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Molecular Endocrinology 17 (4): 589-599
Copyright © 2003 by The Endocrine Society

Isoform-Selective Interactions between Estrogen Receptors and Steroid Receptor Coactivators Promoted by Estradiol and ErbB-2 Signaling in Living Cells

Yongli Bai and Vincent Giguère

Molecular Oncology Group (Y.B., V.G.), McGill University Health Center, and Departments of Biochemistry, Medicine and Oncology (V.G.), Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Vincent Giguère, Molecular Oncology Group, McGill University Health Centre, Room H5-21, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vincent.giguere{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor (ER){alpha} and -ß interact with a variety of coactivator proteins, most notably members of the steroid receptor coactivator (SRC) family, and these interactions have been shown to be regulated by estrogenic ligands and growth factor signaling. Here, using fluorescence resonance energy transfer (FRET), the selectivity of different stimulants on ER{alpha} and -ß interactions with coactivator receptor interaction domains (RIDs) were examined in living cells. We first show that ER{alpha} and ERß homo- and heterodimers form in vivo independently of the presence of 17ß-estradiol (E2) or antiestrogens. We then demonstrate that E2 enhances interactions between ER{alpha} and the RIDs of SRC-1 and SRC-3, whereas the interaction between ER{alpha} with the SRC-2 RID is ligand independent. The transcriptionally inactive mutant ER{alpha}L539A showed no interaction with all three SRC RIDs. Similarly, treatment with the antagonists 4-hydroxytamoxifen and EM-652 abolished all interactions between ER{alpha} and the SRC RIDs. FRET data also demonstrate that, in contrast to ER{alpha}, ERß interacts with all three SRC RIDs in a ligand-independent manner. However, these interactions were further enhanced or stabilized by E2, whereas the antiestrogen EM-652 abolished all interactions. In the presence of both ER{alpha} and ERß, E2 treatment led to the recruitment of SRC RIDs to the nuclei. Finally, expression of the oncogenic activated ErbB-2/Neu protein specifically enhanced ER{alpha} but not ERß interactions with SRC RIDs to an extent similar to E2-stimulated interactions. In summary, using FRET, we demonstrated preferential interactions between ER isoforms and coactivators upon hormonal treatment and activation of a growth factor signal transduction pathway in living cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GENOMIC ACTIONS of natural and synthetic estrogens are predominantly mediated by two members of the nuclear receptor superfamily, namely estrogen receptor (ER){alpha} (NR3A1) and ERß (NR3A2; Refs. 1, 2, 3). In the presence of an estrogenic ligand, the two ERs, either as homo- or heterodimers (4, 5, 6, 7, 8), recognize specific response elements in target genes. Ligand binding also induces conformational changes in the receptors that alter their interactions with coregulatory proteins, a molecular event necessary for the regulation of gene expression by the ligand-receptor complexes (9, 10, 11). A large number of nuclear receptor coregulatory proteins carrying out diverse functions have been identified, and these proteins can be classified as corepressors or coactivators on the basis of their effect on receptor-mediated gene expression (reviewed in Refs. 12 and 13). The best characterized coregulators include silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor as corepressors and p300, cAMP response element binding protein-binding protein, pCAF, and members of the steroid receptor coactivator (SRC) family as coactivators. The SRC family is composed of SRC-1/NcoA1, SRC-2/NcoA2, and SRC-3/NcoA3. The interactions between SRCs and nuclear receptors are mediated through receptor interacting domains (RIDs) containing one or more copies of an {alpha}-helix motif with the consensus sequence LXXLL (14, 15) and a hydrophobic cleft located on the surface of the receptor ligand-binding domain (16). Several studies have shown selective interactions between nuclear receptors, including ER{alpha} and ERß, and SRC RIDs or LXXLL-containing peptides that are dictated by the composition of the LXXLL motifs and surrounding sequences (17, 18, 19, 20, 21, 22, 23, 24).

Activation of growth factor receptor-coupled signaling pathways has been shown to directly modulate nuclear receptor transcriptional activity (25). Growth factor signals that have been shown to enhance ER-regulated gene expression include TGF{alpha} and epidermal growth factor (EGF), insulin and IGF-I, and Heregulin (reviewed in Ref. 26). Heregulin is the ligand for ErbB-4, a member of the ErbB/HER family of receptor tyrosine kinases (27). Within this family, the ErbB2 receptor is of particular interest since its gene is frequently amplified and overexpressed in primary breast cancer, and its presence can impede the antiproliferative effects of hormone therapy (28). Serine phosphorylation was shown to mediate EGF activation of ER{alpha} through the Ras-MAPK signaling cascade (29, 30). Similarly, ERß transcriptional activity can be enhanced by the Ras pathway (31), and phosphorylation of MAPK sites located in the amino-terminal domain of ERß stimulates a ligand-independent interaction with SRC-1 (32). Equally, growth factors can also signal to steroid receptors through direct phosphorylation of SRC proteins and modulate their transcriptional activity (33, 34).

Fluorescence resonance energy transfer (FRET) is a method to monitor two proteins simultaneously and can be applied to study the dynamic behavior of two components of a specific signaling system (35, 36, 37, 38). FRET has recently been used successfully to study ligand-induced ER{alpha}-LXXLL peptide interactions in living cells (39). Here, we have chosen to generate chimeric cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) pairs to examine the selectivity of interactions between ER{alpha} and -ß and coregulatory proteins in response to diverse stimuli, including natural hormones, antiestrogens, and the ErbB-2 pathway. FRET was first tested in regard to the percentage of CFP donor and YFP acceptor captured as FRET signal due to overlapping spectrums. Individual cell differences in FRET signal were compared to establish a suitable level of confidence in the assay. We then examined the formation of ER{alpha} and ERß homodimers and heterodimers in vivo in the absence and the presence of ligand. Third, the specificity of interactions of ER{alpha} and ERß with all three members of the SRC family was determined: 17ß-estradiol (E2) promotes ER binding to the SRCs, whereas treatment with the antiestrogens 4-hydroxytamoxifen (OHT) and EM-652 abolished the interactions of ERs with the coactivators. For both ER{alpha} and ERß, coactivators are recruited to nuclei upon E2 treatment, whereas the transcription-defective ER{alpha}L539A mutant fails to promote coactivator movement to the nuclei and physical binding to ER{alpha}. In addition, we show that ER{alpha} and ERß display preferential binding to SRC proteins in response to E2. Finally, we demonstrate that ErbB2/neu signaling selectively activates ER{alpha} interactions with all three SRC RIDs but has no effect on ERß-SRC RID interactions. From these data, we can conclude that multiple mechanisms exist in cells to coordinate and selectively modulate ER-regulated gene expression in vivo.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CFP Donor and YFP Acceptor Bleed through to FRET Channel
CFP and YFP pairs were chosen for FRET because they meet the basic requirements of FRET analysis. The human kidney 293 cell line was used for all experiments. The emission of CFP overlaps well with the excitation of YFP: CFP has an extinction coefficient (EM) 26,000 cm-1 M-1 (435 nm excitation) and YFP an EM of 36,500 cm-1 M-1 (514 nm excitation) (Fig. 1AGo). A total of 105 CFP or YFP alone or fusion proteins were analyzed in eight independent experiments. These samples comprise the same fusion proteins in three individual cells, different fusion donors or acceptors, and the fusion proteins in the presence of ER ligand. As shown in Fig. 1BGo, CFP bleeds through FRET signal because its emission spectrum crosses over with that of YFP. Thus, exciting CFP can cause the detection of emission at both the CFP emission filter and the YFP emission filter. Fd is the image signal of CFP donor alone using the FRET filter sets (435 ± 10 nm excitation, 535 ± 10 nm emission; F, FRET filters; d, donor alone sample), and Dd is the CFP signal using CFP filter sets (435 ± 10 nm excitation, 480 ± 10 nm emission; D, donor CFP filter sets; d, donor alone sample). The FRET signal (Fd) divided by CFP signal (Dd) is the percentage of CFP bleeding through FRET channel (see Materials and Methods and Ref. 37). As shown in Fig. 1BGo, the slope (Fd/Dd) is relatively stable, although ligand treatment (10-8 M E2) caused some variation, most likely the effect of conformation changes in the receptor (data not shown). The confidence for CFP bleeding through to the FRET channel is 39.38 ± 0.81% (n = 105, P < 0.01).



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  		Figure 1. Bleed Through to FRET Signal by Donor CFP, Acceptor YFP Due to Overlapping Spectrum

A, CFP and YFP spectrum illustration: CFP donor excitation (purple line), CFP emission (cyan line), YFP excitation (green line). B, Donor CFP bleeds through FRET channel. CFP donor-alone transfections (105) were analyzed (P < 0.01). Fd is the FRET signal contributed by CFP when excited at 435 ± 10 nm and detected at 535 ± 10 nm. Dd is the donor CFP signal excited at 430 ± 10 nm with emission filter at 480 ± 10 nm. C, Acceptor YFP bleeds through FRET channel. Fa is the FRET signal of YFP excited at 435 ± 10 nm with emission filter at 535 ± 10 nm. Aa is the acceptor signal excited at 480 ± 10 nm with emission at 535 ± 10 nm. All values are pixel/area after subtracting the corresponding backgrounds.

 
The bleed through of YFP acceptor to FRET signal was also characterized in a similar manner as that of the CFP donor. In this case, the bleed through is due to overlap of the CFP excitation spectrum with that of YFP excitation. In other words, YFP is slightly excited when CFP is excited at 435 nm. Fa is the image signal of YFP donor alone using the FRET filter sets (435 ± 10 nm excitation, 535 ± 10 nm emission; F, FRET filters; a, acceptor alone sample), and Aa is the YFP signal using YFP filter sets (480 ± 10 nm excitation, 535 ± 10 nm emission; A, acceptor YFP filter sets; a, acceptor alone sample). As shown in Fig. 1CGo, YFP confers a very consistent percentage to the FRET channel: 58.36 ± 1.07% (n = 105, P < 0.01). All image signal values (Fd, Dd, Fa, and Aa) have been subtracted from the background in the same image.

The bleed throughs of CFP and YFP stay relatively constant irrespective of different amounts of CFP or YFP molecules in the individual cells. In contrast, when both donor and acceptor are coexpressed, the bleed through of CFP donor alone (Fd/Dd) or YFP acceptor alone (Fa/Aa) to the FRET signal is concentration independent. That is, the different amount of CFP donor or YFP acceptor [Df(Fd/Dd), Af(Fa/Aa) in which Df relates to image shot of both CFP donor and YFP acceptor with donor filters and Af relates to image shot of both CFP donor and YFP acceptor with acceptor filters] in a particular cell changes the percentage of donor or acceptor contribution to the total FRET signals (Materials and Methods and Ref. 37).

FRET Signal of Similar Dimers Varies among Individual Cells
As shown in Fig. 2Go, bleed through of CFP and YFP to FRET signal for the same pair can vary to a certain extent. Fourteen single cells were examined for the same CFP-ER{alpha} and YFP-ER{alpha} pair. Individual cells express varying amounts of donor and acceptor because of differences in cell cycle, microenvironment, and capture of different quantities of transfected DNA. Thus, the changes in donor and acceptor expression in cells cause variations relating to the contribution of donor or acceptor to the FRET signal. However, based on the formula for FRET calculations, a dramatic change in donor or acceptor expression level only leads to a small change in the FRET signal. The contribution ratio is calculated as the following: donor contribution is given by Df(Fd/Dd)/Ff and acceptor by Af(Fa/Aa)/Ff.



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  		Figure 2. Individual Cell Variance of Real FRET and Bleed Throughs from Donor and Acceptor

A, Fourteen pairs of ER{alpha} homodimers in the absence of ligand were used as an example to show single-cell variance for the same pair of dimers. The bleed through from donor and acceptor were calculated as described in Materials and Methods. Donor CFP bleed through is in gray, acceptor bleed through is in white, and real FRET signal is black. B, Distribution of bleed throughs from donor and acceptor for 10 pairs of different ER dimers. Values for each different pair are from three single-cell image processing. The 10 pairs are CFP-ER{alpha}, YFP-ER{alpha} without or with 10-8 M E2 treatment (lanes 1 and 2), CFP-ER{alpha}, YFP-ERß, control or E2 (lanes 3 and 4), CFP-ERß, YFP-ER{alpha} control or E2 (lanes 5 and 6), CFP-ERß, YFP-ERß control or E2 (lanes 7 and 8) and CFP-ER{alpha}L539A, and YFP-ER{alpha}L539A control or E2 (lanes 9 and 10).

 
The variances among different ER homodimers and heterodimers were also compared. A total of 10 pairs of ER dimers were analyzed, and the value for each pair came from three single cells. As shown in Fig. 2BGo (lanes 1–10), the ER dimers are ER{alpha} homodimers in the absence or presence of 10-8 M E2 (lanes 1 and 2), ER{alpha}/ß heterodimers (CFP-ER{alpha} and YFP-ERß, lanes 3 and 4; CFP-ER{alpha} and YFP-ER{alpha}, lanes 5 and 6), ERß homodimers (lanes 7 and 8), and ER{alpha}L539A mutant homodimers (lanes 9 and 10). As shown, the contribution of f donor and acceptor are similar among the homodimers and heterodimers in different cells. Therefore, the FRET signal can be compared when homodimers and heterodimers form in a similar manner.

ERs Form Homodimers and Heterodimers Independently of Ligand in Vivo
ER{alpha} and ERß have been shown to form homodimers and heterodimers with or without a DNA element in vitro, but such dimeric complexes have yet to be observed in living cells (5, 6, 7, 8, 40, 41). Before attempting to monitor ER dimers using FRET, we first wanted to establish that colocalization is necessary, but not sufficient, to observe direct interaction between two proteins. As shown in Fig. 3AGo, CFP and YFP as well as the stably linked CFP-YFP control proteins are colocalized, and no difference can be seen visually. The colocalization is illustrated by overlaying the image obtained with the CFP filter with the YFP image, thus creating the green color (colocalization) from the cyan (CFP) and yellow (YFP) original image colors. However, the normalized FRET values (see Materials and Methods and Ref. 37 for how calculations were made) showed that only the CFP-YFP chimeric protein yields a positive and strong signal (Fig. 3CGo, first two bars). Thus, colocalization does not interfere with FRET measurement and is insufficient for direct interaction.



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  		Figure 3. FRET Analysis of CFP-YFP Stable Binding and ER Dimers

A, Colocalization of CFP and YFP or CFP-YFP. Colors are pseudocolors using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) for visualization of colocalization of donor and acceptor. Overlaying of donor CFP with acceptor YFP in case of colocalization shows green signal images. B, ER{alpha} homodimer colocalization. CFP-ER{alpha} and YFP-ER{alpha} are located in nuclei. C, FRET signals of ER homodimers and heterodimers with CFP-YFP as the positive control. FRET value is calculated with signal values subtracting the background on the same image and the calculation formula as specified in Materials and Methods.

 
We next studied ER dimer formation in living cells. The CFP and YFP filters are designed for each fluorescence group. When the cells expressed only donor CFP, no signal was detectable when YFP filters were used (data not shown). Therefore, the CFP and YFP filters can specifically detect their respective signals due to their narrow range of emission spectrum. An example of captured ER{alpha} homodimer images is shown in Fig. 3BGo. The images showed that the ER{alpha} fusion proteins are colocalized in the nucleus. The normalized FRET results for various ER dimer pairs are shown in Fig. 3CGo. It can be seen that the ERs form both homodimeric and heterodimeric complexes in the absence and the presence of 10-8 M E2, although with the exception of the CFP-ERß/YFP-ER{alpha} pair, dimerization appears to be stabilized in the presence of the hormone. The apparent inhibition of dimer formation in the CFP-ERß/YFP-ER{alpha} pair by E2 probably results from hormone-induced conformation changes that increase the distance between the acceptor and donor groups in this particular heterodimeric complex. ER{alpha}L539A is an ER{alpha} transcription-deficient mutant that is still capable of binding ligand but fails to activate gene expression (7). As shown in Fig. 3CGo, ER{alpha}L539A retains its ability to form homodimers independent of E2 and transcription activity. In addition, treatment with the pure antiestrogen EM-652 (42) at 10-11 M did not change the stability of ER dimers in vivo (data not shown and see below), although it abolished the ER activation of reporter genes.

Preferential Interactions between ER{alpha}, ERß, and RIDs of the SRC Family
The RIDs of all three members of the SRC family were examined for their ability to bind to ER{alpha} and -ß. A representative image file for SRC-1 RID (amino acids 597–781) is shown in Fig. 4AGo. The SRC-1 RID moved to the nucleus from the cytosol when the cells were stimulated with E2 (Fig. 4AGo). This change in cell localization was observed only when the SRC-1 RID was cotransfected with ER{alpha} or ERß as E2 alone did not cause the SRC-1 RID to move from the cytoplasm to the nucleus (data not shown). Thus, the change of localization likely results from the ERs binding to and retaining the SRC-1 RID in the nucleus. To further confirm the specificity of E2 action, OHT at 10-6 M and the pure antagonist EM-652 at 10-11 M were also applied separately to cell medium. As shown in Fig. 4AGo, OHT did not affect the localization of the SRC-1 RID. Similar data were obtained when using EM-652 (data not shown). Furthermore, the transcriptionally inactive ER{alpha}L539A mutant had no effect on the localization of the SRC-1 RID (Fig. 4AGo). Consistent with the localization data, FRET analysis demonstrated that E2 promoted strong ER{alpha} interactions with the SRC-1 RID (Fig. 4BGo). As expected, OHT and EM-652 abolished the binding between ER{alpha} and all the SRC-1 RID (Fig. 4BGo and data not shown for OHT). Furthermore, the transcription-defective ER{alpha}L539A mutant failed to recruit the SRC-1 RID in the presence of E2 (Fig. 4BGo). Similar data were obtained when the interactions between ER{alpha} and the SRD-3 RID (amino acids 547–780) were analyzed, whereas we observed that the interactions between the SRC-2 RID (amino acids 616–806) and ER{alpha} are constitutive but nonetheless abolished in the presence of EM-652 (Fig. 4BGo). In addition, the transcriptionally inactive ER{alpha}L539A mutant also failed to interact with the SRC-2 RID. SRC-3 RID did not interact with this ER mutant either (data not shown). Taken together, these data show that the manner by which ER{alpha} interacts with the distinct SRC RIDs is not equivalent.



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  		Figure 4. FRET Analysis of ER{alpha} Interactions with Coactivator RIDs

A, An example of colocalization of donor CFP-ER{alpha} and acceptor YFP-SRC-1 images under various conditions, including in the presence of 10-8 M E2 and 10-6 M OHT on wild-type ER{alpha} and on the transcription-defective mutant ER{alpha}L539A. B, FRET signals change in response to estradiol or pure antiestrogen EM-652 10-11 M treatment. ER{alpha} homodimers are used as positive controls.

 
We next investigated whether the SRC RIDs and ERß show similar interaction properties to ER{alpha}. An image file of the SRC-1 RID and ERß coexpression is shown in Fig. 5AGo. ERß is predominantly localized in the nuclei whereas the SRC-1 RID displays a broad cellular distribution. As observed with ER{alpha}, treatment with E2 leads to the recruitment of the SRC-1 RID to the nuclei in the presence of ERß. Similarly, OHT and EM-652 did not cause coactivator relocalization to the nucleus when ERß was coexpressed (data not shown). FRET data shown in Fig. 5BGo demonstrate that, in contrast to ER{alpha}, ERß interacts with all three SRC RIDs in a ligand-independent manner. However, these interactions were all enhanced to a different extent by E2 while the antiestrogen EM-652 abolished all ERß binding with SRC RIDs. The formation of ER{alpha} and ERß heterodimers was used here as a positive control. These data demonstrate that ERß interactions with the SRC RIDs are distinct from that of ER{alpha} in living cells.



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  		Figure 5. FRET Analysis of ERß Interactions with Coactivator RIDs

A, An example of colocalization of ERß with SRC-1 RID and B, FRET values of ERß binding to coregulator RIDs in the presence of vehicle alone, 10-8 M E2, or 10-11 M EM-652. ER heterodimers are used as positive controls.

 
ErbB-2/neu Specifically Promotes Binding of SRC RIDs to ER{alpha}
We next studied the effects of aberrant tyrosine kinase signaling generated by expression of the activated ErbB-2/Neu receptor on SRC RID interactions with the two ER isoforms. ErbB-2 pathway has been shown to target the ER and promote hormone-independent growth in human breast cancer cells (43). As shown in Fig. 6AGo, the SRC-3 RID localized to the nucleus in the presence of ErbB-2/Neu, but not in the presence of signaling-defective ErbB-2 protein in which four tyrosine residues are changed to phenylalanine residues (44). FRET analysis showed that ErbB-2/Neu can activate ER{alpha} interactions with SRC-1 and SRC-3 RIDs to an extent similar to that observed in the presence of E2 (Fig. 6BGo). The presence of neu/ErbB-2 did not affect the ligand-independent interaction previously observed between the SRC-2 RID and ER{alpha}. In sharp contrast, the presence of ErbB-2/Neu did not significantly activate ERß interaction with any of the three SRC RIDs tested (Fig. 6CGo), indicating that ErbB-2/Neu may specifically activate ER{alpha}-regulated gene expression in vivo.



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  		Figure 6. FRET Analysis of ER{alpha} and ERß Interactions with Coactivators upon neu/ErbB-2 Cotransfection

A, Neu increases coactivator SRC-3 concentration in the nucleus and CFP-ER{alpha} colocalized with SRC-3 RID. B, FRET analysis of ER{alpha} interactions with SRC RIDs in response to 10-8 M E2 or neu/ErbB-2. C, FRET analysis of ERß interactions with SRC RIDs in response to E2 or neu/ErbB-2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we used three filter sets to obtain images for each pair of proteins, and values for the individual cells were subtracted from the background. The signal values were calculated using the normalized FRET formula (37). With CFP-YFP stable FRET signal as the positive control, the formation of ER{alpha} and ERß homodimers and heterodimers were analyzed for the first time in living cells. Formation of ER dimers occurs in the absence of ligand but is significantly enhanced and/or stabilized in the presence of E2. OHT and the pure antiestrogen EM-652 did not disrupt ER homodimer or heterodimer formation. We also observed that the binding modes of ER{alpha} and ERß to individual SRC RIDs were not equivalent. ER{alpha} recruits SRC-1 and SRC-3 RIDs strictly in a hormone-dependent manner, whereas a ligand-independent interaction was observed with the SRC-2 RID. The antiestrogens OHT and EM-652 abolished all interactions between the RIDs and ER{alpha}. Similarly, the transcriptionally inactive ER{alpha}L539A mutant still formed homodimers but failed to bind to any of the three SRC RIDs. On the other hand, it was observed that ERß interacts with all three SRC RIDs in a ligand-independent manner that can be stabilized by E2 and abolished by antiestrogens. Strikingly, the presence of the activated neu/ErbB-2 promoted interactions between ER{alpha} and the SRC RIDs but had no effect on ERß, suggesting that ERß may be refractory to activation by this specific signal transduction pathway.

Efficient FRET relies on the proper selection of fluorescence donor and acceptor groups and the optimal filter sets to reduce noise. Imaging software using complex mathematics calculation also affects the final FRET signal normalization. The contribution of donor or acceptor alone to FRET is quite consistent, namely 39% for CFP and 58% for YFP. As shown in Fig. 1Go, YFP is more consistent in leaking through the FRET channel. The percentage of bleed throughs was calculated with 105 donor- or acceptor-alone transfections, including cells on the same image as well as image file obtained at different times.

Individual cells can express different amounts of donor and acceptor fusion proteins because transient transfection allows variance in gene expression. This expression pattern does not cause significant change in FRET signal in most cases. One of the major reasons is that the FRET signal is only a small fraction of the visible image signal. Thus, a relatively large change in CFP or YFP amounts in different cells leads only to a small change in FRET signal. Observations also showed that visually stronger signal did not necessarily cause higher FRET values. Most importantly, the orientation and interaction of the two proteins are still the determinant of a high FRET value.

The FRET signal in this study eliminates the bleed through of both donor and acceptor to obtain accurate FRET values. The energy transfer from the donor fluorescence group to the acceptor group is very small. Therefore, the noise contributed by donor and acceptor each alone is very dramatic compared with the actual resonance energy transfer. If either noise is ignored, the total FRET signal (Ff) is not proportional to the actual FRET signal in most cases. If the purpose is to compare the interactions under different chemical treatments, theoretically one of the bleed throughs can be ignored for both if the chemical does not change the amount of CFP donor or YFP acceptor in the cell. However, our observations showed that the CFP, YFP cotransfection and CFP-YFP transfection are the only experimental conditions when two filter sets can be used accurately. For most other applications, although one of the bleed throughs plays a minor role, the FRET is misrepresented because the actual FRET signal is overwhelmed by the bleed through. In our case, YFP has a small bleed through [Af(Fa/Aa)/Ff] when cotransfected as a pair. It can contribute from 8%–36% depending on the pair of interactions analyzed and the amount of YFP fusion proteins expressed in the particular cell. In summary, to minimize the concentration effects of CFP or YFP fusion proteins, the three filter sets were adopted for all pairs, and FRET-normalized values were calculated for each pair.

ER interactions with some of the coactivators such as CBP and SRC-1 have been studied using FRET. However, these studies had some limitations. First, FRET was conducted by labeling purified proteins with fluorescence groups in vitro, equivalent to glutathione-S-transferase pull down methodologically (45). Second, the fusion proteins contained only the ER ligand binding domain or a short stretch of SRC-1 surrounding the LXXLL motifs (18, 46). Third, recent studies on ER{alpha} used a ratio to represent the FRET signal change (39). Here we used the normalized FRET calculations which eliminate the bleed through from both the donor and acceptor (36, 37, 47). Finally, we analyzed interactions of both ER{alpha} and ERß with each of the three SRC RIDs. In addition, the effects of selective ER modulators and the expression of an activated tyrosine kinase receptor were examined on each set of interactions in vivo.

The classical mode of action for steroid hormone receptors dictates that the receptors are sequestered in an inactive multiprotein complex until hormone binding releases the receptor, which then forms homodimers and subsequently binds to its cognate response element. However, several studies have shown not only that ER{alpha} and ERß can form homodimers on DNA in the absence of hormone, but that they also possess the ability to form transcriptionally active heterodimers (5, 6, 7, 8, 41, 48). Using FRET, this study demonstrates for the first time that ER{alpha} and ERß can indeed form heterodimers in living cells, and that formation of both homo- and heterodimers can be observed in an intact cellular context in the absence of ligand. These data suggest that at least a subset of ERs is free of the multiprotein chaperone complex. This subpopulation of receptors may be required to engender responses to ligand-independent stimuli (see below). Our results obtained in living cells also extend previous in vitro experiments demonstrating that antiestrogens do not affect ER dimerization in vitro (48).

Previous studies have clearly established that ER{alpha} and ERß, while sharing a high degree of homology in their ligand-binding domain, can differentially recognize synthetic peptides encoding distinct LXXLL motifs (19, 21, 49). In addition, in vitro experiments using the BIAcore instrument showed that ER{alpha} and ERß have strong affinity preferences for the RIDs of particular coactivators (17). However, chromatin immunoprecipitation experiments have demonstrated that ER{alpha} can interact with all three SRC isoforms when liganded and bound to DNA in cells (50, 51). Here we show that indeed both ER{alpha} and ERß can recognize and interact with the RIDs of all three SRC isoforms in living cells. The major difference is, however, in the manner in which both receptors interact with the RIDs. While the interaction between ER{alpha} and the RIDs of SRC-1 and -3 is clearly ligand dependent, its interaction with the SRC-2 RID is ligand independent. In addition, ERß and all three SRC RIDs display significant levels of ligand-independent interaction that can be further stabilized or enhanced by the presence of E2. Although these results diverge from the classic model of ligand-induced interaction between receptors and coactivators, other evidence supports these findings. First, the initial characterization of the transcriptional properties of ERß demonstrated that this receptor can stimulate transcription in a ligand-independent manner and that this activity could be further enhanced by cotransfection of SRC-1 (31, 32). ERß has also been shown to interact with target promoters in a ligand-independent manner (52). Furthermore, fluorescence recovery after photobleaching studies have shown transient ligand-independent interactions between ER{alpha} and SRC-1 in a live-cell setting (53). Taken together, the data suggest that ligand-independent interactions between receptors and coactivators may play a more important role than previously anticipated in estrogenic signaling.

The amplification and concomitant overexpression of ErbB-2 has been associated with a significant number of breast cancers (54). The gathering of excess ErbB-2 at the cell surface results in constitutive activation of signaling cascades that drive tumor cell growth (27). Although tumors that overexpress ErbB-2 tend to be ER{alpha} negative (54, 55), it has been suggested that up-regulation of growth factor-signaling pathways may be an early event in progression to ER{alpha} negativity, resulting in an intermediate ER{alpha}-positive/ligand-independent aggressive phenotype (56). The biological interactions between ERs and ErbB-2 is indeed complex: although expression of ErbB-2 leads to a sustained decrease in endogenous ER{alpha} expression, it also promotes E2-independent transcriptional activity (43). Conversely, E2 down-regulates ErbB-2 expression in human breast cancer cells (57). Here we have shown, for the first time, that expression of ErbB-2 leads ER{alpha}, but not ERß, to recruit SRCs, thus providing a possible molecular mechanism to explain the transcriptional effects of ErbB-2 on ER activity (43). The ErbB-2-induced interactions between ER{alpha} and SRC RIDs are likely the result of phosphorylation events of either the receptor or the SRCs or both as these proteins have all been demonstrated to be the targets of various kinases (29, 33, 34). The most striking result is perhaps the complete specificity of ErbB-2 action on ER{alpha} and ERß. EGF-induced phosphorylation of specific serine residues within the ERß amino-terminal region was previously shown to promote ligand-independent interactions between the receptor and SRC-1 both in vitro and in vivo (32). These data suggest that EGF and ErbB-2 may act through different mechanisms on each ER. It should also be noted that, in transient transfection assays performed in 293 cells used in this study, introduction of ErbB-2 did not up-regulate the transcriptional activity of either ER{alpha} or ERß, suggesting that essential ER-coregulatory factors present in human breast cancer cells may be absent in 293 cells. Taken together, these results lend support to the concept that the mode and amplitude of ER-regulated gene transcription probably result from the combined effects of differential expression of ER{alpha} and ERß and their coregulators, the targeted genes and the cell context, and thus further demonstrate the complexity and specificity of signaling events in living cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
CFP-C1 and YFP-C1 mammalian expression vectors were purchased from BD Biosciences CLONTECH Laboratories, Inc. (Palo Alto, CA). The CFP-YFP chimera was made by PCR using the following primers: upper, 5'-TCCCCGCGGTAGCCGCCATGGTGAGCAAGGGC-GAGGAGCTG; and lower, 5'-CGGGATCCCTTGTACAGCTCGTCCATGCCGAG. The YFP 717-bp fragment was digested with SacII and BamHI and cloned into the CFP-C1 vector, creating a linker of 21 amino acid residues (SGLRSRAQASNSAVDGTAVAA) between CFP and YFP. All CFP or YFP fusion proteins have a short stretch of sequence between the CFP or YFP and the target protein. CFP-ER{alpha}/YFP-ER{alpha} and CFP-ERß/YFP-ERß were made by PCR using CMXhER{alpha} and CMXhERß as templates, respectively. ER{alpha} primers were as follows: upper, 5'-CGGGGTACCATGACCATGACCCTCCACACCAAAG; and lower, 5'-CGCGGATCCGACTGTGGCAGGGAAACCCTCTGC. ERß primers were: upper, 5'-CCCAAGCTTCGATGAATTACAGCATTCCCAGCAATGTC; and lower, 5'-TCCCCCGGGCTGAGACTGTGGGTTCTGGGAGCCC. CFP-ER{alpha}L539A and YFP-ER{alpha}L539A were constructed in a similar manner with the exception that CMXhER{alpha}L539A was used as template. Plasmid constructs were sequenced to ensure their integrity. The YFP-coregulator RID constructs were engineered with the following primers. hSRC-1 (amino acids 597–791): upper, 5'-CCGCTCGAGCTATGCAACCAGCAAAGGCTGAGTCCA; and lower, 5'-CGGAATTCGACACTTTGACCTTTACGTCATCCAG; hSRC-2 (amino acids 616–806): upper, 5'-CCGCTCGAGCTATGCCCCAGGCGGCCAGCGGGG; and lower, 5'-CGGAATTCGACAAGTTGTCCAGCTCGCTGCCAGG; mSRC-3 (amino acids 547–780): upper, 5'-CCGCTCGAGCTATGAATATAAGCCAGCCAAGTAAAGTG; and lower, 5'-CGGAATTCGACTCGGTCTTAATTTTGGGGTCTTTCTC. The ErbB-2/Neu expression plasmid was a gift from Dr. William J. Muller (McGill University).

Cell Culture and Medium
The human embryo kidney 293 cell line was used for all transfection experiments. The cells were grown in DMEM medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum. E2 and OHT were purchased from Sigma (St. Louis, MO) and EM-652 was a gift from Dr. Fernand Labrie (Laval University, Québec, Montréal, Canada). Transient transfections were performed using the FuGene transfection reagent (Roche Diagnostics GmBH, Mannheim, Germany) according to the manufacturer’s instruction, typically with 0.5 µg of expression plasmids and carrier DNA pBleuscriptKSII for a total of 1 µg. Before treatment with any of the chemicals, cells were grown in phenol red-free DMEM with 10% of charcoal-dextran-stripped fetal bovine serum for at least 24 h. Using ethanol or dimethyl sulfoxide as the vehicle, E2 was applied at 10-8 M, OHT at 10-6 M, and EM-652 at 10-11 M, respectively.

Fluorescence Microscopy and FRET
An Eclipse TE300 microscope (Nikon, Melville, NY) was used in connection with a Hamamatsu camera controller, a CCD camera, and a Dell precision 420 computer. A LAMBDA DG-4 high-speed filter changer from Sutter Instrument Co. (Novato, CA) was used together with a Cermax Xenon laser lamp from ILC Technology (Sunnyvale, CA). The cells were regularly enlarged 600 times. CFP filters: excitation, 435/20 nm; emission, 480/20 nm. YFP filters: excitation, 480/20 nm; emission, 535/20 nm. FRET filters: excitation filter of CFP and emission filter of YFP. The mathematical basis for normalized FRET calculations is from Gordon et al. (37). Background subtractions were conducted for all values using the Inovision Isee 5.5 software (Inovision Corp., Durham, NC). Briefly, donor- and acceptor-only images were obtained to calculate Fd/Dd and Fa/Aa ratios, respectively. Fd and Dd are images of donor with the FRET filters and the donor CFP filters separately. Similarly, Fa and Aa are images of acceptor with the FRET filters and acceptor YFP filters. Coexpressed cell images of donor and acceptor were then taken and processed to obtain the relevant values Ff, Df, and Af from images obtained using the three filter sets. Donor bleed-through percentage is calculated as Df(Fd/Dd)/Ff x 100%, acceptor bleed through is Af(Fa/Aa)/Ff x 100%, and the real FRET is the value subtracting donor and acceptor percentages from 100%. The normalized FRET is calculated using the formula FRET = [Ff - Df(Fd/Dd) - Af(Fa/Aa)]/[Df x Af].


   ACKNOWLEDGMENTS
 
We thank Dr. Fernand Labrie for providing EM-652, Nick Bertos for expert advice on microscopy, and members of the Giguère laboratory for comments on the manuscript.


   FOOTNOTES
 
This work was supported by the Canadian Breast Cancer Research Initiative, the National Cancer Institute of Canada, and the Canadian Institutes of Health Research (CIHR). V.G. is a CIHR Senior Scientist.

Abbreviations: CFP, Cyan fluorescent protein; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; FRET, fluorescence resonance energy transfer; NcoA, nuclear receptor coactivator; OHT, 4-hydroxytamoxifen; RID, receptor interaction domain; SRC, steroid receptor coactivator; YFP, yellow fluorescent protein.

Received for publication October 16, 2002. Accepted for publication December 30, 2002.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Coregulators:   SRC-1  |  GRIP1  |  AIB1
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



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