The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor {alpha} to the plasma membrane -- Data Supplement - HTML Page - 08334SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Text
Supporting Materials and Methods
Reagents.
Tissue culture supplies were obtained from Fisher Scientific. IMEM with or without phenol red (zinc option Richter’s modification) and FBS were products of GIBCO Life Technologies. 17b -estradiol (E2) was obtained from Steraloids (Wilton, NH). ICI 182,780 (ICI) was from AstraZeneca (Wilmington, DE). E2 and fulvestrant were dissolved in ethanol with a final concentration of ethanol in a medium of < 0.01%. The smartpool of double-stranded small inhibitory (si)RNA against Shc (P-002031-01-20), estrogen receptor a (ERa ) (ESR1-NM-00-05), or insulin-like growth factor-1 receptor (IGF-1R) (IGF1R-NM-000875) as well as nonspecific siRNA (D-001206-01-05) were from Dharmacon Tech (Lafayette, CO), which uses a sophisticated algorithm to combine four or more siRNA duplexes in a single pool for each selected protein, to enhance the probability that the siRNA pool reagent will reduce mRNA to low levels. A pool of siRNA corresponding to nonspecific mRNA was used as a negative control. All siRNAs were dissolved in RNase-free buffer based on the manufacturer’s protocol at the concentration of 20 m M. The transfection reagent siPORT lipid was from Ambion (Austin, TX). Cell Culture and siRNA Transfection.
MCF-7 cells growing in 5% FBS-IMEM were routinely maintained in a humidified 95% air, 5% CO₂ incubator at 37°C. Two days before treatment, the medium was changed into phenol red-free IMEM supplemented with 5% dextran-coated charcoal-stripped FBS (DCC-FBS) for 24 h and then IMEM with 1% DCC-FBS for 4 h. Cells were then treated with different agents, as indicated, and collected for assays. For transfection experiments with siRNA directed against Shc, ERa , or IGF-1R, the medium for MCF-7 cells was first changed into antibiotic- and serum-free DMEM without phenol red (GIBCO Life Technologies) for 2 hours. Cells were then transfected with siRNA against mRNA of nonspecific (control), IGF-1R, ERa , or Shc at 50 pmol/ml. The transfection method using siPORT lipid was carried out according to the manufacturer’s instructions. After 6 hours, fresh IMEM containing 5% DCC-FBS was added to bring the volume to 2 ml per well in total. Two days later, cells were extracted for Western blotting. Immunoprecipitation and Immunoblotting.
Immunoprecipitation (IP) and immunoblotting (IB) were carried out as described previously (1). Briefly, cells were washed once with ice-cold PBS containing 1 mM Na₂VO₄ and extracted with binding buffer (50 mM Tris, pH 8.0/150 mM NaCl/5 mM EDTA/5% glycerol/1% Triton X-100/25 mM NaF/2 mM Na₂VO₄/10 m g/ml each aprotinin, leupeptin, and pepstatin). Cell lysates were centrifuged at 14,000 × g for 10 min at 4°C to pellet insoluble material. The protein concentration of the supernatant was determined by using a DC protein assay kit based on the Lowry method (Bio-Rad). Equal amounts of proteins from cell extracts (0.5 mg) of each treatment were immunoprecipitated by using one of the following antibodies: 1 m g of monoclonal anti-IGF-1R domain antibody (3B7, Santa Cruz Biotechnology), 1.2 m g of monoclonal anti-ERa antibody (D-12; Santa Cruz Biotechnology), and 1 m g of monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology). Incubations proceeded overnight at 4°C in the presence of 35 m l of 50% slurry protein-G Sepharose beads (GIBCO Life Technology). The beads were washed three times in cold binding buffer. For Western blotting, the proteins (either eluted from the beads or from the whole cell extracts) were analyzed on 7.5% SDS--polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with one of following primary antibodies, including horseradish peroxidase-conjugated monoclonal antiphosphotyrosine antibody (4G10, Upstate Biotechnology, Lake Placid, NY ), polyclonal anti-ERa antibody (MC-20, Santa Cruz Biotechnology), polyclonal anti-Shc antibody (Transduction Laboratories, Lexington, KY), polyclonal anti-IGF-1R antibody (C-20, Santa Cruz Biotechnology), polyclonal anti-IRS-1 (Upstate Biotechnology), and monoclonal anti-VDR antibodies (9A7, Affinity BioReagents, Golden, CO). After washing the PVDF membrane, the immunoblot was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour (donkey against rabbit IgG was from Pierce; sheep against mouse IgG was from Amersham Pharmacia) and further developed by using the chemiluminescence detection system (Pierce). The phosphorylation status of mitogen-activated protein kinase (MAPK) from whole-cell extract was assayed using an antibody recognizing dual phosphorylation MAPK. Confocal Analysis of Colocalization of ERa , Shc, and IGF-1R on Cell Membrane.
Cells were fixed in paraformaldehyde for 20 min at room temperature, rinsed with PBS, then postfixed in cold acetone for 5 min. The coverslips were then blocked in normal goat serum followed by incubation with appropriate primary antibodies diluted in 1% normal goat serum PBS. Cells were then rinsed three times in PBS and incubated with appropriate secondary antibodies and phalloidin (for localization of F actin). Primary antibodies used were: rabbit anti-ERa (H184, Santa Cruz Biotechnology sc-7207 diluted 1:100), mouse anti-Shc (PG-797, Santa Cruz Biotechnology sc-967 diluted 1:200), mouse anti-IGF-1R (3B7, Santa Cruz Biotechnology sc-967 diluted 1:200), and mouse antivinculin (hVIN-1, Sigma V-9131 diluted 1:200). Anti-mouse and anti-rabbit second antibodies conjugated to the fluorescent dyes Alexa 488 (green) or Alexa 633 (far red, colored as blue) and phalloidin conjugated to Alexa 546 (red) were purchased from Molecular Probes. Colocalization of ERa , Shc, and IGF-1R on the cell membrane was demonstrated by the development of white color due to the overlapping of green, blue, and red pixels. The detailed method on confocal microscopy study was previously described (2). Extended Discussion

Estradiol, like dihydrotestosterone, cortisol, progesterone, and 1,25(OH)₂D₃, is a member of the steroid hormone family, which classically acts through transcriptional regulation at the nuclear level (3). However, an increasing body of evidence has demonstrated that all of these hormones also exert rapid membrane-mediated effects on their target cells (4-8). The rapid effects induced by these steroid hormones require only seconds to minutes, leading to activation of many signaling pathway molecules, such as kinases (9), adapter proteins (1), G proteins (10), metaloproteinase activity (11), release of heparin-binding epidermal growth factor (EGF) (12), and Ca²⁺ (13). All of these actions presumably require translocation of ERa to the region of the plasma membrane. These results led to the conclusion that the rapid effects induced by steroid hormones are cell membrane-dependent events and may be mediated by specific membrane receptors. Controversy exists regarding the identity of the membrane receptor proteins and specifically whether they are classical receptors or other binding proteins.

Regarding the estrogen membrane receptors, evidence has accumulated that the same receptor can be present in the nucleus as well as in the region of the membrane, at least in human breast cancer cells (1), endothelial cells (14), and rat pituitary cells (15). Down-regulation of ERa by selective siRNA in the present study clearly demonstrates the role of ERa itself in mediating the effects of E2 on MAPK activation. Unlike transmembrane growth factor receptors, ERa has no intrinsic transmembrane domain, indicating that it is not a transmembrane protein. We postulated that a third-party protein might mediate its translocation to the region of the plasma membrane. The serine-522 residue in the C-terminal portion of ERa has been reported to be a critical residue linking the receptor to the cell membrane, perhaps through the interaction with caveolin (16). However, expression of this mutant in the ERa -lacking Chinese hamster ovary cells decreased only 60% of ERa on the membrane, suggesting that serine-522 is not the sole residue for ERa membrane association. It is possible that mutation of serine-522 altered the 3D structure of ERa , making ERa less functional with respect to its interaction with partner proteins.

Recently a truncated ERa , ERa p46, has been reported in human endothelial cells. It targeted to the plasma membrane in a palmitylation-dependent manner, but the domain required for membrane interaction was not defined (17). Furthermore, it is noteworthy that ERb shares highly homologous sequences with ERa , particularly in the DNA- and C-terminal ligand-binding domains. The sequences for both receptors have far less homology in the N-terminal regions (18). At the present time, only ERa has been reported to be associated with the cell membrane, but not ERb , even though both receptors are coexpressed in many breast cancer cells (19). Before this study, it was unclear how ERa translocates to the cell membrane and transduces E2 signals to downstream kinase. Our current findings clearly demonstrated that Shc plays a role as a driving force to bring ERa to the membrane by binding on membrane IGF-1R. Although not addressed in our studies, ERa palmitoylation might occur and contribute to the permanent residence of the receptor on the membrane. The hypothesis is currently under active investigation in our laboratory.

Caveolin-1 has been reported to interact with ERa and suggested to serve as a means of holding ERa on the membrane in MCF-7 cells (20). However, ERa in this process is dissociated from caveolin on E2 treatment and would be expected to cause a decrease in amount of ERa on the membrane. In our previous and current studies, an increase of ERa association with the plasma membrane region was observed on E2 treatment. The reason for these possible discrepancies is unknown. We postulate that caveolae might serve to sequester ERa in an inactive form, which could then be released on E2 stimulation. Caveolae serve as part of the plasma membrane structure and contain foci of growth factor receptors, including IGF-1R, that reside there. Perhaps these provide an intermediate station for ERa before its interaction with Shc and IGF-1R.

Transmembrane receptors for growth factors, such as IGF-1 and EGF receptor (EGFR), are believed to be an integral component in the growth response to E2. It was unknown, however, at which step these receptors are involved in rapid E2 action. The requirement for the activation of IGF-1R in E2 rapid action on MAPK activation has been reported (11, 19). The tyrosine phosphorylation of IGF-1R induced by E2 was first reported in uterine epithelial cells (21, 22). Using WT ERa -transfected COS-7 cells, Kahlert et al. (19) further demonstrated that E2-induced IGF-1R phosphorylation required the physical interaction of ERa with IGF-1R, but the molecular basis for this interaction was not clear. Previously we demonstrated a strong interaction between ERa and Shc through N-terminal ERa and PTB/SH2 domains of Shc in MCF-7 cells (1). Shc is a well-defined molecule, which mediates growth factor mitogenic actions by activation of MAPK pathway (23). Activation of IGF-1R will recruit Shc binding on the receptors (24). Regarding IGF-1R, it is noteworthy that a large amount of evidence pointed out that Y950 of IGF-1R is a major site for Shc binding, phosphorylation, and activation in response to IGF-1 (25, 26). IGF-1R functions as a membrane-docking site that recruits the adapter protein Shc after the receptor is activated. Interestingly, the combination of E2 with IGF-1 did not show an additive effect on either IGF-1R phosphorylation or the protein complex formation, indicating that E2 might work on the same Y950 residue on IGF-1R. Nevertheless, more complicated mechanisms existed for the involvement of IGF-1R, because phosphorylated IGF-1R also recruits p85 regulatory domain of PI3K binding to the receptor. This domain has been reported to physically associate with ERa in human vascular endothelial cells and MCF-7 cells (9, 27), which might be explained by our biochemical findings that down-regulation of Shc decreased the level of ERa associated with IGF-1R but did not completely eliminate ERa and IGF-1R interaction. In terms of the biological role of Shc-mediated ERa membrane association, further investigation is required to study the membrane-associated ERa function on E2-induced mitogenic and antiapoptosis actions. At least the current study provides important evidence that Shc may play a pivotal role in mediating E2 action in breast cancer cells.

EGFR, in a fashion analogous to IGF-1R, is also a transmembrane tyrosine kinase receptor that has been engaged in different processes critical for cell proliferation, survival, and tumor invasion. The expression of EGFR in MCF-7 cells is very low, but EGF indeed strongly induced MAPK phosphorylation in MCF-7 cells, indicating that EGFR exists. E2 has been reported to activate MAPK by activation of matrix metalloproteinases-2 and -9, EGF release, and activation of EGFR (10, 11). At the present time, it is not clear whether EGFR is also involved in Shc-mediated ERa membrane association, which is highly likely because EGFR activation requires Shc binding to the receptor. The interaction between the signaling pathways of ERa and the EGFR is known to contribute to the biological effects of E2. Experiments show that EGF antibody prevents E2-induced vaginal and uterine growth (28), implying that crosstalk from ERa to the EGFR at the membrane may be physiologically important. It has been postulated that E2 through ERa utilizes membrane EGFR to rapidly signal through various kinase cascades that influence both transcriptional and nontranscriptional actions of estrogen in breast cancer cells. The requirement for EGFR on E2-induced ERa -membrane association is currently under investigation in our laboratory.

The present study demonstrated a dramatic dissociation between the requirement of Shc for MAPK activation and for dynamic membrane changes. Specifically, knock down of Shc blocked MAPK activation by E2 but not dynamic membrane changes (Figs. 3Bc and 4Bc). The result clearly indicates that Shc appears not to be involved in E2-induced changes in dynamic membrane structures. In contrast, ERa is required as reported previously in studies, demonstrating that ICI blocked E2-indued cell dynamic membrane formation (9). Our working hypothesis to explain this dichotomy of effects is that an ERa /Shc/IGF-1R complex in the plasma membrane may be needed to activate MAPK. On the other hand, the presence of ERa in the cytosol contiguous with dynamic membrane changes may be sufficient to induce dynamic membrane changes. Our prior study demonstrated that E2 caused not only colocalization of ERa with actin in the membrane ruffles but also a dramatic accumulation of ERa in the cytoplasm contiguous with the pseudopodia that formed. Although this is an appealing hypothesis, we have no direct data to support it and plan extensive experiments to determine the mechanism for these dissociated effects of E2 on Shc and cell morphology. Src is known to be involved in E2 action in MCF-7 cells and physically interacts and regulates many focal adhesion molecules, such as p130Cas, AND-34, and FAK kinase (29, 30). Study of the role of Src and the focal adhesion molecules noted above might reveal further clues for E2 action on cell morphologic changes in breast cancer cells.

Previously we demonstrated that ERa , Shc, and Src are all upstream components in E2 action on MAPK phosphorylation in MCF-7 cells, and ERa membrane targeting is required for E2 on MAPK activation (1, 31). Using siRNA against ERa and Shc, we further confirmed our previous findings in this study. Specifically, we demonstrated that IGF-1R also functions upstream of MAPK activation because both siRNA against IGF-1R and the IGF-1R inhibitor AG1024 block MAPK activation. Surprisingly our preliminary data show that PD98059, a MAPK kinase (MEK) inhibitor, also blocked IGF-1R phosphorylation induced by E2 (data not shown). The same observation was also documented by Kahlert et al. (19) using ERa -transfected COS-1 and HEK293 cells. This novel reciprocal crosstalk was also reported between PI3K and Src regulation (9), indicating the complexity in the regulation of signal transduction pathway molecules. The mechanisms for E2-induced IGF-1R phosphorylation are unknown. Sato et al. (32) recently reported that Shc directly activates c-Src, which has been long known to phosphorylate both Shc and IGF-1R. It seems that Shc and Src can mutually activate each other, depending on the upstream stimulators. Based on our findings, we propose a model for E2-induced ERa membrane association and its function on MAPK activation (Fig. 10). In this model, liganded ERa interacts with Shc, leading to Src activation. Src in turn phosphorylates IGF-1R, which recruits Shc. Shc acts as a translocator bring ERa to the cytoplasmic membrane. Association of Shc with membrane IGF-1R serves two functions; one is to lead to ERa membrane association, and the other activates MAPK in an IGF-1R-dependent manner.

In this study, we exercise guarded caution and refer to ERa as being translocated into the region of the plasma membrane. Our confocal microscopy data demonstrated colocalization of ERa with actin in a focal pattern, but this technique requires only that both proteins are close, within 800 Å of each other, and not strictly directly interacting. In addition, our prior studies showed that E2 can cause accumulation of ERa in the "fist" of the pseudopodia induced by E2 administration (9). The "fist" contains cytoplasm in its interior, and this is the region where the ERa appears to accumulate. For these reasons, we use the term "in the region of the plasma membrane." An approach using fluorescence resonance energy transfer (FRET) studies will now be necessary to determine directly whether ERa resides in the membrane itself, as suggested by its binding to IGF-1R.

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