Reviews of Reproduction (1998) 3, 164–171

Oestrogen receptor beta (ERβ)

Philippa T. K. Saunders

MRC Reproductive Biology Unit, 37 Chalmers Street, Edinburgh, EH3 9EW, UK

Steroid action is mediated by specific intracellular receptors, which are shifted to a transcrip- tionally active state after ligand binding. In 1996, the cloning of a new member of the nuclear receptor superfamily from the rat prostate was reported. Ligand-binding experiments have shown that this receptor binds specifically to oestrogens and it has been named oestrogen receptor beta (ERβ) to distinguish it from the oestrogen receptor (ERα) cloned from uterus in 1986. The alpha and beta forms of the oestrogen receptor have identical numbers of exons, and the cDNAs cloned from humans, rats and mice all share significant sequence homologies especially within their DNA and ligand-binding domains. Splice variants of ERβ have been identified. ERβ mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, brain, heart and the gonads and genital tracts in both males and females, and in some, but not all, tissues the pattern of expression is distinct from that of ERα. Studies in vitro have demonstrated that ERα and ERβ can exist as hetero- or homodimers and that these forms may interact differentially with response elements on genes. The identification of ERβ has made us rethink the potential sites of action of both endogenous oestrogens and exogenous natural and synthetic oestrogens and anti-oestrogens and is currently the subject of intensive research efforts.

Steroid hormones (for example, oestrogens, androgens and individual domains of the receptors have been exchanged, or progestagens) regulate cell function via specific intracellular activities of receptors modified by site-directed mutagenesis receptors expressed in their target tissues. The cloning of the (for examples, see Carson-Jurnica et al., 1990). The cDNAs en- cDNAs encoding a number of steroid receptors and com- coding ERβ in humans (Mosselman et al., 1996; Enmark et al., parison of their sequences has revealed that, together with 1997), rats (Kuiper et al., 1996) and mice (Tremblay et al., 1997) the receptors for thyroid hormones, they belong to a large have all been cloned and shown to have significant sequence superfamily of genes that function as ligand-activated tran- homology (Fig. 1). The open reading frame predicted from the scription factors (reviewed by Carson-Jurnica et al., 1990). A ERβ cDNAs encodes a protein of 485 amino acids with a pre- cDNA encoding a receptor that bound oestrogens in vitro was dicted molecular weight of approximately 54 000 which con- isolated from a human breast cancer cell line in 1986 (Green trasts with the size of ERα (approximately 67 000) detected by et al., 1986) and homologous receptors, all with significant western blotting (Furlow et al., 1990; Grohe et al., 1998). sequence homology, were swiftly isolated from rat, mouse and The most conserved region of the alpha and beta forms chicken (for example, see White et al., 1987). However, despite of ER is the DNA-binding domain (C); the positions of the an intensive period of research, and identification of many new cysteine residues that co-ordinate the two zinc fingers of the C members of the steroid–thyroid hormone receptor superfamily domain are conserved in both ERα and ERβ. The sequence (reviewed in Mangelsdorf et al., 1995), it was 10 years after the EGCKAF (hERβ amino acids 122–127) at the base of the first identification of the original oestrogen receptor (ER) that zinc finger, which has been shown to be essential for specific the identification of a second oestrogen-specific receptor was binding to oestrogen response elements (ERE) on target genes reported (Kuiper et al., 1996). This novel ER, first cloned from a (Forman and Samuels, 1990) and the region of the second zinc rat prostate cDNA library, has been named oestrogen receptor finger believed to contribute to specific receptor dimerization beta (ERβ) to distinguish it from the previously identified ER (CPATNQC, hERβ 143–150), is identical in both ERα and ERβ. which has now been re-named oestrogen receptor alpha (ERα; Domain E, which defines the ligand specificity of the different for review, see Kuiper and Gustafsson, 1997). steroid receptors, is located in the C-terminal portion of the protein and also includes a region that contributes to receptor dimerization (Forman and Samuels, 1990). Alignment of the E Structure of ERβ and homology to ERα domains of ERα and ERβ revealed two stretches of the protein All the members of the steroid receptor family share a common with significant sequence homology: hERβ 260–343/hERα arrangement of five structure–function domains, denoted A–F 353–431 and hERβ 418–453/hERα 511–547 (Enmark et al., 1997) (Mangelsdorf et al., 1995) and ERβ is no exception (Fig. 1). Like and, therefore, these regions of the protein are likely to be those ERα and other members of the steroid hormone receptor that determine the specificity of the receptors for oestrogens family, the hERβ gene has been shown to be encoded by eight (Carson-Jurnica et al., 1990). Kuiper et al. (1997) have detailed exons (Enmark et al., 1997). The functional roles of the domains similar regions of homology within the ligand-binding do- of steroid receptors have been defined by experiments in which mains of rat ERβ and rat ERα, and characterization of the © 1998 Journals of Reproduction and Fertility 1359-6004/98 $12.50 Downloaded from Bioscientifica.com at 09/25/2021 12:13:06PM via free access Oestrogen receptor beta (ERβ) 165

(a) hERβ 485 AÐB C D E F

(b) rERβ 485 79.6 98.5 85.6 93.4 98.6

(c) mERβ 485 80.6 98.5 84.4 91.9 78.6

(d) hERα 595 17.5 97.0 30.0 59.1 17.9

Fig. 1. Domain structure of oestrogen receptor β (ERβ). The locations of the different domains (A–B, C, D, E and F) are shown for (a) hERβ (Enmark et al., 1997) and the percentage amino acid homology between ERβ in the human and the homologous receptors identified in (b) rats (Kuiper et al., 1996) and (c) mice (Tremblay et al., 1997) are shown. (d) Homology between hERβ and hERα (Green et al., 1986) is shown for comparison. Note that ERβ from all three species share significant homology and that the DNA-binding domain (C) is well conserved in both α and β forms of receptor. structural relationship of the ligand-binding domains of ERβ oestrogen antagonist, ICI 182,780, to protect the binding of and ERα expressed in bacteria have confirmed that they are human ERα and ERβ to DNA at higher temperature also closely related (Witkowska et al., 1997). New data suggest that, suggest that this compound interacts differently with the two at least in rats, mRNA splice variants of ERβ containing an receptors (Pace et al., 1997). The identification of a variant of insertion of 54 nucleotides in the ligand-binding domain (ERβ2) ERβ containing an 18 amino acid insertion within the ligand- (Chu and Fuller 1997; Petersen et al., 1998), with or without binding domain (ERβ2, Chu and Fuller 1997; Petersen et al., the presence of the 117 nucleotides encoding the second zinc 1998) has added considerably to the potential complexity of finger of the DNA-binding domain (Petersen et al., 1998), are ligand activation via ERβ. ERβ2 was found to have a signifi- expressed in several tissues. cantly lower affinity for oestradiol than ERβ lacking the ad- The greatest sequence diversity between ERβ and ERα is ditional amino acids, and some differences in the binding of present in the N-terminal A–B domain, the hinge domain (D) specific oestrogens (for example, genistein) to the two forms and in the C-terminal domain (F) (Fig.1). Therefore, prep- of ERβ was recorded (Petersen et al., 1998). arations of specific ERβ cDNA probes, primers and antibodies have tended to be targeted to these regions (for example, see Formation of ligand-activated steroid receptor dimers Brandenberger et al., 1997; Couse et al., 1997; Kuiper et al., 1997; Saunders et al., 1997). Depending upon whether one or both of the receptors are pres- ent in a cell, after interaction with ligand, the ER subtypes can exist as either homodimers (ERα–ERα or ERβ–ERβ) or hetero- Interaction with natural and synthetic oestrogens dimers (ERα–ERβ; see Fig. 2) (Cowley et al., 1997; Pettersson Clone 29 (isolated by Kuiper et al., 1996) was identified as an et al., 1997). The binding affinity of ERα–ERα and ERα–ERβ ER-encoding cDNA and renamed ERβ after the demonstration dimers for a consensus DNA oestrogen hormone response el- that recombinant protein 29 bound specifically to oestrogens ement (ERE) is reported to be higher than that of the ERβ–ERβ with high affinity (oestradiol > diethylstilboestrol > oestriol > homodimer (Cowley et al., 1997), although both full length re- oestrone). Additional data have shown that ERβ and ERα recep- ceptors contain identical sequences in their zinc fingers at the tors expressed in vitro have similar specificity for a wide range positions believed to be critical for interaction with DNA (see of ligands including a number of synthetic oestrogens (Kuiper above). Paech et al. (1997) have shown that both forms of recep- et al., 1997). Scatchard analysis indicates that ERβ has a lower tor homodimer induced similar transactivation profiles in vitro affinity for oestradiol than does ERα; rat ERβ 0.4–0.6 nmol l–1 when a luciferase reporter gene linked to an ERE was used (for compared with rat ERα 0.1 nmol l–1 (Kuiper et al., 1996, 1997), example, Fig. 2, gene X) and both forms of receptor homodimer mouse ERβ, 0.5 nmol l–1 compared with mouse ERα 0.2 nmol l–1 were activated with oestradiol or diethylstilboestrol. However, (Tremblay et al., 1997). Both forms of ER are inactivated by the they also observed that ERα–ERα and ERβ–ERβ homodimers anti-oestrogen EM-800; however, the agonist effects of tam- complexed with oestradiol or several anti-oestrogens signalled oxifen appear to be different depending on the ER subtype in opposite ways when they interacted with Fos and Jun pro- (Tremblay et al., 1997). Differences in the ability of another teins to modulate gene activation via an AP-1 site (Paech et al.,

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1997). Both forms of homodimer and ERα–ERβ heterodimers have been amplified by others from these tissues using RT– are able to bind the co-activator SRC-1 (Cowley et al., 1997), PCR (Kuiper et al., 1997), which reflects the lack of sensitivity of although in the case of ERβ–ERβ this appeared to occur in the the northern blot analysis. In addition, using probes directed absence as well as in the presence of ligand (Tremblay et al., both against the N-terminal domain of rat ERβ (Byers et al., 1997). Additional complexity in the activation of gene ex- 1997) and 550 bp of the ligand-binding domain of mouse ERβ pression may occur when ERβ splice variants are expressed, (Tremblay et al., 1997), multiple mRNA transcripts, reported as as Petersen et al. (1998) have shown that both ERβ and ERβ2 ranging in size from approximately 1.0 to 10 kb, have been de- can dimerize either with each other or with ERα before binding tected in ovarian RNA samples obtained from rats and mice, to an ERE. respectively. In contrast, in the same experiments, mRNA en- coding ERα was only detected as a single transcript of approxi- mately 6.5 kb. It has not been fully elucidated whether all ERβ Pattern of expression of mRNA encoding ERβ transcripts encode functional protein, how they are generated, Messenger RNAs encoding ERβ and ERα have been amplified and if all transcripts are expressed in the same abundance in all from cell and tissue extracts using reverse transcription fol- tissues. However, it is interesting to note that four splice vari- lowed by polymerase chain reaction (RT–PCR). This method ants of rat ERβ (see above) have already been identified (Chu has the advantage of being very sensitive but does not give any and Fuller 1997; Petersen et al., 1998). Data suggest that the idea of the concentrations of mRNAs encoding ERβ and ERα in relative abundance of the two variants that differ only in the in- the cell types within complex tissues. What these studies have sertion within the ligand-binding domain (E) may vary among revealed is that mRNA encoding ERβ is present in a very wide different tissues (Chu and Fuller 1997; Petersen et al., 1998). range of tissues. Kuiper et al. (1997) amplified mRNA encoding In situ hybridization has proved useful in the identification ERβ from the testis, pituitary, ovary, uterus, bladder, lung, thy- of cellular sites of expression in complex tissues, including the mus, adrenal, heart and multiple regions of the brain, but not ovary and brain. In the first report of the cloning of rat ERβ, from spleen, liver, stomach or kidney, using rat tissue extracts Kuiper et al. (1996) showed that silver grains were localized and N-terminal specific primers. In human fetal tissues re- over the granulosa cells of follicles at different stages of de- covered between week 16 and week 23 of gestation, the highest velopment and to the epithelium of the prostatic alveoli after concentrations of mRNA encoding ERβ were detected in the tissue sections were incubated with radiolabelled antisense testes, ovaries, adrenals and spleen, with moderate to low con- oligonucleotide probes directed against domain D and the centrations in uterus, thymus, pituitary, skin, lung and kidney 3’ untranslated region of the receptor mRNA. In situ hybrid- (Brandenberger et al., 1997). In the same study, comparison of ization data obtained using a radiolabelled antisense riboprobe the abundance of mRNAs encoding ERβ and ERα by semi- prepared from a cDNA encoding part of ERβ (A–B domain, quantitative RT–PCR suggested that the expression profiles of accession number Y09372) isolated by RT–PCR from prostate the two receptors were different. In the male genital tract, Hess mRNA prepared from the common marmoset (Callithrix et al. (1997) detected mRNA encoding ERβ in the efferent jacchus) is shown (Fig. 3). Silver grains, consistent with the ductules, all regions of the epididymis, the vas deferens and expression of mRNAs encoding ERβ, are localized to granulosa prostate of adult male rats. Consistent with the documented cells within the ovary and cells lining the prostatic alveoli, effects of oestrogens on bone physiology (Turner et al., 1994), as reported in rats. Byers et al. (1997) have presented additional expression of mRNAs encoding ERα and ERβ have been de- in situ data for the rat ovary showing a low concentration of tected in rat osteoblasts isolated from 1-day-old rat bone (Onoe mRNA in some corpora lutea and claiming that the amount et al., 1997) and a human osteoblast cell line (Arts et al., 1997). In of mRNA encoding ERβ is specifically downregulated on the both rat and human osteoblasts, expression of mRNA encoding evening of pro-oestrus in the granulosa cells of preovulatory ERβ detected by semi-quantitative PCR appeared to be differ- follicles. The pattern of expression of mRNAs encoding ERβ in entially regulated compared with mRNA encoding ERα (Arts the ovaries was shown to be distinct from that of ERα, which et al., 1997; Onoe et al., 1997). mRNA encoding ERβ has also was not regulated during the ovarian cycle and was not been detected in cells from human breast tumours, some, but specifically localized to granulosa cells but uniformly dis- not all, breast cancers and breast cancer cell lines (Dotzlaw tributed throughout the ovary (Byers et al., 1997). The use of et al., 1997; Enmark et al., 1997) in the vasculature (Iafrati et al., in situ hybridization has also led to the localization of ERβ to 1997) and muscle (Petersen et al., 1998). specific regions of the brain (Shughrue et al., 1996). mRNAs en- The low expression of mRNA encoding ERβ in most cell coding ERα and ERβ are both highly expressed in the postero- types has required the loading of up to 5 µg poly A+ mRNA dorsal nucleus but were found to be differentially expressed in for analysis of transcript sizes on northern blots, and so this other subregions of the amygdaloid complex, where differ- method has been used only on a limited number of samples ential regulation of the two mRNAs by oestradiol treatment compared with the number of samples investigated using was observed (Osterlund et al., 1998). On sections of frozen RT–PCR. On multi-tissue northern blots of poly A+ mRNA iso- human tissues, mRNAs encoding ERβ have been localized lated from adult human tissues, mRNAs encoding ERβ were to the mucosa of the duodenum and rectum, the kidney detected in ovary and testis, and the major transcript size was (especially the cortex), the fetal lung, breast cancer cells, lymph approximately 7 kb with a less abundant mRNA of approxi- nodes, the prostate and cells within the testicular seminiferous mately 9–10 kb; smaller transcripts (2.4 and 1.3 kb) were also epithelium (Enmark et al., 1997). detected in the testis (Mosselman et al., 1996; Enmark et al., RNase protection assays have been undertaken with a 1997). On these blots, mRNAs encoding ERβ were not detect- variety of cRNAs specific for ERβ and ERα on cell and tissue able in samples from heart, prostate and lung, although they RNAs and provide a quantitative comparison of the abundance

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ERα + Ligand ERβ (E2)

Change in conformation/dimerization α/α β/β

ERE X α/β ERE X

ERE X α/α β/β

API A API A

Fig. 2. Formation of homo- and heterodimers between ligand-activated forms of oestrogen receptor β (ERβ) and ERα. Formation of heterodimers between the alpha and beta forms of ER have been demonstrated in vitro (Cowley et al., 1997). Homo- and heterodimeric forms will bind to a consensus oestrogen response element (ERE; Cowley et al., 1997) and activate transcription of a reporter gene (X). ERα and ERβ homodimers were shown to signal in opposite ways when complexed with Fos and Jun at an AP-1 enhancer element (AP-1) shown coupled α β to a reporter gene (A). Interaction of oestradiol (E2) with ER homodimers activates transcription whereas, with ER homodimers, the same ligand results in inhibition of transcription (Paech et al., 1997). of mRNAs for the two receptor types (for example, see Couse RNase protection assays and concluded that the absence of et al., 1997; Enmark et al., 1997). The use of cRNAs specific for ERα expression did not have a significant effect on the amount the splice variants of ERβ will also be useful for determining of mRNA encoding ERβ in the male and female reproductive the expression of multiple species of mRNA encoding ERβ tracts. Distribution of mRNA encoding ERβ in the brain of the (Petersen et al., 1998). Calculation of the ratio of mRNAs en- ERKO mouse studied using in situ hybridization has revealed coding ERβ and ERα extracted from human tissues (Enmark that the absence of ERα does not preclude region-specific et al., 1997) has confirmed that ERβ is the ER form exclusively expression of ERβ which appears comparable with expression expressed in granulosa cells but that mRNAs encoding ERα in the wild type mouse but slightly different from that of rats may be more abundant in endometrial cells. In mice, Couse (Shughrue et al., 1996, 1997). Data on the potential of oestrogens et al. (1997) reported that mRNAs encoding ERβ were more to suppress the vascular injury response via an ERα-independent abundant than ERα in the ovary and lung. pathway, and the expression of mRNAs encoding ERβ in the A number of studies on the expression and localization of aorta has also been generated by comparison of wild type and mRNAs encoding ERβ have been carried out using samples ERKO mice (Iafrati et al., 1997). from the ERα knockout (ERKO) mouse which lacks expression of a full-length functional mRNA encoding ERα (Lubhan et al., Cellular sites of expression of ERβ protein 1993). The ERKO female mice are infertile (Lubhan et al., 1993) and the males have reduced fertility (Eddy et al., 1996). Couse Data on the immunoexpression of ERβ protein have so far been et al. (1997) investigated expression of ERβ in ERKO mice using more limited than those reporting the expression of the mRNA.

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Fig. 3. Expression of mRNA encoding ERβ in the ovary and prostate of the Marmoset monkey identified by in situ hybridization. Images are shown under dark field illumination, the cells expressing mRNA encoding ERβ contain silver grains (white dots) formed after hybridization of an antisense riboprobe specific from the A–B domain of marmoset ERβ (accession number Y09372) labelled with [35S]UTP according to established methods (Millar et al., 1993). Controls prepared using sense strand probes contained the same density of silver grains as the slide background (not shown). In (a) the positions of two large ovarian follicles (F1 and F2) are indicated; silver grains are localized to granulosa cells lining the follicles (arrows) and cumulus cells around the oocyte (arrowhead) which are clearly visible in F1. Some silver grains were also present over the stroma (S). (b) A section of the prostate of a marmoset with silver grains concentrated in the epithelial cells surrounding the alveoli (av). Exposure time was 4 weeks; scale bars represent 100 µm.

In rats, specific immunolocalization has been detected in and epithelial cells of the bladder, heart, lung, adrenal and multiple cell types, including granulosa (see Fig. 4a) and seminal vesicle (Saunders et al., 1997, 1998) and in the brain corpora luteal cells of the rat ovary, uterus, smooth muscle (Aves et al., 1997; Simonian and Herbison, 1997). In the ovaries

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Fig. 4. Immunolocalization of oestrogen receptor β (ERβ) to cell nuclei. ERβ was immunolocalized to ovaries (a,b), testes (c) and prostate (d) from rats. Sections were prepared from fixed tissues embedded in paraffin wax and immunohistochemistry performed as described by Saunders et al. (1997). Note that in the ovary from an adult rat (a), ERβ are present in follicles containing only one or two layers of granulosa cells (arrowheads) as well as in a medium sized follicle containing an antrum (asterisk). A small follicle (b) is shown at higher magnification to illustrate the presence of intense immunostaining of ERβ in granulosa cell nuclei. In the adult rat testis (c), ERβ was present in nuclei of Sertoli cells (arrowheads) and in some germ cells, for example, pachytene spermatocytes (asterisks). In the rat prostate (for example day 14; d) cells lining the alveoli were immunopositive. Within the stroma, some ERβ-positive cell nuclei were observed but not all cells contained receptor protein (receptor negative nuclei stained blue by haematoxylin). Scale bars represent 100 µm (a,d), 20 µm (b) and 50 µm (c).

Downloaded from Bioscientifica.com at 09/25/2021 12:13:06PM via free access 170 Philippa T. K. Saunders of rats, the most intense immunostaining for ERβ is detected in The author is grateful to colleagues who have contributed to studies granulosa cells in both preantral and antral follicles (Fig. 4a,b). on ERβ at the MRC Unit; particular thanks are due to Mike Millar, Joe In the prostate of rats (days 14, 35 and adult), immunopositive Gaughan and Julie Wilson. The diagrams in this paper were skillfully cell nuclei were detected in the epithelial cells lining the alveoli prepared by Edward Pinner. and sporadic immunopositive cells were found within the stroma (Fig. 4c). A similar pattern of expression is seen in a References photograph of a prostate illustrated in an article by Pennisi (1997), although the species and specificity of the antibody Key references are identified by asterisks. Arts J, Kuiper GGJM, Janssen JMMF, Gustafsson J-A, Lowik CWGM, used are not specified. Immunolocalization has demonstrated Pols HAP and Van Leeuwen JPTM (1997) Differential expression of expression of ERβ protein in multiple testicular cell types, in- estrogen receptors α and β mRNA during differentiation of human osteo- cluding Sertoli cells, Leydig cells and some germ cells (sper- blast SV-HFO cells Endocrinology 138 5067–5070 matocytes and spermatogonia) within the adult rat (Saunders Aves SE, Lopez V, McEwen BS and Weiland NG (1997) Differential co- localization of estrogen receptor β (ERβ) with oxytocin and vasopressin et al., 1998), marmoset and human (not shown). The pattern of β in the paraventricular and suproptic nuclei of the female rat brain: an expression of ER in the ovary and testis is distinct from that immunocytochemical study Proceedings of the National Academy of Sciences of ERα, which is not expressed in the granulosa cells of the USA 95 3281–3286 ovary (Hild-Petito et al., 1988; Saunders et al., 1997) and only Brandenberger AW, Tee MK, Lee JY, Chao V and Jaffe RB (1997) Tissue α β expressed in Leydig cells of the testis (Fisher et al., 1997). distribution of estrogen receptors alpha (ER- ) and beta (ER- ) mRNA in β the midgestation human fetus Journal of Clinical Endocrinology and Expression of an ER protein of 54 kDa has been detected in Metabolism 82 3509–3512 neonatal rat cardiac myocytes by western blotting (Grohe et al., *Byers M, Kuiper GGJM, Gustafsson J-A and Park-Sarge O-K (1997) 1998). In the brain of rats, ERβ was detected in selected nuclei Estrogen receptor-β mRNA expression in rat ovary: down regulation by within the paraventricular nucleus (PVN) and supraoptic nu- gonadotropins Molecular Endocrinology 11 172–182 Carson-Jurnica MA, Schrader WT and O’Malley BW (1990) Steroid receptor cleus, which are regions that do not contain ERα (Aves et al., β superfamily: structure and functions Endocrine Reviews 11 209-220 1997). Dual labelling studies have identified expression of ER Chu S and Fuller PJ (1997) Identification of a splice variant of the rat estrogen in 35–50% of neurones expressing oxytocin in the PVN, receptor beta gene Molecular and Cellular Endocrinology 132 195–199 whereas expression of ERβ was variously reported as low or Couse JF, Lindzey J, Grandien K, Gustafsson J-A and Korach KS (1997) α α undetectable in cells expressing arginine–vasopressin (Aves Tissue distribution and quantitative analysis of estrogen receptor- (ER ) and estrogen receptor β (ER) messenger ribonucleic acid in the wild-type et al., 1997; Simonian and Herbison, 1997). and ERα-knockout mouse Endocrinology 138 4613–4621 Cowley SM, Hoare S, Mosselman S and Parker SG (1997) Estrogen re- ceptors alpha and beta form heterodimers on DNA Journal of Biological More excitement for the future? Chemistry 272 19 858–19 862 β Dotzlaw H, Leygue E, Watson PH and Murphy LC (1997) Expression of Studies on ER have demonstrated that the receptor is very estrogen receptor-beta in human breast tumors Journal of Clinical widely expressed in the body and have prompted a complete Endocrinology and Metabolism 82 2371–2374 re-evaluation of the cellular targets of oestrogen action. The Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB potential for the formation of homodimers of each ER type as and Korach KS (1996) Targeted disruption of the estrogen receptor gene well as heterodimers between ERβ and ERα, which can have in male mice causes alteration of spermatogenesis and infertility Endocrinology 137 4796–4805 differential effects on gene activation, makes the determination *Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, of the exact cellular sites of expression of the different types of Fried G, Nordenskjold M and Gustafsson J-A (1997) Human estrogen ER critical in understanding cell-specific effects of oestrogen receptor β-gene structure, chromosomal localization, and expression pat- action. An understanding of tissue-specific responses to dif- tern Journal of Clinical Endocrinology and Metabolism 82 4258–4265 Erenburg I, Schachter B, Mira y Lopez R and Ossowski L (1997) Loss of an ferent oestrogens will also need to take account of the new α β estrogen receptor isoform (ER delta3) in breast cancer and the conse- splice variants of ER that have been identified in rats (Petersen quences of its re-expression: interference with estrogen-stimulated proper- et al., 1998), for example, whether they are translated in all ties of malignant transformation Molecular Endocrinology 11 2004–2015 tissues, if they are expressed in all species and how they in- Fisher J, Millar MR, Majdic G, Saunders PTK, Fraser HM and Sharpe RM α fluence gene activation. Do the alternative transcripts of ERβ (1997) Immunolocalisation of oestrogen receptor-alpha (ER ) within the testis and excurrent ducts of the rat and marmoset monkey from perinatal contribute to cancer cell proliferation as has been suggested for life to adulthood Journal of Endocrinology 153 485–495 ERα in breast cancer cells (Erenburg et al., 1997)? After the Forman BM and Samuels HH (1990) Interactions among a subfamily of many interesting studies generated from ERKO mice (Lubahn nuclear hormone receptors: the regulatory zipper model Molecular et al., 1993; Eddy et al., 1996), a full examination of the pheno- Endocrinology 4 1293–1301 Furlow JD, Ahrens H, Mueller GC and Gorski J (1990) Antisera to a type of the ERβ knockout mice (Gustafsson, 1998) is eagerly β α synthetic peptide recognize native and denatured rat estrogen receptors awaited as are the results of any ER –ER negative offspring Endocrinology 127 1028–1032 that might be generated from the different mouse lines. Will Green S, Walter P, Kumar V, Krust A, Bornert J-M, Argos P and Chambon P these mice lack all evidence of oestrogen receptors or lead to (1986) Human oestrogen receptor cDNA: sequence, expression and hom- the identification of further ER subtypes? There is more to be ology to v-erb-A Nature 320 134–139 β Grohe C, Kahiert S, Lobbert K and Vetter H (1998) Expression of oestrogen discovered about ER -specific gene activation, for example, receptor α and β in rat heart role of local oestrogen synthesis Journal of whether ERβ–ERα dimers activate a different gene cascade Endocrinology 156 R1–R7 from homodimers, and details of the specific interaction of ERβ Gustafsson J-A (1998) Novel insights into estrogen mechanism of action β with chaperone proteins, including those of the heat shock through ER Xth International Congress on Hormonal Steroids Quebec City Hess RA, Gist DH, Bunick D, Lubahn DB, Farrell A, Bahr J, Cooke PS and family (see Smith and Toft, 1993). 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