoinositide-3 kinase [74, 75]. PIK3R1 as a substrate of IGF1R and EGFR not only transduces IGF1R signals to activate AKT1, but also forms a protein complex with ESR1 and CSK [60], leading to estrogen-induced CSK activation [60]. In this complex, CSK SH2 interacts with pY-537 of ESR1, and CSK SH3 with a polyproline sequence in the linker region of PIK3R1 [63, 76]. However, the interaction domains between ESR1 and PIK3R1 are currently unknown [67].

Role of SHC1, G Protein, GPCR, and Caveolins

The adapter protein SHC1, the G proteins (Gs and Gi), the GPR30, and caveolin-1 have all been involved in estrogen-induced MAPK1/3 activation by association with ESR1 [12, 52, 77–79]. Of these proteins, SHC1 was demonstrated to directly interact with ESR1 in MCF-7 cells, in which the SH2 domain of SHC1 physically interacts with ESR1 A/B region [12]. SHC1 has no intrinsic kinase domain. It transduces signals on the basis of 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 (Fig. 5) [80]. Interestingly, several laboratories have recently demonstrated another novel mechanism of CSK activation induced by SHC1 and G proteins in various cell types (human epidermal carcinoma A431, murine SFY, and COS-7) [81–84]. For example, the N-terminus of SHC1 associates with an activating loop (amino acids 401–435) in the CSK kinase domain (Fig. 5), altering its conformation, which leads to Y-416 phosphorylation and, thus, activation of CSK. This mechanism, unlike the one previously described, does not require the dephosphorylation of pY-527.

Both G proteins and caveolin-1 might also be potential candidates in the formation of the activating particle mediating the rapid effects of E2, because they have been reported to be in the complex with ESR1 [39, 68]. However, so far there is no evidence showing that they directly interact with ESR1 in a cell-free system. Currently, the physiological significance of this protein-protein interaction is unclear. GPR30 was recently reported to specifically localize to the endoplasmic reticulum and bind to estrogen [78]. Furthermore, MAPK1/3 and AKT1 activation were also reported to occur by an estrogen-induced sequential event involving the activation of G proteins and matrix metalloproteinase 2 and 9, release of heparin-binding EGF, and eventually activation of EGF receptor on the cell membrane [34, 79].

	FUTURE PERSPECTIVES

TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 
The data reviewed suggest that an ordered series of events coordinates the formation of complexes that allow signaling through the MAPK1/3 pathway, the AKT1 pathway, and, potentially, other pathways. Minimal attention has been directed to the precise order of events. With respect to localization of ESR1 to the membrane region in MCF-7 cells, SHC1 binding to ESR1 seems to be an early event. When ESR1 complexes with CSK, PIK3R1, PELP1, and G proteins, it is not known if the complex formation is also cell-type dependent. The precise molecular sites on each protein that allow complex formation are incompletely understood.

Taken together, we have devised a model for the formation of a multiprotein complex (the activating particle) involving ESR1, CSK, PELP1, PIK3R1, SHC1, and G proteins in the case of ESR-responsive breast cancer cells (Fig. 1). In this protein complex, PELP1, PIK3R1, SHC1, and G proteins all act to mediate ESR1-induced CSK activation. It should be noted that the term "signalsome" has recently been suggested, which also describes the formation of protein complexes involving IGF1R/SHC1/ESR1 or EGFR/G protein/CSK/ESR1 [7]. These "signalsomes" form rapidly in response to E2 treatment in MCF-7 cells. Practically speaking, a better understanding of why ESR1-mediated, nongenomic estrogen action requires such a large protein complex to be formed is a question that deserves high priority. Agents disrupting this complex might serve as targets to counteract the membrane-initiated effects of E2. In the meantime, we also need to deduce how this complex can form and activate signaling pathways in a timely fashion. As both genomic and nongenomic actions of estrogen can regulate cell proliferation and apoptosis, the protein interactions discussed above may be the clue to developing therapeutic manipulations of specific targets that might be equally effective for the treatment of estrogen-dependent and estrogen-independent breast cancer. A combination of drugs targeting different proteins may provide synergistic benefits to prolong a patient's life.

	FOOTNOTES

 
¹ Supported by the Department of Defense Breast Cancer Research Program (grant DAMD 17–02–1–0610 to R.X.S.) and the NIH (grant CA 65622 to R.J.S.).

² Correspondence: Robert X.-D. Song, Division of Endocrinology, University of Virginia Health Science Center, Charlottesville, VA 22908. FAX: 434 924 1284; rs5wf{at}virginia.edu

Received: 5 December 2005.

First decision: 14 January 2006.

Accepted: 24 March 2006.

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TOP ABSTRACT INTRODUCTION IDENTITY OF MEMBRANE E2... INTERACTIONS OF ESR1 WITH... MECHANISM OF MEMBRANE... ROLE OF THE IGF1R... MECHANISM OF ESR1-MEDIATED... ESR1-INTERACTING MOLECULES FUTURE PERSPECTIVES REFERENCES

 

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