Heterodimerization of ERs.
Previous studies showed that coexpression of the human “short” ERβ of 477-amino-acid with the human WT ERα leads to the formation of the ERα/β heterodimer in vitro and in situ (4
). We wanted to address whether the WT ERβ (ERβ) that contains an additional 53 amino acids at the amino terminus also heterodimerizes with WT ERα (ERα) when cosynthesized. In order to facilitate the examination of biochemical and functional properties of heterodimer receptors, we initially examined the abilities of ER homodimers synthesized in vitro and in situ to interact with a 32
P-end-labeled DNA fragment bearing the consensus ERE derived from the vitellogenin A2 gene by EMSA. ERα and ERβ cDNAs contain sequences that encode a Flag epitope at the amino terminus. [3
H]methionine was used to quantify in vitro-synthesized ERs. Fluorography indicated that ERα migrates at molecular masses of 67 and 46 kDa, the latter being a minor species synthesized at various levels among experiments and not always observable (Fig. , 3
H). ERβ displayed an electrophoretic migration of 60 kDa. The detection of proteins by Western blot analysis by using ERα (HC-20 and HC)- or ERβ (PA1-313 and PA1)-specific antibody or the M2 (Flag) antibody for both receptors confirmed the identity of receptors. The absence of detection of the minor species of ERα migrating at 46 kDa by the Flag antibody either in vitro or in cell extracts from the transiently transfected ER-negative CHO cells suggests that this is an amino-terminally truncated ERα isoform. Two distinct ERα species were also detected by HC in Western blots of transfected CHO or ERα-positive T47-D cells derived from breast ductal carcinoma. ERβ showed a 60-kDa band by the PA antibody in cell extracts from transfected CHO or ERβ-positive PA-1 cells of ovarian teratocarcinoma.
FIG. 1. In vitro and in situ synthesis of ERα and ERβ. (A) For in vitro synthesis of ERs, 1.5 μg of linearized pBS-KS bearing no cDNA or a cDNA for an ER was transcribed by T3 polymerase and translated by rabbit reticulocyte lysate in (more ...)
Despite similar amounts of receptors as assessed by the Flag antibody in Western blots (Fig. , WB), the extent of interaction of the in vitro-synthesized ERβ with an equal concentration of ERE (Fig. , EMSA) was significantly lower than the in situ-synthesized ERβ whether or not a saturating concentration (10−7
M) of E2 was present (data not shown). Moreover, the ERβ synthesized in vitro showed a diffuse ERE-binding pattern that gradually formed a distinct complex as a result of increasing receptor concentration. In contrast, the ERβ synthesized in situ interacted with ERE efficiently. ERβ synthesized in S2 (data not shown) cells or in Sf9 insect cells (45
) also binds efficiently to DNA. Although it is not clear, differential processing of ERβ species in vitro versus in situ could lead to differences in the extent of homodimerization of ERβ and/or stability of the ERβ homodimer. This, in turn, could be manifested as differences in the amount and pattern of ERβ interaction with DNA.
ERα synthesized in vitro formed three distinct ERE-bound complexes as detected by EMSA in the absence or presence of the HC antibody: a major complex and two minor complexes (Fig. , In Vitro), as reported previously (4
). The slowest-migrating complex (complex 3) likely represents the WT ERα homodimer. The fastest-migrating band, the least prominent complex, corresponds to the amino-terminally truncated ERα homodimer (complex 1). The intermediate ER-ERE complex appears to be the heterodimer of the WT and the truncated ERα species (complex 2). This conclusion is suggested because the Flag antibody specific to the amino-terminal Flag epitope fails to retard the migration of the complex 1 (lane 4). A similar ERE-binding pattern was also observed with the ERα synthesized in situ (Fig. , CHO). The ERβ synthesized in vitro, which we used fivefold more compared to ERα to obtain similar amount of binding, forms a single ER-ERE complex that migrates somewhat faster than the complex 2 of the ERα, whereas the ERE-bound in situ-synthesized ERβ migrates slightly faster than the complex 3 of ERα.
To examine the formation of a functional ERα/β heterodimer, we used a constant amount of the ERα expression vector, together with increasing amounts of ERβ expression vector in transcription-translation reactions or in transfection into CHO cells. The relative amounts of ER proteins were assessed in Western blots of equal aliquots of the in vitro reactions (Fig. , WB, In Vitro) or of CHO cell extracts (CHO) with the Flag antibody. The results indicated that increasing amounts of ERβ were cosynthesized with a constant amount of ERα. To assess the formation of a functional heterodimer, we used ERα without the Flag epitope to distinguish ER subtypes with the use of the Flag antibody. The cosynthesis of ERs in vitro or in situ led to the formation of a functional ERα/β heterodimer that showed an electrophoretic migration between ERα-ERE complex 3 representing the ERE-bound ERα homodimer and the ERβ homodimer in EMSA (Fig. , EMSA). The extent of heterodimer formation was correlated with a gradual decline in the ERα-ERE complex. It is also evident that residual ER homodimers were still present.
FIG. 2. Coexpression of ERα and ERβ in vitro and in situ leads to formation of ERα/β heterodimer. For Western blotting of in vitro-synthesized ERs, 0.3 μg of pBS-KS vector bearing the Flag ERα cDNA was coexpressed (more ...)
Thus, the coexpression of WT ERβ, as shown previously for the “short” ERβ (4
), with ERα leads to the formation the ERα/β heterodimer, the extent of which is dependent upon the relative amount of the contributing partners.
Construction of fusion ERs.
To study the effects of ERα/β heterodimer in genomic estrogen signaling, we used a genetic conjugation approach (30
) to circumvent the pivotal dimerization step in receptor action. Single-chain ERs were produced by joining the 3′ end of the coding sequence of one ERα or ERβ cDNA to the 5′ coding sequence of another to encode two ER monomers in tandem (Fig. ) without or with a Flag epitope at the amino terminus. Analysis of radiolabeled proteins by fluorography revealed that the homofusion α-α and β-β and the heterofusion α-β and β-α displayed electrophoretic migrations with molecular masses of 120 to 136 kDa that correspond to two ER molecules (Fig. , 3
H). The detection of proteins by Western blot analysis with ERα (HC)-, ERβ (PA1)-, or Flag epitope-specific antibody (Flag-InV) confirms the identity of receptor species. Since radiolabeled counterparts and the Flag antibody show synthesis at comparable levels, differences in the extent of detection of α-β and β-α by the receptor-specific antibodies in Western blotting are likely due to a position-dependent alteration in the epitope recognition site.
FIG. 3. Construction and synthesis of fusion ERs. (A) Schematics of ER fusion receptor cDNAs. A PCR-generated NdeI restriction enzyme site at the 5′ or 3′ end of an ER was used to genetically fuse two ER cDNAs in tandem. (B) Synthesis of fusion (more ...)
Similarly, the fusion receptors synthesized in transiently transfected CHO cells migrated with molecular masses ranging from 120 to 136 kDa assessed by the Flag antibody, whereas ERα and ERβ migrated with molecular masses of 67 and 60 kDa, respectively (Fig. , Flag-CHO).
Characterization of biochemical properties of the fusion ERs. (i) DNA binding.
We examined by EMSA whether single-chain receptors synthesized in vitro (Fig. ) or in situ (Fig. ) interact with the consensus ERE. Equal amounts of the in vitro-synthesized ERα and fusion receptors retarded the electrophoretic migration of labeled ERE. Due to inefficient ERE binding of the ERβ synthesized in vitro, we used a fivefold greater concentration of receptor to obtain similar amounts of ERE-bound receptors. The ERE-bound homo- and heterofusion ERs showed migrations comparable to those of the ER dimers. The interaction of receptor species with ERE is specific, because the protein-ERE complex was further retarded by the Flag antibody. Similarly, equal amounts of cell extracts of transfected CHO cells synthesizing an ER or a single-chain receptor interacted efficiently with the consensus ERE in vitro.
Since ERs bind to EREs as dimers, similar migration among ERE-bound WT ERs and fusion species suggests that the fusion receptors interact with DNA as monomers. We also observed that the ERE-bound homo- and heterofusion proteins showed slightly faster electrophoretic migrations than ERα, possibly reflecting differences in size and in conformation among ER species. We took advantage of these differences in the electrophoretic mobility to test the conclusion that the fusion receptors indeed bind to ERE as monomers and, implicitly, do not dimerize. We used the ERα expression vector together with the same (1:1) or a twofold-greater (1:2) amount of the expression vector bearing the α-β or the α-α (data not shown) cDNA in transcription-translation reactions with radiolabeled Met (Fig. , upper panel). The ERα cDNA lacks the amino-terminal Flag epitope to discern ERE-bound receptor species by the use of Flag antibody. Reactions were subjected to EMSA (Fig. , lower panel). The Flag antibody shifted only the ERE-bound α-β without altering the migration of ERα. This result demonstrates that the fusion receptors bind to ERE as monomers.
FIG. 4. (A) The fusion ERs bind to ERE as monomers. The expression plasmids bearing ERα and the same (1:1) or a twofold-greater (1:2) concentration of the expression vector bearing the heterofusion Flag-α-β were cosynthesized in the presence (more ...)
Each DBD of ER consists of two zinc finger-like modules that form a single functional domain. Each module contains a zinc ion with tetrahedral coordination by four Cys residues (8
). Substitution of two of the zinc-coordinating Cys with His residues of the first zinc finger in the DBD of ERα prevents the receptor from interacting with DNA (21
). If the fusion receptors bind to ERE as monomers, analogous mutations in the DBD of one α monomer should prevent the interaction of the variant fusion ERs with an ERE. Although synthesized at comparable levels, as assessed by fluorography (Fig. , upper panel), the mutant homofusion α*-α and heterofusion α*-β failed, just as the DNA-binding defective ERα* dimer had failed, to bind to ERE in EMSA in contrast to the WT counterparts (Fig. , lower panel). This result is consistent with the conclusion that fusion receptors bind to DNA as monomers.
The dimerization of ERα is primarily mediated by a surface located within the LBD of each monomer (21
). The absence of dimerization also suggests that two ER monomers in a single-chain receptor fold into a compact structure whose formation may or may not be dependent upon interactions between LBD dimerization surfaces. To address this point, we generated dimerization defective ERα. A previous study showed that changing of three Leu residues to Glu in the helix 11 of the LBD of the murine ERα prevents dimerization (41
). We made analogous mutations by changing three Leu residues at positions 504, 508, and 511 to Glu. Similarly, we produced variant homofusion α-α that bears these same mutations in either (αdd
-α or α-αdd
) or both (αdd
) ERα monomers, and we produced the heterofusion αdd
-β that has mutations in the ERα monomer. The synthesis of mutant and WT fusion receptors was assessed by fluorography (Fig. , upper panel). EMSA revealed that the dimerization defective ERαdd
, as expected, did not significantly interact with an ERE (Fig. , lower panel). Although the mutant fusion receptors (αdd
-α is shown in Fig. ), bound to an ERE, the interaction of these receptors with the ERE was also compromised (~3-fold) compared to the WT counterparts when the binding was assessed at low concentrations of receptors (Fig. ). This indicates that the interactions between dimerization surfaces of both ERα monomers of the single-chain species increase the efficiency with which the single-chain receptor interacts with ERE.
Moreover, the αdd-β heterofusion, or homofusion αdd-αdd, as its WT counterpart, did not dimerize with the cosynthesized ERα (data not shown). Thus, fusion receptors fold to allow complementary interactions of the dimerization interfaces in the two LBDs, despite the head-to-tail fusion of two ER monomers in a single-chain configuration.
(ii) DNA-binding specificity.
Efficient binding of single-chain receptors to ERE in a manner similar to the ER dimers implies that the DNA-binding specificity of ER is also preserved in fusion receptors. Therefore, we assessed the interaction of receptor species with DNA fragments that contain various test sequences (Fig. ) by EMSA (Fig. ). Previously, we (45
) and others (27
) have shown that ERα and ERβ bind to various ERE sequences with similar preference and affinity. Likewise, WT and fusion receptors bound the consensus ERE and ERE sequences derived from the estrogen-responsive pS2, complementary 3 (C3), oxytocin (Oxy) genes, and the thyroid hormone responsive element (TRE). Receptors had very little, if any, interaction with GRE and vitamin D responsive element (VDRE). There was no observable interaction with half-ERE (1/2ERE) or a mutant ERE sequence that bears three nucleotide substitutions in the consensus (mERE).
FIG. 5. (A) Upper strand of the sequence containing the consensus ERE (in brackets), pS2, and Oxy ERE, GRE, VDRE, and TRE responsive elements and 1/2 ERE and mERE with the underlined five- and three-nucleotide changes from the ERE, respectively. The central base (more ...)
Conservation of the DNA-binding specificity of ERα in single-chain receptors also suggests that the fusion receptors utilize both half-sites in an ERE for binding. We tested this prediction by using the missing nucleoside hydroxyl radical assay that allows the analysis of DNA-protein interaction at a single-nucleotide resolution. It is expected that the protein binding to DNA would be adversely affected if a nucleoside important for binding were missing (13
). A low-intensity or missing band in the lane containing ERE-bound ERs (B) or, conversely, a high-intensity band in the lane containing free ERE (F), identifies a nucleoside important for the formation of the ER-ERE complex. A high ratio of free to bound ERE is represented by a long horizontal bar in Fig. ; the length of a bar represents the strength of contacts with ERs. The results revealed that the fusion receptors, just as for the ERα dimer, occupied both half-sites of the consensus ERE by contacting the same nucleosides with similar strength (Fig. ).
Moreover, the fusion receptors bound to the consensus, pS2 and Oxy ERE sequences with affinities similar to those of the ERα dimer as assessed by DNA competition assay (http://dbb.urmc.rochester.edu/labs/muyan/figure.htm
). Likewise, all receptor species bound to E2 with comparable affinities (http://dbb.urmc.rochester.edu/labs/muyan/figure.htm
). We also found that stoichiometry of E2 binding among ER species was primarily conserved. For example, as we reported previously (30
), 1 mol of ERα binds 1 mol of E2, whereas 1 mol of α-α or α-β binds 1.86 ± 0.11 or 1.77 ± 0.19 (n
= 3) mol of E2, respectively. Thus, the folding of the fusion ERs allows the receptors to bind to ERE and E2 efficiently.
(iii) Cofactor interactions.
The p160 family of coregulators interacts with the agonist-nuclear receptor complex through a LXXLL nuclear receptor interacting motif (NRIM) (14
). Upon E2 binding, the LBD undergoes a conformational change by which the helix 12 located at the carboxyl terminus of the LBD is realigned over the ligand-binding pocket (3
). Since the conjugation approach uses a fusion between the amino terminus of one monomer and the carboxyl terminus of the other, the critical alignment of helix 12 could be compromised despite the fusion receptors bind to E2 efficiently.
To address this issue, we examined the abilities of the fusion receptors to interact with cofactors by using EMSA. We (45
) and others (1
) showed that the p160 family of cofactors, including TIF-2 and SRC-1, interact with ERα and ERβ in an agonist-dependent manner. Equal molar concentrations of fusion receptors synthesized in vitro and the ERα dimer were preincubated without or with a saturating concentration (10−7
M) of E2. Samples were then incubated with an end-labeled ERE. GST alone or the GST fusion polypeptide of TIF-2623-986
containing NRIM was added into the reaction mixtures at increasing concentrations. Reactions were subjected to EMSA (Fig. ). GST alone at any concentration had no effect on the electrophoretic mobility of the ERE-ER complexes in the absence or presence of E2 (data not shown). The electrophoretic mobility of ERE-bound ERα was quantitatively retarded by TIF-2 in response to E2 (lanes 7 to 10). E2 also enhanced the ability of the fusion receptors to recruit TIF-2 with affinities about twofold lower than that of the ERα dimer. SRC-1219-399
(SRC-1) interacted with the E2-bound ERα-ERE complex. The SRC-1 was also recruited by the fusion receptors, however, with efficiencies ~5-fold lower than that observed with the ERα dimer. Apparent differences in the extent of SRC-1 recruitment compared to TIF-2 by the fusion receptors also suggest differences in the binding affinities of cofactors for fusion receptors.
FIG. 6. (A) Interactions of cofactors (CF) with fusion ERs as assessed by EMSA. Equal molar concentrations of in vitro-synthesized receptors were preincubated in the absence (−, lanes 1 to 5) or presence (+, lanes 6 to 10) of 10−7 M E2 (more ...)
In addition to NRIM, TIF-2 through a glutamine-rich region (Q) also interacts efficiently with the amino terminus of ERα, but not of ERβ, independent of E2 (38
). To examine whether this receptor-subtype specific interaction is preserved in the heterofusion ERs, the interaction of the GST fusion TIF-2 containing residues 1125 to 1325 (TIF2-Q) was tested in EMSA with receptors synthesized in vitro (Fig. ). The results revealed that increasing concentrations of the cofactor gradually retarded the electrophoretic migration of the EREbound ERα and α-α in the absence or presence (data not shown) of 10−7
M E2. The α-α bound to the cofactor with ~2-fold-lower affinity than the dimer ERα. The TIF-Q, on the other hand, did not affect the electrophoretic migration of ERβ and β-β. TIF2-Q interacted quantitatively with the heterofusion receptors, however, with affinities twofold lower than those observed with the homofusion α-α.
Overall, these results suggest that cofactor interacting surfaces in the fusion receptors are altered.
Biological activities of the fusion ERs. (i) ERE-dependent ER signaling.
We next examined whether the homofusion α-α and β-β simulate the transactivation properties of the parent ER dimers. Furthermore, we attempted to determine whether the heterofusions α-β and β-α display novel functions by incorporating functional properties of both contributing partners in genomic estrogen signaling pathways. ERβ has considerably less transcription potency than ERα in ERE-dependent signaling pathways independent of the promoter and cell context (11
). The expression vector without or with cDNA for an ER was cotransfected into CHO cells, together with a reporter plasmid bearing no (TATA), one (1ERE), or two (2ERE) consensus EREs placed upstream of a simple TATA box (TATA) promoter that drives the expression of the firefly luciferase cDNA as reporter. Normalized activity from each reporter construct was compared to the basal activity from the reporter bearing no ERE in response to the parent expression vector (V) in the absence of E2, with the latter value set to one. Proteins were synthesized at similar amounts (Fig. , Flag-CHO) and localized to the nucleus in the absence (Fig. ) or presence (data not shown) of a physiological concentration (10−9
M) of E2. As we showed previously (44
), ERα, but not ERβ, induced transcription synergistically from two ERE-containing reporter constructs in CHO cells (Fig. ). Although α-α increased luciferase activity synergistically in response to E2, albeit to a lesser extent than that observed with ERα, β-β had only an additive effect on luciferase activity. On the other hand, heterofusion α-β or β-α induced synergy at levels comparable to that observed with the α-α. These results show that the homofusion α-α and β-β emulate the activities of the parent receptors and suggest, as proposed previously (40
), that ERα is the dominant partner in the ER heterodimer when tested from a simple TATA box promoter construct bearing only tandem consensus EREs.
FIG. 7. (A) Intracellular localization of fusion ERs was examined by immunocytochemistry. The expression vector without (V) or with a cDNA for Flag ERα, ERβ, or fusion α-β was transiently transfected into CHO cells. The proteins (more ...)
The expression of estrogen-responsive genes is the result of integrated effects of various trans-acting factors that are critical for gene expression. We examined the biopotencies of the fusion ERs from constructs bearing the enhancer-promoter region of estrogen-responsive pS2 and C3 genes that drive the expression of the luciferase enzyme cDNA. To accomplish this, we transiently transfected CHO cells with an expression vector bearing a receptor cDNA (Fig. ). Normalized activity from each reporter was compared to the promoter activity in response to the parent expression vector (V) in the absence of E2, with the latter value set to one. ERα and the homofusion α-α in response to 10−9 M E2 augmented the luciferase activity from both the pS2 and C3 promoters ~6-fold, whereas ERβ and β-β increased transcription ~2-fold. The heterofusion ERs showed biopotencies that were similar to those observed with ERα and α-α from the C3 promoter. The biopotencies of the heterofusions were, on the other hand, intermediate between the homofusion receptors that simulated the transcription activities of the parent ERα and ERβ when tested with the pS2 promoter construct.
In HeLa cells, the heterofusion ERs also induced synergy with biopotencies that simulate that of α-α, which showed less potency than the ERα dimer (Fig. ). The ERβ dimer and β-β had little effect on luciferase activity. The magnitude of transcription from the C3 promoter by the heterofusion ERs in response to 10−9 M E2 was similar to that of α-α, which had significantly lower potency than the ERα dimer (Fig. ). Both ERβ and β-β, on the other hand, had minimal effects on luciferase activity. The heterofusion receptors showed transcription capacities comparable to those of α-α and ERα when tested from the PS2 promoter.
HeLa cells were transiently transfected as described for Fig. . The data represent the means ± the SEM of three experiments performed in duplicate.
These results suggest that, although the biopotencies of the fusion receptors are dependent upon the promoter and cell context, the heterofusions α-β and β-α emulate the activity of α-α rather than the β-β when tested with estrogen-responsive gene promoters. Thus, it appears that ERα dictates the mode of transcription of the ERα/β heterodimer in the ERE-dependent ER signaling pathway.
(ii) Repression of ERα transactivity by ERβ.
Previous reports indicated that ERβ functions as a dominant inhibitor of ERα transcriptional activity in the ERE-dependent signaling pathway in transfected mammalian cells (12
). Indeed, cotransfections of an expression vector bearing ERα cDNA, together with increasing amounts of ERβ cDNA in transiently transfected HeLa cells, revealed that ERβ effectively inhibits the E2-ERα induced transcription from the C3 or the pS2 gene promoter (Fig. , left panels). At the concentration that induced maximal repression, the inhibition of ERα-induced transcription by ERβ was independent of the E2 amount regardless of the promoter-type (data not shown).
FIG. 9. Effects of coexpression of ERs and fusion receptor on transcriptional responses. (Left) HeLa cells were transiently transfected with a constant amount (in nanograms) of ERα expression vector and increasing amounts of the ERβ expression (more ...)
This inhibitory capacity of ERβ on ERα-induced transcription in coexpression systems could occur at multiple levels. These include heterodimerization, competition for binding to DNA, and/or transcriptional silencing through squelching. Since the fusion receptors do not dimerize, hence generating homogenous populations of receptor species, we reasoned that coexpression of the fusion receptors would allow us to address the underlying mechanism. To accomplish this, we performed a cotransfection assay using an expression vector bearing the α-α cDNA with an expression vector carrying the β-β or heterofusion α-β or β-α cDNA (Fig. , right panels). The results revealed that the cotransfected β-β repressed the transregulatory activity of α-α. On the other hand, the heterofusion α-β or β-α augmented the luciferase activity induced by α-α. This finding also suggests that the repression of the α-α activity by β-β is independent of sequestration of factors required for transactivation under the condition we are testing. Since the heterofusion ERs simulate the activities of the ERα dimer and α-α, these results also imply that ERβ as a homodimer represses the ERα function in the ERE-dependent signaling pathway. Indeed, the DNA-binding-defective β-β (β*-β*) bearing two mutant ERβ monomers did not affect luciferase levels induced by α-α. Thus, an effective repression of ERα-mediated transcriptional responses by ERβ occurs through competition for ERE binding.
(iii) ERE-independent and DNA-dependent ER signaling.
The ligand-ER complex regulates the expression of the Col and RARα genes through the DNA-dependent and ERE-independent signaling pathway. The functional tethering of the AF-1 and/or AF-2 domains of both ERs to the Jun/Fos family of proteins bound to the AP-1 element in the promoter of the Col gene provides the basis for gene responsiveness to ligand-ER signaling (22
). Similarly, the functional interaction of the activation domains of ERα, but not ERβ, with the Sp-1 transcription factor bound to the GC box provides responsiveness for the RARα gene expression (36
). Using a reporter plasmid that contains the Col or RARα promoter as a model for the DNA-dependent and ERE-independent signaling, we examined whether or not the heterofusion ERs emulate the ligand-mediated effects of ERα. The expression vector with cDNA for an ER was cotransfected into cells with a reporter plasmid with the Col or RARα promoter that drives the expression of the firefly luciferase cDNA. Ligand-mediated responses from the reporter plasmid by an expression vector were compared to the activity in the absence of ligand, the latter value being set to one.
The ligand-mediated responses from reporter vectors were cell type dependent. Although ligand ER did not alter luciferase activity from either the Col or the RARα promoter in transiently transfected CHO cells, the homofusion ERs simulated the transactivation abilities of the parent dimers in response to ER ligands in HeLa cells (Fig. ). ERα and α-α induced the transactivation from both the Col and the RARα promoters in response to 10−7 M ICI (see also Materials and Methods), whereas ERβ or β-β had no effect on luciferase activity in the presence of ICI. The heterofusion ERs induced transcription in response to ICI in a manner similar to the ERα dimer and α-α. E2 at any concentration tested, shown at 10−9 M, had little effect on luciferase activity from the Col or the RARα promoter.
FIG. 10. Transcriptional responses to fusion ERs from the ERE-independent signaling pathway. HeLa or MDA-MB-231 cells were transiently transfected with 125 ng of expression vector bearing no cDNA (V) or a cDNA for an ER, together with 125 ng of reporter vector (more ...)
E2, on the other hand, suppressed reporter enzyme activity mediated by ERα and α-α in the ER-negative MDA-MB-231 cell line derived from a breast adenocarcinoma; whereas ICI had little effect on luciferase activity (Fig. , lower panel). Both the α-β and the β-α heterofusions emulated the E2-mediated effects of ERα and α-α on transcription from the Col promoter. The ligand-receptor complexes had no effect on responses from the RARα promoter in this cell line (data not shown). Thus, as observed with the ERE-dependent signaling, it appears that ERα dictates the mode of transcriptional properties of the heterofusion proteins in the DNA-dependent and ERE-independent signaling pathway as well.