Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 363 The ER� and ER� proteins are composed of six functional domains, labeled A–F, a signature characteristic of members of the superfamily of steroid/thyroid hormone nuclear receptors (Fig. 1). The N�-terminal A/B domain is the least conserved among all members and demonstrates only 17% identity between the human ER� and ER� (64). In contrast, the C domain is the most highly conserved among the different members of the family. It possesses two zinc fingers forming a helix-loop-helix motif and primarily functions in tightly binding the receptor to the DNA hormone response elements. The sequences encoding the two zinc fingers possess 97% homology between the ER� and ER� genes and are located in separate exons (exons 3 and 4) in each (50, 64, 79). The E domain, or ligand-binding domain, confers ligand specificity to the receptor and is moderately conserved among the members of the superfamily. The ER� and ER� proteins possess 60% conservation of the residues in the E domain; however, each binds estradiol with nearly equal affinity and exhibits a very similar binding profile for a large number of natural and synthetic ligands (80). The D domain possesses signals for nuclear localization of the receptor and exhibits approximately 30% identity between the two human forms of ER (64). The C�-terminal F domain is unique to the ER among the nuclear receptors for the gonadal and adrenal hormones (6) but is not well conserved among the ERs of different species nor between the ER� and ER�, which share approximately 18% homology (64). Studies using forms of the ER� missing the C� terminus have indicated a role for the F domain in modulating transactivational activity of the ER� when complexed with mixed agonist/antagonist ligands, possibly via influencing coregulatory function and/or dimerization of the receptor (81, 82). There are also functional domains that span those boundaries described above. Residues involved in the dimerization of the receptors are located in the second zinc finger of the C domain as well as in the major dimerization surface in the E domain (83, 84). Furthermore, two domains critical to the transactivational function of the ER are the AF-1 in the N� terminus and AF-2 in the C� terminus. These two domains may function independently or interact during the process of transactivation, depending on the cell type, target promoter, and the presence and/or type of ligand (52). The AF-2 domain is critical to the ligand-dependent transactivational activity of the receptor and may be involved in the recruitment of coregulator proteins, whereas the AF-1 is thought to be a region of site-specific phosphorylation involved in ligandindependent activity of the receptor (reviewed in Refs. 31 and 52). Recent studies have also suggested the presence of a third domain, AF-2a, within the ligand-binding domain of the human ER� (85). The discovery of the ER� has introduced a new level of complexity to the current model as well. To date, there exist no data indicating a physiological response solely mediated by ER�. In contrast, the �ERKO mouse has confirmed the requirement for ER� in mediating several actions of estradiol, as will be discussed in this review. Nevertheless, in vitro experiments from several laboratories have indicated the possibility of cooperative activity between the two receptors, FIG. 1. Drawing of the mouse ER proteins, cDNAs, and genes as well as the targeting scheme employed to generate the ERKO mice via homologous recombination. Shown are the common functional domains of the ER� and ER� receptor proteins, indicating the residues involved in DNA and ligand binding. The common structure of the cDNAs and genomic genes for the ERs is illustrated, indicating the exon sequences that encode the functional domains of the receptor. Generation of the �ERKO mouse involved the targeted insertion of a 1.8-kb NEO sequence into exon 2 of the ER� gene such that the translational reading frame (indicated by the direction of the arrow) of the genes was the same (see Ref. 46). Generation of the �ERKO mouse involved a similar scheme, in which a 1.8-kb NEO sequence was inserted into exon 3 of the ER� gene; however, in this case the NEO gene is in the reverse orientation (see Ref. 47). The schematic drawing of the genomic DNA was adapted from that of the human ER� gene (Ref. 79). Drawing is not to scale.
364 COUSE AND KORACH Vol. 20, No. 3 acting in the form of heterodimers (50, 62, 65, 86). These studies generally report a tendency of ER� to form homodimers whereas ER� prefers to heterodimerize with ER�. However, Giguere et al. report (87) that the heterodimer is the preferred state when both mouse ERs are present. The transactivational activity of the heterodimer when assayed in in vitro mammalian cell transfection assays appears to lie between that of the more active ER� homodimer and the less active ER� homodimer (50, 62, 86). A major consideration when evaluating the possible physiological functions of an ER�/ER� heterodimer is evidence of coexpression of the two receptors in the same cell, which has not yet been definitively reported (88). To this end, studies and reagents are only now becoming available to directly assess this question. Several functional characteristics of the two ERs are similar. The residues critical to function of the AF-2 domain appear to be identical in the mouse ER� and ER� (87). Tremblay et al. (50, 89) demonstrated that a tyrosine residue critical to the function of the AF-2 domain was conserved in both the ER� and ER� and that mutation of this amino acid resulted in similar constitutive, ligand-independent transactivational activity in both receptors. In contrast, the N�-terminal AF-1 domain shows no significant regions of similarity between the two ERs (87). However, a potential activation site of the mitogen-activated protein (MAP)-kinase pathway previously shown for ER� is present and active in the ER� (50, 89). Additionally, when acting on a basal promoter linked to a consensus estrogen response element, both ER� and ER� were able to recruit the coactivator SRC-1 and were equally susceptible to inhibition by the antiestrogens raloxifene, ICI 164,384, and EM-800 (50). However, as studies continue, distinct differences at the molecular level and in the transactivational capacities between ER� and ER� have been described. Two separate studies have demonstrated the specificity of the agonist activity of 4-hydroxytamoxifen to be unique to ER�, although this appears to be highly dependent on the cell and promoter context as well as experimental design (50, 90). Furthermore, Paech et al. (91) reported that when interacting with DNAbound AP-1 transcription factors, the in vitro transactivational activity of estrogen agonists and antagonists was quite different depending on which form of ER was present. Whereas antagonists, such as raloxifene, tamoxifen, and ICI 164,384, were able to block the stimulatory activity of the ER�/AP-1 complex, these same compounds acted as potent agonists when bound to an ER�/AP-1 complex (91). Further experimental support for the existence of distinct structural and functional differences between ER� and ER� was recently provided by Sun et al., who showed that certain nonsteroidal ligands were receptor selective in their binding and agonist/antagonist activities (92). Perhaps the most significant disparity lies in the tissue distribution of the two receptors. Studies employing the techniques of RT-PCR and/or ribonuclease protection assay (RPA) have indicated that ER� mRNA is predominant in the uterus, mammary gland, testis, pituitary, liver, kidney, heart, and skeletal muscle, whereas ER� transcripts are significantly expressed in the ovary and prostate (Fig. 2) (63, 80, 93, 94). These same studies have indicated relatively equal levels of mRNA for the two receptors in the epididymis, thyroid, adrenals, bone, and various regions of the brain (80, 93, 95–97). However, as more studies are reported, several discrepancies in the expression patterns of ER� and ER� among different species are becoming apparent (80, 93, 96). For example, whereas ER� mRNA is easily detectable in the pituitary of the rat (70, 98, 99), human (100), and rhesus monkey (96), levels in the pituitary of the mouse appear low to undetectable (93). A similar difference in expression is apparent in the mammary gland, in which normal and neoplastic human tissue and cell lines express detectable ER� mRNA (64, 68, 73, 101, 102), although the mammary gland of the mouse appears to predominantly express ER� (93). Furthermore, even in those tissues expressing both ERs, there is often a distinct expression pattern within the heterogeneous cell types composing the tissue. In the ovary, ER� is apparently localized to the granulosa cells of maturing follicles, whereas ER� is detectable in the surrounding thecal cells (69, 103, 104). In the prostate of the rat, expression of ER� and ER� is detectable in the stroma and epithelium, respectively, but does not appear to be colocalized in any portion of the tissue (49). However, through the combined use of immunohistochemistry and in situ hybridization, Shughrue FIG. 2. RT-PCR for ER� and ER� mRNA in various tissues of the wild-type mouse. RT-PCR was carried out on 0.5 �g of total RNA pooled from adult wild-type mice using primers specific for the mouse ER� and ER� transcripts (see Refs. 93 and 123). Equal amounts of the individual RT-PCR reactions were then fractionated on an agarose gel. Note the broad tissue distribution of ER� mRNA, whereas ER� transcripts are primarily expressed in the ovary, hypothalamus, lung, and male reproductive tract. RT-PCR for �-actin was carried out as a positive control. (�RT) indicates a negative control, i.e., PCR on total ovarian RNA minus reverse transcriptase, indicating the specificity of the ER primers for cDNAs generated by the reverse transcriptase enzyme.
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