Estradiol-receptor Complex Research Articles

Kinetics of estradiol entry into uterine cells.

The mechanism by which 17β-estradiol (E2) enters uterine cells remains unclear; facilitated transport and passive diffusion have been proposed. These conflicting conclusions were drawn from experimental data obtained with intact uterine tissue, where the initial rates of estrogen uptake could not be accurately measured for a variety of reasons which will be discussed in this report. To clarify this issue, we have studied the kinetics of E2 entry into suspended uterine cells by measuring the initial rates of formation of the intracellular receptor-estradiol complex (R-E2), under conditions where the formation of cytoplasmic R-E2. and its nuclear translocation were not rate limiting. Thus, the initial rates of formation of R-E2 should be a function of the quantity of E2 which entered the cells. Data are presented demonstrating that at 37 C, E2 uptake is extremely rapid, with 20% of the intracellular receptor sites being saturated within the first 15–30 sec at extracellular E2 concentrations higher than 10 n...

Antibodies to estrophilin: Comparison between rabbit and goat antisera

The immunochemical similarity of mammalian estrophilins has been demonstrated by the ability of immunoglobulin (Ig-i) obtained from rabbits and a goat immunized with purified estradiol-receptor complex of calf uterine nuclei to cross-react with all radioactive estradiol-receptor complexes (E ∗R) tested to date, as determined by sucrose gradient, gel filtration and double antibody precipitation techniques. Rabbit antibodies to the nuclear receptor of calf uterus react with 5.2 S nuclear E ∗R from calf, rat, rabbit and sheep uterus, as well as from rat endometrial and pituitary tumors to give two more rapidly sedimenting E ∗R-Ig-i complexes (7–8 S and 10–12 S). Extranuclear E ∗R (4 S) from uteri and tumors of several mammalian species, and from rat and human mammary cancers, as well as nuclear and extranuclear E ∗R (3.5 S) from MCF-7 human breast tumor cells, give a single major sedimentation peak (7–8 S) with rabbit Ig-i. The hormone specificity of rabbit Ig-i has been demonstrated by the absence of any interaction between Ig-i and androgen receptors of rat prostate, or mammalian and non-mammalian progesterone receptors. Also, there is no reaction with free estradiol, with non-specific binding components in human breast cancer cytosol, or with 125I-labeled mouse or rat a-fetoprotein. Goat antibodies to calf uterine E ∗R react with nuclear and extranuclear E ∗R of all tested tissues, including uterus, tumor and human breast cancer, to give a major new entity sedimenting at 11–14 S. The titer of antibodies to estrophilin in goat Ig-i is considerably higher than in rabbit Ig-i. Unlike rabbit antibody, interaction of goat Ig-i with extranuclear estrophilin causes a decrease in affinity and a reduction in the number of binding sites for estradiol. Fab fragments from both rabbit and goat Ig-i react with nuclear E ∗R from calf uterus to give a product sedimenting slightly ahead of the original E ∗R complex.

Estrogen Action in the Mouse Uterus: Characterization of the Cytosol and Nuclear Receptor Systems

Cytosol receptor binding of 17β-estradiol in the mouse uterus was demonstrated in immature, ovariectomized, and intact animals. Quantification of specific binding by protamine sulfate precipitation was approximately 0.70 pmol estradiol bound/mg cytosol protein for all of the groups studied. Nuclear binding levels, assessed by the nuclear exchange assay, were 0.11–0.15 pmol/100 μg DNA. Stability of the nuclear receptorestradiol complex was evaluated at 25, 30, and 37 C and was found to be inversely related to the incubation temperature. Linearity of the binding assays was demonstrated for cytosol and nuclear receptors compared to milligrams of cytosol protein or micrograms of DNA, respectively. The mouse uterine cytosol receptor-estradiol interaction was shown to be second order by association rate analysis (k1 = 2.0 × 105 M-1 sec-1). Dissociation of estradiol from the receptor complex (k-1) appeared to be first order, with a constant of 2.3 × 10-5 sec-1. Sucrose gradients performed in low salt buffer (10 ...

Estrogen receptor forms in human myometrial cytosol.

Abstract The estrogen receptor forms in human myometrial cytosol have been characterized in a low salt medium (without KCl) in the absence and presence of the protease inhibitor, diisopropylfluorophosphate (DFP). The sedimentation coefficients of the receptors were 9.0 ± 0.1 S, 5.4 ± 0.16 S and 4.3 ± 0.17 S. The amount of the receptor forms present was dependent on the time of the DFP addition. Obviously a 9 S → 4.3 S receptor transformation was catalyzed by the protease in the cytosol while the 5.4 S receptor form seemed to be unaffected. The estradiol binding capacity of the receptors in cytosol without DFP was increased compared to cytosol prepared in the presence of DFP. The protease catalyzed transformation which resulted in a reduction in the amount of estradiol bound to the 9 S receptor form was accompanied by a two-fold increase in the amount bound to the 4.3 S receptor. No change in the dissociation constant of estradiol receptor complex was observed by the dextran-charcoal technique. Either the proteolytic 9 S → 4.3 S transformation has to uncover hidden estradiol binding sites or then part of the estradiol bound to the 9 S receptor dissociates rapidly so that it cannot be detected under the conditions used.

Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins.

During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.

A re-examination of the interaction of estradiol with target cell receptors

Two intranuclear estradiol receptor complexes can be distinguished by sucrose density gradient ultracentrifugation of the immature rat uterus following either in vivo or in vitro administration of radiolabelled estradiol. Time course studies demonstrate that the earliest detectable complex sediments as a 4 S form whereas a 5 S form accumulates during prolonged exposure to hormone (> 10 min). Pulse chase experiments suggest that the 5 S complex arises from the lighter 4 S complex in a precursor-product manner. The properties of both the intranuclear 4 S and 5 S forms are shown to be those of authentic estrogen receptors. These observations are not in agreement with the generally held view that receptor 4 S to 5 S transformation occurs in the cytoplasm and is a necessary prerequisite to nuclear translocation.

Evidence for two kinds of chromatin binding sites for the estradiol-receptor complex

Evidence for two types of chromatin binding sites for the estradiol-receptor complex was found in target nuclei using micrococcal nuclease digestion. Nuclei from rat uteri, which had been incubated for 1 hr at 37°C in Dulbecco's modified Eagle's medium containing 10 −8 M 3H-estradiol, produced monomer (nu 1) and short oligomer nubodies which contained significantly higher specific activities of 3H-estradiol/nu-body than the bulk chromatin. The specific activities decreased from nu 1 to nu 2 to nu 3, and more extensive digestion produced nu-bodies with lower specific activities. 3H-estradiol-containing nu-bodies sedimented slightly faster than bulk particles. The bound estradiol could be released from the nu-bodies with 400 mM KCl, producing a 5.3S estradiol-receptor complex similar to the “activated” form of receptor released by salt from uterine nuclei of steroid-treated animals. Concomitant with the appearance of 3H-estradiol-enriched nu-bodies, micrococcal nuclease also released from the chromatin an estradiol-receptor complex which sedimented at 6.9S in low salt and 5.3S in high salt. This moiety may be bound to a small fragment of DNA since receptor released from chromatin by DNAase I sedimented at 6.0S rather than at 6.9S. It is possible that the binding of estradiol to chromatin, which is known to turn on specific gene transcription in vivo, induces or reflects a local conformational alteration which we detect as an enhanced sensitivity to nuclease, and which is recognized intracellularly as a start signal for transcription.

Sex Steroid Receptors in the Perinatal Rat Brain

Many sex differences in the regulation of reproductive function in rats are the result of a differentiation of the brain which occurs neonatally. Although injections of either androgens or estrogens are capable during the neonatal period of altering hypothalamic systems involved in reproductive behavior and gonadotropin regulation, the physiological role of each type of hormone has not been clearly established. In both sexes, circulating estrogens are normally kept from interacting with estrogen receptors in the limbic brain by the high levels of alpha-fetoprotein in the blood. The local aromatization of androgens in the brain could circumvent alpha-fetoprotein, since androgens do not bind to this serum protein. The “paromatization hypothesis” states that testosterone, which is abundant in neonatal male circulation but absent in females, is locally converted to estradiol in the limbic brain. There it binds to estrogen receptor proteins to produce tissue differentiation. The ontogeny of estradiol binding proteins in limbic areas is consistent with the aromatization hypothesis, with a rapid increase in receptor levels occurring shortly after birth. Also, the presence of endogenous estradiol-receptor complexes has been demonstrated in the cell nuclei of male neonates but not female neonales. Furthermore, the presence of estradiol binding proteins in other regions of the neonatal male and female brain suggests an additional role of estradiol. unknown as of yet. Several studies with agents which block the aromatization of androgens to estrogens or the binding of estrogens to their receptors are consistent with the aromatization hypothesis, since these agents prevent the differentiation of intact neonatal males. However, specific androgen binding proteins are also present in neonatal brains, and androgen-receptor complexes can be found in cell nuclei of neonates after an injection of tritiated androgen. The possible involvement of these receptors in sexual differentiation of the brain is suggested by the finding that an antiandrogen inhibits both the binding of androgens (but not estrogens) and the differentiation of males.

Studies on the binding of the estradiol-receptor complex to rat DNA fragments

The activation by estradiol of a series of reactions in hormone target tissues provides a model for the study of gene regulation in eukaryotic cells. We have examined the interaction of the estradiol-receptor complex with the various frequency classes of rat DNA to determine whether specificity can be detected at this level of chromosome organization. Interaction of the 8S estradiol-receptor complex with DNA was assayed by a change in the sedimentation rate of the complex. The receptor was found to bind to high molecular weight rat DNA as well as to 450 nucleotide long fragments of rat DNA. When the sheared DNA was separated by denaturation and reassociation into three frequency classes, the unique and moderately repeated sequences bound the receptor almost as effectively as the total sheared DNA. The resulting DNA-receptor complexes were sensitive to pancreatic DNAase. The highly repeated class of sequences bound the receptor less effectively than the other fractions. This did not result from the presence in this fraction of an inhibitor of binding, since addition of unique DNA fragments resulted in formation of a normal DNA-receptor complex. Nor was nuclease activity responsible since intact DNA could be isolated from the incubation mixture. An increase in length of the highly repeated DNA to 800 nucleotide long fragments caused them to bind the receptor almost as effectively as the other DNA fractions. These studies suggest that there may be sequences in eukaryotic DNA that preferentially bind the estradiol-receptor complex.

Antiestrogen modulation of the salt-resistant nuclear estrogen receptor.

Studies were performed in vivo to determine the ability of the antiestrogen U-11.100A (UA) to modulate the binding of the estrogen receptor to uterine nuclei. UA (100 μg) injected at 30, 60, or 120 min into immature female rats arrested the rapid estradiol receptor complex (ERC) clearance from the nuclei after the 1-h accumulation which normally occurs after a single 5-μg injection of 17β-estradiol (E2). The UA receptor complex was then accumulated in the nuclei and retained for at least 48 h. As there is no indication that there is any significant replenishment of the cytoplasmic estrogen receptor within the first 12 h after a UA (100 μg) injection or after an E2 (5 μg) injection followed by a subsequent UA (100μg) injection at 30, 60, or 120 min, the UA appears to be recapturing estrogen receptors initially translocated to the nuclei. However, by 24 h after a UA injection, there is replenishment of the cytoplasmic estrogen receptor, and subsequent E2 injections at this time will cause the nuclear transl...

Cytosol estradiol receptor in human mammary carcinoma: An assay based on isoelectric focusing in polyacrylamide gel

Limited trypsin digestion of the estradiol-receptor complex in human mammary cancer cytosol changed the sedimentation coefficient of the complex from 8 to 3–4 S. Gel filtration in high ionic strength on Sephadex columns calibrated with reference proteins showed that, after trypsinization, the Stokes radius of the estradiol-receptor complex decreased from 2.8 to 1.9 nm. Isoelectric focusing analysis indicated that the 8 S form of the steroid-receptor complex precipitated during electrofocusing in glass columns, whereas the trypsinized receptor focused as a sharp peak at pH 6.6. Based on these findings a method was developed for isoelectric focusing of the partially trypsin-digested estradiol-receptor complex in human mammary cancer cytosol on slabs of polyacrylamide gel. With this technique, the focusing time is 2 hr, at least eight different tumors may be analyzed on one gel, and sample volumes up to 300 μl are easily applied. Furthermore, electrofocusing of the estradiol-receptor complex was compared to sucrose gradient centrifugation analysis in low ionic strength and was found to be more sensitive and more specific. We therefore suggest that the described technique of isoelectric focusing should be considered as an alternative to sucrose gradient centrifugation for estradiol receptor assays.

Estrogen Receptors and Post-Receptor Markers in Human Breast Cancer: A Reappraisal

Several major defects in the estrogen receptor pathway have been evidenced in most human breast cancers by an immunofluorescence tracing of estradiol receptor complexes at the single cell level. Endogenous peroxidase seems a reliable postreceptor marker for estrogen-sensitive breast cancer cells. Since almost all human breast cancers appear to include both hormone-sensitive and autonomous cell populations, a combined use of endocrine and cytotoxic regimens is urged. The hormonal regulation of tumor growth parameters could be exploited in order to achieve a maximum recruitment of synchronized tumor cells at risk to chemotherapy.

Characteristics of the estrogen receptor system of the guinea pig uterus

Physico-chemical parameters of estradiol-receptor interaction in guinea pig uterine cytosol (velocity constants of association and dissociation, half-life time of estradiol-receptor complex, and the value of free energy change) were studied. The number of receptor sites per cell was calculated. It was shown that under conditions of different degree of approximation to the equilibrium, the affinity percentage in the system under study remained unchanged for the majority of steroids, this pointing to the achievement of equilibrium in these cases. The steroid affinity to the analyzed R-system depended on presence of intact 3- and 17beta-hydroxils with somewhat greater significance of 3-(phenolic) hydroxil of steroid molecule.

Evidence of a small molecule in mouse Leydig cell tumors which inhibits the conversion of estrogen receptor from 4S to 5S.

The activation process of cytosol estrogen receptor (RE) from mouse Leydig cell tumors was investigated. When RE from tumors which were affected in their growth by estrogen was heated at 25 C for 30 min in high ionic strength buffer it was converted from native 4.0S to a transformed (5.3S) state. Dialysis of the cytosol from these tumors at 0-4 C in either the presence or absence of estradiol also resulted in conversion to 5.3S when estradiol was added before the sample was applied to the sucrose density gradient. When the dialyzed cytosol was applied to a sucrose density gradient without estradiol, the peak of estrogen binding capacity tested by adding E2 to the fractions from the gradient, occurred at approximately 4S. The dialysate of cytosol had an inhibitory effect on activation by warming. Dialysis of cytosol in the presence of estradiol enhanced the translocation of estradiol-receptor complex to nuclei. Molecular weight (approximately 128,000) and frictional coefficient (f/f0 1.62) of the estrogen-receptor complex dialyzed in the presence of high salt concentration were identical with those of warmed RE complex, but quite different from the native RE (approximately 73,000 daltons, frictional coefficient 1.48). This evidence indicates the presence in the cytosol of one or more small molecules which, when noncovalently bound to RE, inhibit the conversion of the native cytosol RE to the transformed state R'E2RE, having an additional protein unit.

Effect of colchicine on estrogen action II. Translocation of 17 β-estradiol cytosol receptor complex into the nucleus

Colchicine has previously been shown in our laboratory to inhibit 17 β-estradiol stimulation of uterine water uptake in the immature rat measured 6 h after administration of the agents. We sought to determine whether this effect was mediated through colchicine action on translocation of estradiol receptor complex into the uterine cell nucleus. The time course of estradiol effect on uterine water uptake was followed with and without concurrent colchicine administration up to 6 h after administration. At no time during this period did there appear to be any influence of colchicine on translocation of the estradiol receptor complex into the nucleus. Examination of physical chemical characteristics of the nuclear estradiol receptor complex after estradiol and estradiol plus colchicine treatments revealed no observable differences. Thus, colchicine inhibition of estradiol-stimulated uterine water retention does not appear to be mediated through inhibition of nuclear translocation of estradiol-receptor complex nor to be due to any reduced retention time of estradiol-receptor complex in uterine nuclei.

Oestradiol-receptor complexes in subnuclear fractions of rat uterine tissue.

The subnuclear distribution of 3H-oestradiol-receptor complexes was studied in uterine tissue of ovariectomized adult rats. Nuclei were sonically disrupted and 8 different subnuclear fractions were isolated by discontinuous sucrose density gradient centrifugation. 3H-Oestradiol-receptor complexes, measured by hydroxylapatite column chromatography, were localized in a light chromatin fraction as well as in a heavy chromatin fraction. Using the hydroxylapatite chromatography technique it was possible to demonstrate three classes of oestradiol-receptor complexes which differ in affinity for the chromatin. Oestradiol-receptor complexes with a high affinity for the chromatin were predominantly localized in the heavy chromatin fraction, whereas complexes with a lower affinity for their acceptor sites were present in the lighter chromatin fraction.

Analysis of [ 3H]estradiol binding to nuclei prepared from epididymides of sexually immature intact rabbits

The estrogen receptor that is present in the epididymis of sexually immature rabbits is capable of entering nuclei in the time- and temperature-dependent manner characteristic of such reactions. Nuclear translocation reaches a maximum after 1 h of incubation at 23°C. Even short incubations (15 min) at 37°C destroy the cytoplasmic receptor and as a consequence nuclear translocation at this temperature is precluded. We have noted that a small, but demonstrable, amount of receptor enters nuclei at 0°C. Nuclear translocation is absolutely dependent upon estradiol binding to the receptor as shown by the fact that inhibitors of estradiol binding to the receptor (unlabeled estradiol, estrone and estriol) prevent translocation. Compounds that do not prevent estradiol binding to the receptor (5α-dihydrotestosterone, progesterone and cortisol) have no effect on the translocation of the estradiol-receptor complex to nuclei. Nuclei incubated with [ 3H]estradiol in the absence of receptor did not take up the hormone. Nuclear binding of estradiol falls into two categories: (a) that which is readily extractable with 0.4 M KCl; and (b) that which is resistant to KCl extraction. The resistant fraction is further subdivided into a fraction from which label can be readily extracted with ethanol and an ethanol-resistant residual fraction. The KCl-soluble fraction represents about 30% of the total radioactivity associated with nuclei. Acceptor sites for the estradiol-receptor complex are not limited to target cell nuclei since the complex is apparently bound to nuclear constituents in two presumptive nontarget tissues (spleen and skeletal muscle). However, the number of acceptor sites in target cell nuclei is demonstrably greater than in nontarget cell nuclei.

Estradiol receptor of calf uterus: interactions with heparin-agarose and purification.

Heparin attached covalently to agarose beads binds the "native" form of the estradiol receptor with very high affinity. Chondroitin sulfate does not bind to the receptor. When the receptor is complexed with hormone, the affinity is at least 10 times higher. Only the "native" and not the "nuclear" or the "derived" (i.e., after activation by a calcium-dependent enzyme) forms of the estradiol receptor interact with heparin. The "native" estradiol-receptor complex is purified to homogeneity after chromatography on columns of heparin-agarose, Sephadex G-200, and DEAE-cellulose, followed by two more Sephadex G-200 columns. The purified molecule is a single polypeptide of molecular weight 69,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The sedimentation coefficient on sucrose gradients is 4.3 S, the Stokes radius from gel filtration is 36.5 A, and the isoelectric point is 6.4. The purified [3H]estradiol-receptor complex exchanges the radioactive hormone with estradiol or other estrogenic steroids, but not with testosterone, 5alpha-dihydrotestosterone, or progesterone.

Development of a microassay for estradiol receptors.

The calculation of equilibrium binding constants for a specific receptor-hormone interaction requires the exact determination of the unbound ligand concentration and the specifically bound concentration at equilibrium. These determinations usually require corrections for the contribution of non-specific binding. We introduce several parameters allowing equilibrium concentrations to be calculated from experimental concentration values; these parameters can be measured for the particular receptor assay procedure used. These parameters are used in a microassay of estradiol-receptor complex by selective retention on DE-81 cellulose paper.

Antibodies to estrogen receptor: Immunochemical similarity of estrophilin from various mammalian species

Immunoglobulin obtained from the serum of rabbits immunized with a highly purified preparation of estradiol-receptor complex from calf uterine nuclei has been shown to contain specific antibodies to the receptor protein (estrophilin) by four criteria: (a) precipitation of the radioactive steroid upon addition of goat antibody against rabbit gamma globulin to a mixture of the tritiated estradiol-receptor complex and the immunoglobulin, (b) adsorption of the estradiol-receptor complex by the immunoglobulin linked to Sepharose, (c) adsorption of the estradiol-receptor complex in the presence of the immunoglobulin by Staphylococcus aureus protein-A linked to Sepharose, and (d) the ability of the immunoglobulin to increase the sedimentation rate of the estradiol-receptor complex. Antibodies to calf nuclear estrophilin were shown to crossreact with the nuclear receptor of rat uterus, as well as with the extranuclear receptor of calf, rat, mouse, and guinea pig uterus and of human breast cancer. The antibodies do not react with either the nuclear or extranuclear dihydrotestosterone-receptor complexes of rat prostate or with the extranuclear progesterone-receptor complex of chick oviduct. These findings indicate an immunochemical similarity among estrophilins from several mammalian species, as well as between extranuclear and nuclear forms of the receptor, but not among receptor proteins for different steroid hormones.