Estrogen Receptor Characterization Following Selective Sedimentation Separation of Estrogen-Binding Components In Immature Rat Uterine Cytosol

Abstract

Characterization of a specific estrogen receptor (ER) in fetal and early postnatal rat uterine cytosol is complicated by the presence of other high-affinity estrogen-binding components, such as alpha-fetoprotein (AFP). In an attempt to circumvent their influence, we have employed the selective sedimentation of unlabeled cytosol through sucrose gradients, followed by the analysis of [³H]estradiol binding to a pool of fractions comprising the ER region, as well as to individual gradient fractions. As the amount of AFP present in 21-day-old rats is sufficiently low to permit ER characterization by conventional methodology, we have validated the selective sedimentation method by comparing its results with those obtained conventionally. Though conventional gradient analysis revealed only one estrogen-binding component, saturation and binding inhibition analyses indicated the presence of multiple components, identified as AFP and the ER. These conclusions were supported by results from labeling individual gradient fractions obtained following selective sedimentation of unlabeled cytosol. Further, when unlabeled 7–9 S gradient fractions were pooled and assayed by saturation and binding inhibition analyses, only one binding component, with ER characteristics, was revealed. These results validate selective sedimentation as an effective method for separating multi-component estrogen-binding systems and suggest its applicability to similar systems.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.     

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed.Besides the 8 S cytosol estrogen $\text{receptor}$, there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α₁-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the $\text{receptor}$ increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α₁-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components.During the course of these experiments, it has been observed that the increase of the estradiol $\text{receptor}$ is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.     

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.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.     

The in vivo regulation of the progesterone "receptor" in guinea pig uterus: dependence on estrogen and progesterone

An assay for quantifying the high affinity progesterone binding protein in guinea pig uterine cytosol was developed using Florisil to separate bound and free steroid. The activity of the progesterone binding protein increased between 4–12 hours following estrogen administration and by 4 days of treatment was 10-fold higher than castrate controls. When estrogen administration was discontinued the progesterone binding activity declined slowly with a half-life of 3 days. By contrast, progesterone treatment caused a rapid decline of binding activity within 3 hours. These studies suggest that the antagonistic actions of estrogen and progesterone determine the quantity of available progesterone binding sites in guinea pig uterine cytosol.     

Further characterization of estrogen binding to rat testis cytosol

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.     

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90,000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.     

A specific glucocorticoid binding macromolecule of rabbit uterine cytosol

A high affinity (K_d=2.7 × 10^?10M at 0°) dexamethasone binding macro-molecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 17β. The binding component has a sedimentation coefficient of 8S and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4S. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol.     

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime — bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound protein was recovered by incubating the adsorbent with 2-mercaptoethanol. Crude antibody and uterine cytosol was prepared for affinity chromatography in buffer containing 10^?3 to 10^?2M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S ag SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200–300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.     

Effect of active immunization against 17 beta-estradiol on cytosolic estrogen receptors in the rat uterus

Following active immunization of female rats against estradiol-17 beta, the amount of specific binding sites for estrogen decreased in uterine cytosol as a function of antiserum titres. They were undetected when antibodies titres were higher than 1/2000. Moreover, a binding protein specific for estradiol-17 beta appeared. Estradiol binding was not displaced with an excess of unlabeled DES nor precipitated with protamine sulfate. The sedimentation coefficient of the hormone-protein complex (7-8 S) was not modified in medium of high ionic strength (0.4 M KCl). That protein represented antibodies to Estradiol-17 beta which could be precipitated with antiserum to rat IgG.     

Comparative analysis of the interaction of various estrogens with the estrogen-receptor system of the uterus

Equilibrium binding of labeled estron, estradiol, estriol and DES was studied in uterine cytosol of immature Wistar rats. The dissociation kinetics of the ligand complexes with specific high affinity sites suggested the homogeneity of estrogen receptors in rat uterine cytosol. The feasibility of intracellular regulation of estrogen action in target cells both at the receptor and post-receptor levels is discussed.     

Interaction of estrogen receptor of calf uterus with a monoclonal antibody: probing of various molecular forms

A monoclonal antibody to estrogen receptor (JS34/32) is able to recognize, in the calf uterine cytosol, a protein (approximately 65 000 daltons) giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two molecules of this antibody are able to simultaneously interact with the native 8S form of the receptor present in the calf uterine cytosol ("twin antibody" assay). This indicates the presence of two antigenic determinants on the "low-salt" 8S form of the receptor. This form of the receptor shows an increase in Mr from 345 000 to 665 000 after interaction with the soluble antibody. Dissociating agents that induce the dissociation of the 8S form to smaller forms also induce the dissociation of the two antigenic determinants. The 4S "high-salt" form of the estrogen receptor has one determinant per molecule, appearing to be the smallest form of the receptor not containing repetitive structures associated with the steroid binding site. The nuclear receptor also shows the presence of more than one antigenic determinant on its molecule.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Differences in the form of the salt-transformed estrogen receptor when bound by estrogen versus antiestrogen

Our laboratory has previously reported that antiestrogen binding to molybdate-stabilized non-transformed estrogen receptor results in a larger form of the receptor in 0.3 M KCl when compared with estrogen bound receptor. Estradiol promoted the formation of monomers in the presence of 0.3 M KCl whereas antiestrogen appeared to promote dimer formation. We have extended these studies examining the rabbit uterine salt-transformed estrogen receptor partially purified by DEAE-cellulose chromatography. We previously demonstrated that estrogen receptor prepared in this way bound to different sites on partially deproteinized chromatin subfractions or reconstituted chromosomal protein/DNA fractions when the receptor was complexed with estrogen vs antiestrogen. Analysis of these receptor preparations indicated that DEAE-cellulose step-elution resulted in a peak fraction which sedimented as a single 5.9S peak in 5-20% sucrose density gradients containing 0.3 M KCl for receptor bound by the antiestrogens H1285 and trans-hydroxytamoxifen. However, receptor bound by estradiol sedimented as 4.5S. These receptor complexes bound DNA-cellulose indicating that these partially purified receptors were transformed. DEAE rechromatography or agarose gel filtration of the partially purified antiestrogen-receptor complexes resulted in significant dissociation of the larger complex into monomers. Incubations of 5.9S antiestrogen-receptor complexes with antibodies against nontransformed steroid receptor-associated proteins (the 59 and 90 kDa proteins) did not result in the interaction of this larger antiestrogen-receptor complex with these antibodies (obtained from L. E. Faber and D. O. Toft, respectively). Our results support the concept that antiestrogen binding induces a different receptor conformation which could affect monomer-dimer equilibrium, thus rendering the antiestrogen-receptor complex incapable of inducing complete estrogenic responses in target tissues.     

A manganese catalyzed covalent binding of estradiol - 17 beta to cytosol proteins of rabbit uterus.

Studies of the temperature sensitivity of estradiol receptor binding in rabbit uterine cytosol have revealed the existence of an enzyme which catalyzes the covalent binding of estradiol to cytosol proteins. A fraction, prepared by chromatography on Biogel P-200 and incubated at 37 degrees C in the presence of Mn++, exhibited a time-dependent, temperature-sensitive, oxidative binding of estradiol not seen in the crude cytosol preparation. Although the activity of this enzyme was shown to be independent of estradiol binding by the high affinity estrogen receptor, its presence may complicate studies of estrogen receptor action which involve the use of elevated temperatures.