A conserved region in the hormone‐dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti‐estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen receptor in intact cells and modulates its transcriptional activity in the presence of estrogen, but not the anti‐estrogen 4‐hydroxytamoxifen. In view of its widespread expression in mammalian cells, RIP140 may interact with other members of the superfamily of nuclear receptors and thereby act as a potential co‐activator of hormone‐regulated gene transcription.

The ligand binding domain of the estrogen receptor contains a hormone-dependent transcriptional activation function. To investigate the mechanism by which it stimulates transcription, we have expressed fusion proteins containing either the wild-type or a transcriptionally defective form of this domain fused to glutathione-S-transerase and searched for proteins that specifically interact in viro. By far-Western blotting, three proteins of 160, 140, and 80 kDa expressed in different mammalian cells (HeLa, ZR75-1, and COS-1) were shown to asiate directiy with the wild-type receptor in the presence of estrogen. Two additional proteins appeared to interact indirectly with the hormone binding domain since they were detected only by a pull-down assay. All of these interactions were abolished by antiestrogens, such as 4-hydroxytamoxifen, ICI 164384, or ICI 182780, which inhibit hormone-dependent transcription. Moreover, they were not observed with the tranptionally defective form of the receptor even in the presence of estrogen. Thus, since the ability of these proteins to interact with the hormone binding domain correlates with its transcriptional activity, one or more of them may contribute to hormone-dependent transcriptional activation by the estrogen receptor.

The ability of estrogens to stimulate the transcriptional activity of the estrogen receptor can be inhibited by a diverse range of estrogen antagonists. Here we show that the antiestrogen ICI 164,384, N-(n-butyl)-11-[3,1713-dihydroxyestra-1,3,5(10)-trien-7a-yl]N-methylundecanamide, rapidly reduces the levels of receptor protein transiently expressed in cells without affecting receptor mRNA abundance. The reduction in the levels of receptor protein is dose dependent, reversible by estradiol, and mediated by the hormone-binding domain of the receptor. Pulse-chase experiments indicate that the half-life of the receptor is reduced from -5 hr in the presence of estradiol to <1 hr by ICI 164,384. A similar reduction in estrogen receptor levels is demonstrated in human breast cancer cells treated with ICI 164,384. We discuss the possibility that the increased turnover of the receptor might be a consequence of impaired receptor dimerization.Estrogens regulate cell growth and differentiation by binding to specific receptors that function as transcription factors (1-3). Hormone binding is required for the dissociation of an inactive oligomeric receptor complex to allow receptor dimerization and high-affinity DNA binding (4,5) and to produce the full transcriptional activity of the receptor (6-9). Stimulation of transcription is mediated by two transcriptional activation functions (TAF)-namely, TAF-1 and TAF-2, the activity of TAF-2 being dependent upon estrogen binding. Since estrogens have been found to act as mitogens in at least 30o of breast cancers, a large number of estrogen antagonists have been developed as therapeutic agents. One type, which includes the nonsteroidal breast cancer drug tamoxifen (10), can also act as partial agonists in a number of physiological responses; while another type, which includes ICI 164,384, has been reported to be devoid of agonist activity (11,12). ICI 164,384 is N-(n-butyl)-11-[3,17,3-dihydroxyestra-1,3,5(10)-The mechanism of action of some steroid hormone antagonists is beginning to emerge. It has been shown that tamoxifen promotes high-affinity DNA binding of the receptor but fails to induce the formation ofTAF-2 in the hormone-binding domain, which accounts for its antagonist activity (7, 9). The agonist effect of tamoxifen has been proposed to be derived from TAF-1 in the N-terminal domain of the receptor, which is constitutively active (13). The effect of the so-called pure antiestrogen ICI 164,384 is controversial. In our studies with the mouse estrogen receptor (MOR), we demonstrated that ICI 164,384 inhibited DNA binding of the receptor and proposed that this may be a consequence of impaired receptor dimerization (14). This impairment might result from steric interference produced by the large 7a-alkylamide extension present on ICI 164,384 (12), since we have shown that the steroid binding pocket is at or near the dimer interface (15). However, other workers have found that ICI 164,384 did not inhibit DNA binding and proposed that it also interferes with a subsequen...

We have characterized two distinct binding sites, called site 1 and site 2, in the nuclear protein RIP-140 which interact with the ligand binding domain of the estrogen receptor both in solution and when the receptor is bound to DNA. Both sites are capable of independently interacting with other nuclear receptors, including the thyroid hormone and retinoic acid receptors, but they are not identical since the interaction with retinoid X receptor is mediated primarily by site 1. The interaction is enhanced by agonists but not by antagonists, and the in vitro binding activities to a number of mutant receptors correlate with their abilities to stimulate transcription in vivo. When RIP-140 is fused to heterologous DNA binding domains, it is able to stimulate the transcription of reporter genes in both yeast and mammalian cells. Thus, RIP-140 is likely to function as a bridging protein between receptors and the basal transcription machinery and thereby stimulate the transcription of target genes.The nuclear receptor superfamily of transcription factors binds to DNA and activates or represses the transcription of genes in higher organisms (31,35). The activities of some of these receptors depend on the binding of hormonal ligands, including steroids, retinoids, and thyroid hormone, but the activating ligand has yet to be identified for the majority of them. Nevertheless, the modular structure of the entire superfamily seems to be conserved, since they all consist of three structural domains: an N-terminal domain containing an activation function, AF-1; a DNA binding domain; and a C-terminal ligand binding domain containing an additional activation function, AF-2 (6, 13). The activities of AF-1 and AF-2 depend on the promoter and cell type, and in some cases, both are required for full transcriptional stimulation (45). While the sequence of the N-terminal activation domain, AF-1, varies considerably in different nuclear receptors, that for AF-2 contains a highly conserved C-terminal amphipathic ␣-helix, which is essential for ligand-dependent transcriptional activity (4,11,12,40).The ability of nuclear receptors to stimulate transcription is likely to involve the recruitment of the basal transcription machinery into a preinitiation complex (16, 37). Although receptors bind directly with a number of basal transcription factors in vitro, including the TATA box-binding protein (41), TFIIB (3,19), and human TAF II 30 (20), the interactions are unaffected by ligand binding or by mutations in the AF-2 amphipathic ␣-helix that abolish transcriptional activity, suggesting that receptors are likely to interact with alternative proteins. Furthermore, the observation that AF-2 activity can be inhibited by overexpressing the hormone binding domain in squelching experiments (43) suggests that AF-2 is likely to interact with target proteins that are distinct from basal transcription factors. Several candidate target proteins have been identified; RIP-140 and RIP-160 (8, 9), ERAP-140 and ERAP-160 (17), TIF-1 (28), a number of isoforms...

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, (25,26), and this is accompanied by an inhibition of nuclear uptake of the receptor during nucleocytoplasmic shuttling (27).In this paper, we demonstrate that certain point mutations in the ligand-binding domain of the ER alter dramatically the responsiveness of the mutated ERs not only to estrogen but more importantly to antiestrogens. Two mutants are described whose activation by estrogen is abolished but are instead strong transcriptional activators in response to stimulation by both 4-OHT and the pure antiestrogen ICI 164,384. MATERIALS AND METHODSReceptor Expression Vectors and Reporter Gene Constructs. The isolation of mouse ER (MOR) cDNA clones, the construction of mutants thereof, and their transfer into the pJ3f expression vector have been described (28,29). Missense mutants of the full-length protein [MOR-(1-599)] were generated and named according to the position and identity of the substituted amino acid and the substituting residue-e.g., L543A indicates replacement of leucine at position 543 by alanine. Deletion mutants are described by the remaining amino acids-e.g.,

We have previously demonstrated that androgens are potent inhibitors of breast cancer cell proliferation under both basal and estrogen-induced incubation conditions, while they suppress expression of the estrogen and progesterone receptors. To better understand the mechanisms responsible for the antagonism between androgens and estrogens in breast cancer and to obtain a new tumor marker for the actions of these two steroids, we have investigated the effects of androgens and estrogens on expression of the major protein found in human breast gross cystic disease fluid, namely GCDFP-24. This study was performed in ZR-75-1 and MCF-7 human breast cancer cells. After a 9-day incubation period, physiological concentrations of 17 beta-estradiol stimulated proliferation of ZR-75-1 and MCF-7 cells by 2- to 3.5-fold while simultaneously exerting a marked 70-90% inhibition of GCDFP-24 secretion. The estrogenic effects on GCDFP-24 secretion and cell proliferation were both competitively blocked by simultaneous incubation with the new steroidal pure antiestrogen EM-139. On the other hand, a maximal concentration (10 nM) of the nonaromatizable androgen dihydrotestosterone decreased by 50% the proliferation of ZR-75-1 cells; the half-maximal inhibitory effect was exerted at 0.01 nM. The androgen exerted a 3- to 4-fold stimulatory effect on GCDFP-24 secretion at an EC50 value of 0.01 nM. The effect of dihydrotestosterone on these parameters was competitively blocked by simultaneous incubation with the pure antiandrogen OH-flutamide. The present data show that the effects of estrogens and androgens in ZR-75-1 cells on GCDFP-24 secretion and cell growth are opposite. Similarly, in MCF-7 cells, estrogens stimulate cell growth, while GCDFP-24 secretion is inhibited. The present data also suggest that GCDFP-24 could well be a good biochemical marker for monitoring the response to androgenic and antiestrogenic compounds in the therapy of advanced breast cancer.

The hormone binding domain of the estrogen receptor is required not only for binding estradiol but also to form stable homodimers of the protein and mediate transcriptional activation by the receptor. Residues that are essential for estrogen binding are also involved in dimerization, suggesting that the hormone-binding pocket is at or near the dimer interface. Distinct hydrophobic and charged residues are essential for hormone-dependent transcriptional activation, and these appear to be conserved by other members of the nuclear receptor family. We have found that the pure antiestrogens ICI 164384 and ICI 182780 increase the turnover of the receptor compared with that in the presence of estradiol. Because it is likely that the pure antiestrogens bind to a similar region of the receptor as that of estradiol, we propose that they inhibit receptor dimerization by means of their 7 alpha alkyl-amide extension. It appears that as a consequence nuclear uptake is inhibited and the receptor more rapidly degraded in the cytoplasm.

The effect of the adrenal steroids androst-5-ene-3 beta,17 beta-diol (delta 5-diol) and androstenedione (delta 4-dione) was studied on the growth of mammary carcinoma induced in the rat by dimethylbenz[a]anthracene (DMBA). The plasma levels of the two steroids were maintained at values within the range of those found in the circulation of post-menopausal women by constant release from osmotic pumps in ovariectomized animals. delta 5-diol and delta 4-dione, at the daily release rate of 500 micrograms, led to plasma levels of 1.26 +/- 0.19 and 1.72 +/- 0.75 ng/ml, respectively. At these physiologically relevant plasma concentrations, both delta 5-diol and delta 4-dione caused a marked stimulation of tumor growth while having minimal or no effect on uterine weight or on plasma prolactin and LH levels. Concomitant treatment with the aromatase inhibitor aminoglutethimide completely blocked the stimulatory effect of delta 4-dione released from silastic implants on tumor growth, while simultaneous administration of the antiandrogen flutamide had no significant effect. On the other hand, when aminoglutethimide was administered with delta 5-diol, the stimulatory effect of the adrenal steroid on tumor growth was not affected. Such data indicate that, under the present experimental conditions, transformation of delta 4-dione into androgens plays a minor role, the predominant effect of the adrenal steroid being stimulation of tumor growth through conversion into estrogens, while delta 5-diol exerts a direct estrogenic effect independent from aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)