Interaction of calf uterine estrogen receptors with chicken target cell nuclei

The calf uterine estrogen receptor (ER) was used to study the capacity and the characteristics of the acceptor sites in chicken target cell nuclei. The temperature-activated ER is bound at 0 degrees C with a high affinity to all chicken cell nuclei tested (Kd = 0.4-1.0 nM). The nuclear binding displayed tissue specificity: oviduct greater than liver, heart greater than spleen greater than erythrocytes and was salt-dependent. ER binding to liver nuclei measured in 0.15 M KCl varied between 3000 and 6000 acceptor sites per nucleus. Liver nuclei isolated from estrogen-treated cockerels showed a 2-fold lower binding capacity than nuclei from non-treated chickens. When nuclei were incubated with [3H]ER from embryo liver and increasing concentrations of uterine non-radioactive-ER a progressive inhibition of the binding of the liver ER was found. These experiments suggest that liver and uterine ER compete for a common acceptor site. Liver nuclei charged in vitro with calf uterine ER were digested at 0 degree C with DNAase I and micrococcal nuclease. Both enzymes excised the ER in the form of a chromatin-ER complex. A considerable portion was associated with nucleosomal subunits and a minor fraction was associated with a nuclease-sensitive, protein-poor fraction of the chromatin.     

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.     

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Properties of the estrogen receptors contained in the MtTF4 tumor whose growth is inhibited by estradiol: 17 beta-estradiol, 17 alpha-estradiol and DNA bindings

The steroid and the DNA bindings of the estrogen receptor of the MtTF4 tumor whose growth is inhibited by estradiol where characterized and compared to those of uterine estrogen receptors. In the tumor cytosol: E protects its binding sites against thermal denaturation, depending on the effects of sodium molybdate upon the dissociation rate of [3H]E at 20 degrees C and the ability of receptor to bind to DNA, the activation (or transformation) process, supposed to be necessary for the full action of estrogen ligand, occurs on estrogen receptor complexes and the calf thymus DNA interacts with estrogen receptor with an affinity similar to that of uterine estrogen receptor. Kinetic and equilibrium studies with 17 alpha-[3H]E both in uterus and tumor indicate that this ligand is fast-associating, fast-dissociating and that its affinity for ER is 2- to 4-fold lower than that of 17 beta-[3H]estradiol one. Competition experiments between 17 beta-[3H]estradiol and the unlabelled 17 alpha epimer reveal, in both uterus and tumor, a time-dependent decrease of the apparent potency of 17 alpha-E to inhibit the binding of [3H]E. It is concluded that the estrogen receptors are very similar in MtTF4 tumor and uterus and the diversity of the response of cell growth to E is due rather to differences at the post-receptor level.     

Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites.

The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.     

Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Elution of ER was obtained by chelating agents or by imidazole, thus indicating that histidine residues on the ER molecule are involved in the interaction with the metal. Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentrations. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. Furthermore, if added to pretransformed ER-hormone complex, they strongly inhibited the binding of the complex to isolated nuclei. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is therefore speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.     

The nuclear matrix is the site of glucocorticoid receptor complex action in the nucleus

Binding of highly purified glucocorticoid receptor complexes to nuclear matrix was evaluated. Extraction of purified nuclei with 2M potassium chloride and brief deoxyribonuclease digestion leaves a matrix structure containing 1% of nuclear DNA and 6-12% of nuclear proteins. The nuclear matrix retained two binding sites for receptor complexes, a high affinity, low capacity site and a low affinity, high capacity site. These sites have affinities and capacities consistent with those reported for binding of these complexes to intact nuclei. More extensive deoxyribonuclease treatment of the matrix resulted in a marked reduction of high affinity complex binding. Furthermore, the DNA binding form of the receptor complex but not the unactivated receptor complex bound to DNA fibers anchored to nuclear matrix as visualized by 18 nm gold particle receptor complexes. The data suggest that the nuclear matrix is the major site for coordinating glucocorticoid hormone action in the nucleus.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Effect of ribonuclease on the physico-chemical properties of estrogen receptor

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3-4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25 degrees C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.     

A method for the determination of estrogen receptor concentration in calf uterus and other tissues using an aqueous two-phase system

An assay for the cytoplasmic estrogen receptor in calf, human and rat uterus has been developed. The method is based on partial separation of free and bound estradiol (E2) by means of an aqueous two-phase system containing dextran and poly(ethylene glycol), respectively, in the two phases. Low-speed supernatant from uterus homogenate is equilibrated with E2 and [3H]E2. A two-phase mixture is then added and bound E2 will partition into the lower phase while free E2 is distributed in both phases according to its partition coefficient. The amounts of bound and free E2 are calculated and the receptor concentration and association constant are obtained from a Scatchard plot. No dissociation of bound E2 in the phase system could be demonstrated at 4 degrees C. The interassay coefficient of variation for receptor concentration at 4 degrees C was 20 and 14% for calf and human uterus, respectively. The intraassay variation for receptor concentration in calf uterus determined at 4 degrees C and 23 degrees C was 7.1 and 4.1%, respectively. The influence of freezing the tissue and supernatant preparation was examined and results from supernatant preparations obtained with different centrifugations were compared. The method is simple and rapid, permitting large numbers of samples to be handled efficiently by a single technician.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

Estrogen dependent induction of low affinity binding sites in the nuclear fraction of rat uterus

Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.     

Nuclear Binding of Hydrocortisone-Receptors in the Embryonic Chick Retina and its Relationship to Glutamine Synthetase Induction

Binding of hydrocortisone (HC) to cytoplasmic receptors andof the resulting receptor-hydrocortisone complex (R-HC) to nuclearacceptor sites has been studied in neural retina cells of thechick embryo, in which this hormone induces glutamine synthetase(GS). These studies were done in a cell-free system, as wellas in intact retina tissue in culture. Optimal conditions, specificity, and the quantitative aspectsof R-HC binding to nuclei in the cell-free system were determined.Isolated nuclei retained their binding specificity for R-HCprepared from retina cytosol; at saturation, the total numberof nuclear acceptor sites for R-HC was estimated to be in therange of 1500 per nucleus. These sites were resistant to RNAsebut sensitive to DNAse. When retina tissue was cultured in the presence of progressivelyhigher doses (0–90 nM) of HC, increasing amounts of receptorswere translocated from the cytoplasm to the nuclei. Followingincubation of retina with 9–90 nM HC, the cytoplasm wasalmost completely depleted of receptors and the nuclei becamesaturated with R-HC. From the amount of R-HC bound to nucleiat saturation, the estimated total number of R-HC acceptor sitesper nucleus was comparable to that derived from the cell-freesystem. Increases in the level of GS induction in the retinacorrelated well with amounts of R-HC complex bound by nuclei.     

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.     

Structure and dynamics of the estrogen receptor

To evaluate the structure and function of estrogen receptor (ER) in various mammalian systems, the cytosolic forms of receptor from calf uterus and from MCF-7 human breast cancer cells have been purified to virtual homogeneity by sequential selective adsorption to estradiol-Sepharose and heparin-Sepharose. In both cases, the purified steroid-receptor complex appears to exist as an activated 5S homo- or heterodimer of mol. wt 65,000 (4S) steroid-binding subunits. Purified ER has high affinity for DNA and serves as a substrate for phosphorylation by a purified rat brain kinase. Several monoclonal antibodies prepared against affinity-purified MCF-7 cytosol ER have been used to localize receptor by an indirect immunoperoxidase technique in fixed, frozen sections of human breast tumors, human uterus, rabbit uterus and in other mammalian reproductive tissues and cancers, as well as in fixed MCF-7 cell cultures and in paraffin-embedded sections of breast tumors and human endometrium. In all cases, we have observed only nuclear localization of immunoreactive receptor in tissues and whole cells, even under conditions in which virtually all of the receptor is found in a low-salt extract (cytosol) of the target cells. Treatment of cells or tissues in vivo or in vitro with estradiol alters the intensity but not the distribution of specific staining for ER. By immunoelectron microscopy, receptor was localized in the euchromatin, but not in the marginated heterochromatin or nucleoli of MCF-7 nuclei and epithelial and stromal nuclei of postmenopausal human endometrium. These observations suggest that the majority of the unoccupied receptor may actually reside in the nucleus, rather than in the cytoplasm as previously thought. Thus, hormone action may involve binding of the steroid directly to receptor loosely associated with nuclear components, followed by conversion of the steroid-receptor complex to an activated form which becomes more tightly associated with chromatin.     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.