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The estrogen receptor (ER) is expressed in two forms, ERα and ERβ. Here we show that ERα and ERβ, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (Kd of approximately 2 nm) similar to that of ERα and greater than that of ERβ homodimers. Mutation analysis of the hormone binding domain of ERα suggests that the dimerization interface required to form heterodimers with ERβ is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ERα and ERβ in tissues and the difference in DNA binding activity between ERα/ERβ heterodimers and ERβ it seems likely that the heterodimer is functionally active in a subset of target cells.
). They are poorly conserved in the N-terminal domain but ERβ, like ERα, appears to contain a similar activation domain, activation function 1 (AF-1) sensitive to a mitogen-activated protein kinase pathway (
). Upon estrogen binding, the receptor forms homodimers which then interact with response elements in the vicinity of target genes and modulate rates of gene transcription. In view of the similarity of the ligand binding domain of ERα and ERβ we investigated the possibility that the two receptors may form functional heterodimers in target cells. ERα and ERβ were capable of forming heterodimers on DNA that could bind the coactivator, SRC-1, and appeared to stimulate transcription of a reporter gene. Moreover, we demonstrate that while the region of ERα required for homodimerization overlaps with that required for heterodimerization the two regions are not coincident.
The DNA binding activity of ERα and ERβ was tested usingin vitro translated receptors and a consensus estrogen response element in a gel shift assay. Both ERα and ERβ bound to the element, and the mobility of ERα, but not that of ERβ, was retarded in the presence of the hERα antibody, H226 (Fig.1). When the two receptors were cotranslated we were able to detect a complex with an intermediate mobility corresponding to ERα/ERβ heterodimers, in addition to ERα homodimers (Fig. 1, tracks 7–12). Their mobility was retarded by H226 consistent with the presence of ERα in both complexes. Their relative amounts varied depending on the input ratio of the two receptors but it is noteworthy that ERβ homodimers were barely detected even when ERβ was expressed in 2-fold excess over ERα (Fig. 1, track 11). Their affinity for DNA was then determined by carrying out gel shift experiments over a wide range of probe concentrations (Fig. 2). We found that ERα homodimers and ERα/ERβ heterodimers bound to DNA with a similar Kd of approximately 2 nm whereas that of ERβ homodimers was about 4-fold greater.
We next analyzed the DNA binding activity of ERα and ERβ when they were expressed in COS-1 cells. When ERα alone was expressed, we observed two complexes, a major upper complex, corresponding to the ERα homodimer, and an additional complex that is probably generated by proteolysis. It seems to lack N-terminal sequences since it is recognized by a monoclonal antibody specific for the C-terminal F region (
). As expected, the mobility of ERα but not ERβ was retarded in the presence of the specific hERα antibody, H226. When equivalent amounts of ERα and ERβ expression vector were coexpressed, heterodimers were the predominant form observed, and ERβ homodimers were not detected (Fig.3A). We then used these extracts to compare the effect of 17β-estradiol and 4-hydroxytamoxifen on the DNA binding activity of the three dimeric forms (Fig. 3B). As previously demonstrated for ERα (
), the DNA binding activity of both ERβ and ERα/ERβ heterodimers was unaffected by ligand binding, but their mobilities were slightly increased in the presence of 17β-estradiol (Fig. 3B,tracks 2, 5, and 8). Thus, we conclude that ERα/ERβ heterodimers, expressed in intact cells, are capable of forming on DNA and that ERα homodimers and ERα/ERβ heterodimers are preferentially formed.
Previous work with a series of ERα mutants has identified a region within the ligand binding domain of the estrogen receptor which is required for both receptor dimerization and high affinity DNA binding (
). We have used these mutants to determine whether the region required to form homodimers with a truncated version of ERα (mouse estrogen receptor (MOR)-182–599) is similar to that required to form heterodimers with ERβ on DNA. We find that the ability of R507A to form either ERα homodimers or ERα/ERβ heterodimers is markedly reduced (Fig. 4, compare tracks 4 and 11) while L511R and I518R, which show negligible homodimer formation, retain some ability to form ERα/ERβ heterodimers (Fig. 4, compare tracks 6 with 13and 7 with 14). In contrast, mutation of Q510A had no affect on the dimerization of either receptor. A series of other mutations in this region of the receptor (A509R, L512V, L513G, I514R, L515G, L516A, H517A, R519G) was then screened to attempt to identify additional residues which could discriminate between homo- and heterodimerization, but all the mutants retained their DNA binding activity both as ERα homodimers and ERα/ERβ heterodimers (data not shown). We conclude that the region of ERα required for homodimerization overlaps that required for heterodimerization, but the two regions are not coincident.
We next assessed the transcriptional activity of ERα/ERβ heterodimers in transiently transfected COS-1 cells using the pEREBLCAT reporter gene. ERα and ERβ expression vectors were tested individually or in combination at a ratio of 1:1 or 1:2 to minimize the relative amount of ER homodimers formed (see Fig. 3). The ability of ERα to stimulate transcription was slightly greater than that of ERβ (Fig. 5A), as previously reported for this reporter (
). Coexpression of ERα and ERβ resulted in an intermediate level of transcription that was blocked by the addition of the antiestrogens, 4-hydroxytamoxifen and ICI 182780. Similar results were obtained in HeLa cells (Fig. 5B). Therefore, since the heterodimer is the major dimeric form of the receptor under these conditions, it appears to retain its ability to stimulate transcription.
To obtain additional evidence to support our suggestion that ERα/ERβ heterodimers are capable of stimulating transcription we tested whether they were able to bind the coactivator, SRC-1, as previously demonstrated for ERα (
As shown in Fig.6, when ERα, ERβ, or both were incubated with increasing amounts of GST-SRC-(570–780) in the presence and absence of ligand we could detect additional complexes in the gel shift assay. The interaction of SRC-(570–780) with ERα was dependent on the presence of ligand (Fig. 6, compare tracks 7 and8) whereas there was an appreciable interaction with ERβ in the absence of ligand (Fig. 6, compare tracks 13 and14). The interaction of SRC-1 with the heterodimer was enhanced in the presence of ligand (Fig. 6, compare tracks 19 and 20). As a control, we showed that these retarded complexes were not due to the binding of SRC-1 directly to an ERE (Fig.6, tracks 1 and 2) but required the presence of receptor. Thus ERα/ERβ heterodimers, bound to DNA, are capable of recruiting SRC-1.
The main conclusion from our study is that human ERα and ERβ are capable of forming functional heterodimers on DNA. The relative distribution of ER homodimers and heterodimers will, at least in part, be dependent on the relative expression of the two receptors which varies widely in different cell types. Both ERα and ERβ have been detected in many tissues by reverse transcription-PCR or in situ hybridization (
) but the relative amounts of receptor protein in specific cell types have yet to be determined. Nevertheless this preliminary analysis suggests that the expression of ERα may be greater than that of ERβ in epididymis, testis, pituitary ovary, uterus, adrenals, and heart. Given that ERα homodimers and ERα/ERβ heterodimers are preferentially formed over ERβ homodimers it seems that heterodimers are more likely to be formed than ERβ homodimers in these tissues. On the other hand, ERβ is expressed at higher levels in prostate, bladder, lung, thymus, and certain hypothalamic cells (
), and so ERβ homodimers may be formed in these tissues.
The molecular basis for the reduced DNA binding activity of ERβ compared with that of ERα and ERα/ERβ heterodimers is unclear, but recent work indicates that the mouse ERβ also binds to an ERE less strongly than ERα (
). Comparison of the sequence of the corresponding region in ERα and ERβ indicates that helix 10 is similar (13/18 residues are identical) but the loop region and helix 9 are poorly conserved in the two receptors. Thus, it seems likely that the residues required to form the dimer interface in ERα and ERβ homodimers are distinct. Although the precise dimerization interfaces in these receptors have yet to be identified, functional analysis of a series of mutations in helix 10 of ERα indicates that the residues required for the formation of ERα homodimers are similar but not identical to those required for ERα/ERβ heterodimerization.
Both ERα and ERβ are capable of stimulating the transcription of reporter genes in transfected cells and the activation functions, AF-1 and AF-2 characterized in ERα (
). When ERα and ERβ are expressed, under conditions when heterodimers are the predominant dimeric species, transcription of an ERE reporter gene is stimulated to an intermediate level compared with that of either homodimer suggesting that ERα/ERβ heterodimers are transcriptionally active. This is supported by our observation that ERα/ERβ heterodimers are capable of binding the coactivator SRC-1 (
). We found that SRC-1 interacts with all three dimeric states of ER bound to DNA although its interaction with heterodimers was less dependent on ligand than that observed with ERα homodimers. The interaction of ERβ homodimers with SRC-1 was dependent on ligand in solution (
The discovery of a second estrogen receptor raises many questions, most notably relating to their respective functions. The ability of ERα and ERβ to form heterodimers suggests that estrogen receptor may function in different dimeric states, and it is possible that they could be activated by selective ligands. In view of the similarity of their DNA binding domains it is doubtful whether different forms bind to distinct response elements, but they could activate different genes in different target cells given their distinct expression patterns.
We thank I. Goldsmith for oligonucleotides, A. Wakeling (Zeneca Pharmaceuticals) for 4-hydroxytamoxifen, and C. Nolan (Abbott Laboratories) for the monoclonal antibody H226. We are also extremely grateful to C. Dickson, H. Hurst, E. Kalkhoven, R. White, and members of the Molecular Endocrinology Laboratory for discussions and comments on the manuscript.