Activation of [3H]estradiol--and [3H]H1285-receptor complexes: effect of salt versus ATP on molybdate-stabilized estrogen receptors

The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes.     

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.     

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.     

The effects of the high affinity antiestrogen, H1285, on uterine growth and morphology

Experiments were performed to compare the agonistic and antagonistic properties of the high affinity antiestrogen, H1285, and the low affinity antiestrogens CI-628 and tamoxifen on rat uterine growth and morphology. Myometrial and stromal areas and endometrial epithelial cell heights were calculated. Whereas all antiestrogens displayed some degree of estrogenic response, only H1285 at 5 or 0.5 micrograms was an effective inhibitor of E2 induced proliferative responses in the myometrium, stroma, and endometrial epithelium. All antiestrogens caused tremendous endometrial hypertrophy.     

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin.

Various aspects of the interaction of oestrogen-receptor complexes with calf uterine chromatin covalently coupled to cellulose were analysed. Partially purified [3H]oestradiol-receptor complexes were bound to intact, or partially deproteinized, chromatin resins. Proteins were removed from the chromatin-cellulose resins by extraction with high molarities of salt, including NaCl/urea, guanidine hydrochloride and guanidine thiocyanate. After extensive washing to remove the salt, [3H]oestradiol-receptor-complex solutions were added to the resins and the degree of binding was determined. The extent of [3H]oestradiol-receptor-complex binding to chromatin was enhanced by extraction of chromosomal proteins. By varying the molarity of the salt, and consequently the extent of protein removal, it was possible to resolve [3H]oestradiol-receptor-complex binding to guanidine thiocyanate-extracted chromatin into two components. Similarly, [3H]oestradiol-receptor-complex binding to guanidine hydrochloride-treated chromatin included three regions of enhanced binding capacity. The [3H]oestradiol-receptor-chromatin interaction was saturable with respect to both intact and salt-extracted resins. Thus uterine chromatin may contain three or more specific classes of acceptors for the oestrogen-receptor complex.     

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.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

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.     

Inability of [3H]estriol to induce maximal cooperativity of the estrogen receptor

The interaction of [³H]estriol with the partially purified estrogen receptor from calf uterus shows positive cooperativity that is dependent upon receptor concentration and temperature. At a receptor concentration of 1 nM and 25°C the [³H]estriol-receptor cooperativity was low, the Hill coefficient (n_H) was 1.03 ± 0.02; however, with increasing receptor concentrations the receptor's cooperativity increased until at approximately intracellular receptor concentration (20 nM) the n_H = 1.20 ± 0.04. At 0°C and a receptor concentration of 10 nM the [³H]estriol-receptor interaction was highly cooperative, the Scatchard plot was convex and n_H = 1.58 ±0.04 while at 30°C the Scatchard plot approached linearity and n_H = 1.03 ± 0.02. In comparison, [³H]estradiol was capable of inducing, at 0 or 30°C and at a receptor concentration of 1 nM or greater, maximal receptor cooperativity, n_{H = 1.63}.These data demonstrate: (a) the receptor's conformation and binding mechanism change in a specific manner with temperature, so that receptor analysis at 0°C does not necessarily reflect the receptor's properties at biologically relevant temperatures; (b) the dependence of the receptor's cooperativity on receptor concentration, which suggests interaction between dissociable subunits; and (c) the lower cooperativity induced by estriol, in comparison with estradiol, which indicates estriol is less efficient in shifting the receptor toward a higher affinity or the activated state of the receptor.     

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.     

Sequence analysis of the nonsteroid binding component of the calf uterine estrogen receptor

Microbore reversed-phase high performance liquid chromatography has been utilized to fractionate and purify a number of tryptic peptides generated from the 90K nonsteroid binding component of the calf uterine estrogen receptor. Sequence analysis was performed on six peptides yielding 78 unique amino acid assignments, this corresponds to approximately 10% of the molecule. These peptides share sequence similarities with three heat shock proteins, Drosophila hsp 83 (83% homologous), yeast hsp 90 (55%) and chicken hsp 108 (32%). The amino acid composition of the protein indicates a prevalence of charged amino acid residues.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

Interaction of serine protease inhibitors and substrates with human uterine estrogen receptor

The human uterine estrogen receptor has a site which regulates estrogen binding and which structurally resembles the substrate binding site of chymotrypsin. The hormone binding capacity and the affinity of the receptor is decreased in the presence of 4 mM serine protease inhibitors tosyl-lysine chloromethyl ketone and diisopropylfluorophosphate and the protease substrates tryptophan methyl ester and toluene sulphonyl-arginine methyl ester. The protease inhibitors tosylamide-phenylethyl-chloromethyl ketone and phenyl methyl sulphonyl fluoride caused an increase in the binding capacity whereas the affinity was decreased.     

Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus

The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.     

Interaction of cycloalkanoprogesterones with mammalian progesterone receptor: binding of pregna-D'-pentaranes in the calf uterine cytosol

Pregna-D'-pentaranes (pentaranes) are modified progesterones with demonstrable progestational activity and contraceptive effect. We have examined the steroid binding characteristics of the two newly synthesized progesterone analogs, Pentarane A (16, 17-cyclohexanoprogesterone) and Pentarane B (6-methyl, 16, 17-cyclohexanoprogesterone), and studied the nature of their interaction with progesterone receptor (PR) from the chicken oviduct and the calf uterine cytosols. Pregna-D'-pentaranes exhibited no affinity for the chick PR but interacted with the calf uterine PR as did R5020. The pentaranes, however, bound PR less tightly. R5020- or pentarane-bound PR sedimented as an 8S moiety in 8–30% linear glycerol gradients. Thermal transformation of receptor resulted in the reduction of the 8S form, and caused an increase in the binding of R5020-and progesterone-bound PR complexes to DNA-cellulose. The pentarane-bound PR bound poorly, if at all, to DNA-cellulose. Our data suggest that pentaranes exhibit both similarities and differences with natural and synthetic progestins with respect to their interaction with calf uterine PR. The lack of pentarane binding to chicken PR is reminiscent of the general phenomenon that antiprogestins (RU486, ZK98299, and Org 31710 and Org 31806) do not interact with chicken PR. Pentaranes, therefore, represent unique steroid analogs to investigate the molecular mechanism of steroid hormone action.Abbreviations DMSO Dimethyl sulfoxide - DTT Dithiothreitol - E Estradiol - EDTA Ethylene-diaminetetraacetate - F Cortisol - IA Iodoacetamide - MER -mercaptoethanol - MTG Monothioglycerol - NEM N-ethylmaleimide - Org 31710 (6, 11, 17)-11-(4-dimethylaminophenyl)-6 methyl-4, 5-dihydro[estra-4, 9-diene-17, 2(3H')-furna]-3-one - Org 31806 (7, 11, 17)-11-(4-dimethyl-aminophenyl)-7 methyl-4, 5-dihydro[estra-4, 9-diene-17, 2(3H)-furan]-3-one - P Progesterone - Pentarane A 16, 17-cyclohexanoprogesterone - Pentarane B 6-methyl, 16, 17-cyclohexanoprogesterone - PMSF Phenylmethylsulfonyl Fluoride - PR Progesterone Receptor - R5020 17, 21-dimethylpregna-4, 9(10)-diene-3     

Characteristics of the calf uterine androgen receptor

The highest molecular weight form of the calf uterine androgen receptor separates as an 11S form in glycerol gradients. This "cytosolic" receptor, prepared in the presence of molybdate, polyethyleneimide and low ionic strength, dissociates into 9S and 7.2S forms with increasing KCl concentration. A 4.5S androgen binding component appears as the predominant form of the receptor in the absence of polyethyleneimide and this unit quantitatively converts to a stable 3.5S form in the absence of molybdate. Renaturation of partially purified protein, separated by SDS-PAGE electrophoresis, demonstrates the presence of an androgen binding component in the 110 kDa region of the gel. This renatured protein separates as a 4.5S component in glycerol gradients and has a Stokes radius of 6 nm. Photoaffinity labelling of partially purified receptor preparations, followed by SDS-PAGE electrophoresis, reveals the presence of an androgen binding component having a molecular weight of 115 kDa. The binding characteristics and specificity of the receptor binding to R1881 have been studied and a DHT-affinity chromatography resin used to purify the receptor.