Ligand-selective interdomain conformations of estrogen receptor-alpha

Selective estrogen receptor modulators (SERMs) inhibit estrogen activation of the estrogen receptor (ER) in some tissues but activate ER in other tissues. These tissue-selective actions suggest that SERMs may be identified with tissue specificities that would improve the safety of breast cancer and hormone replacement therapies. The identification of an improved SERM would be aided by understanding the effects of each SERM on the structure and interactions of ER. To date, the inability to obtain structures of the full-length ER has limited our structural characterization of SERM action to their antiestrogenic effects on the isolated ER ligand binding domain. We studied the effects of estradiol and the clinically useful SERMs 4-hydroxytamoxifen and fulvestrant on the conformation of the full-length ERalpha dimer complex by comparing, in living human breast cancer cells, the amounts of energy transfer between fluorophores attached to different domains of ERalpha. Estradiol, 4-hydroxytamoxifen, and fulvestrant all promoted the rapid formation of ERalpha dimers with equivalent interaction kinetics. The amino- and carboxyl-terminal ERalpha domains both contain activation functions differentially affected by these ligands, but the positions of only the carboxyl termini differed upon binding with estradiol, 4-hydroxytamoxifen, or fulvestrant. The association of a specific ERalpha dimer conformation with the binding of ligands of different clinical effect will assist the identification of a SERM with optimal tissue-selective estrogenic and antiestrogenic activities. These studies also provide a roadmap for dissecting important structural and kinetic details for any protein complex from the quantitative analysis of energy transfer.     

A monoclonal antibody to the estrogen receptor inhibits in vitro criteria of receptor activation by an estrogen and an anti-estrogen

The effect of an IgM class monoclonal antibody (B36) (Greene, G. L., Fitch, F. W., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 157-161) raised against the calf uterine estrogen receptor was tested in vitro on certain parameters of estrogen receptor activation by estradiol or 4-hydroxytamoxifen, a potent anti-estrogen. The following results were obtained. The antibody prevented the decrease in the dissociation rate of the receptor-estradiol complex which results from activation of the complex, whereas it did not affect the dissociation rate of the receptor-4-hydroxytamoxifen complex, which remains unchanged upon activation. The antibody also increased the dissociation rate of the preactivated receptor-estradiol complex. The antibody protected the naked estrogen receptor against heat-inactivation. B36 partially inhibited the binding of the estradiol- and 4-hydroxy-tamoxifen-receptor complexes to DNA adsorbed onto cellulose, but did not reverse the receptor-DNA binding. This inhibition was not overcome by higher DNA concentrations and was more pronounced for the receptor interacting with estrogen than with anti-estrogen. All these effects were specific since they were related to antibody/antigen recognition and were dose-dependent. These results indicate that the binding of the antibody to the estrogen-activated receptor induces a conformational change in the receptor and that the antibody can prevent and overcome the effect of activation whatever its mechanism. They also confirm that the conformations of the estrogen receptor differ when bound to estradiol or to 4-hydroxytamoxifen.     

Hydrodynamic characterizations of estrogen receptors complexed with [3H]-4-hydroxytamoxifen: evidence in support of contrasting receptor transitions mediated by different ligands

Size-exclusion high-performance liquid chromatography was used to characterize the hydrodynamic molecular properties of estrogen receptors complexed with estradiol and the antiestrogen 4-hydroxytamoxifen. Cytoplasmic estrogen receptors complexed with [3H]-4-hydroxytamoxifen did not undergo reductions in hydrodynamic size after exposure to KCl or urea. Nuclear receptors complexed with 4-hydroxytamoxifen eluted as hydrodynamically larger molecules than nuclear receptors complexed with estradiol. Because identical hydrodynamic characterizations were obtained with the covalent ligand [3H]tamoxifen aziridine, these differences in chromatographic behavior are due to differences in ligand-mediated receptor properties and are not the result of ligand dissociation. When estrogen receptors, complexed with either [3H]estradiol or [3H]-4-hydroxytamoxifen, were exposed to trypsin, the receptors complexed with 4-hydroxytamoxifen eluted as larger hydrodynamic forms than receptors complexed with estradiol. These observations are interpreted to indicate that estradiol and 4-hydroxytamoxifen mediate contrasting transitions in the molecular orientation of estrogen receptors. The consequences of the transitions mediated by 4-hydroxytamoxifen appear to be that intermolecular associations become difficult to disrupt with KCl or urea and that the accessibility of trypsin-sensitive proteolytic sites becomes altered. Chromatin fractionation using DNase I and hypotonic Mg2+ solubilization identified a chromatin region that was less readily penetrated by receptors complexed with 4-hydroxytamoxifen than receptors complexed with estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies

Two monoclonal antibodies (D547 and H222), obtained against the estrogen receptor from MCF-7 breast cancer cells, were used to study the estrogen receptor from fetal guinea-pig uterus bound to estradiol or to the antiestrogens tamoxifen and 4-hydroxytamoxifen. The estradiol-receptor complex binds partially to the monoclonal antibody D547, shifting its sedimentation coefficient in high salt sucrose density gradients from 4.5S to 7.5S. Recently, we demonstrated that the form selectively recognized by this monoclonal antibody is the activated form of the receptor. The estrogen receptor complexed with tamoxifen or 4-hydroxytamoxifen is also partially recognized by this monoclonal antibody but the fraction of total receptor bound to the antibody is significantly less than for the receptor complexed with estradiol. Another series of experiments showed that the monoclonal antibody H222, which recognizes a different antigenic site on the receptor molecule, binds all the estradiol-receptor complex (independently of the degree of activation), shifting its sedimentation coefficient to 7.5S. However, even if all the 4-hydroxytamoxifen-receptor complex is bound by this antibody, only a fraction of the receptor is recognized when it is complexed with tamoxifen. These data show different interactions between the estradiol-, tamoxifen- and 4-hydroxytamoxifen-receptor complexes and the two monoclonal antibodies tested and suggest that these compounds induce different conformational modifications of the estrogen receptor molecule.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Similarities and differences of the binding of estradiol and 4-hydroxytamoxifen (an antiestrogen) in the chick oviduct cytosol

The binding characteristics of [³H]estradiol and 4-[³H]hydroxytamoxifen (a powerful estradiol antagonist) in the chick oviduct cytosol was analyzed by sucrose gradient centrifugation and dissociation kinetics experiments at 28°C. Heating the cytoplasmic estradiol-estrogen receptor complexes led to the ‘transformation’ of the receptor; as with the estrogen receptor in other target tissues and species, the transformed receptor sedimented in the 5 S region of sucrose gradients containing 0.4 M KCI and had a slower rate of dissociation of bound estradiol. Upon heating, the cytoplasmic 4-hydroxytamoxifen complexes also appeared to undergo similar changes in their physical states as analyzed by sedimentation rates and dissociation kinetics, and we conclude that antiestrogen can transform the receptor. Sodium molybdate inhibited the temperature mediated changes with both estrogen and antiestrogen complexes. Slight but consistent differences in the sedimentation coefficient and rate of ligand dissociation were observed between the complexes formed by estradiol and 4-hydroxytamoxifen but the relevance to opposite biological activities remains unknown.     

Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites

New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.     

Binding of estradiol-peroxidase conjugate to estrogen receptor

The binding of estradiol-horseradish peroxidase conjugate to rat uterine cytosolic estrogen receptor was studied. The conjugate having a steroid to enzyme ratio of 2.8:1 was allowed to bind to protamine precipitated receptor in presence or absence of 100-fold excess of free estradiol. The bound enzyme activity was measured and the data subjected to Scatchard analysis to obtain the dissociation constant and the number of binding sites. Although the binding parameter so obtained differed from values obtained using radiolabelled estradiol, the method may be used for comparative studies.     

Identification of uterine nuclear type II estrogen binding sites in estrogen treated rats

Uterine nuclear fractions from estrogen-treated rats contain both the estrogen receptor and a lower affinity estrogen binding site (type II site). In Scatchard plots of estrogen binding, two types of curves are seen. The hook-shaped form is composed of a linear component (the estrogen receptor) and a convex component (the type II site) while the curvilinear form is resolvable into two linear binding species (the estrogen receptor and a secondary site). To clarify the relationship between the two forms, we examined the curvilinear form from immature rats injected for 4 days with estradiol (E2) for type II site properties. Like the hook-shaped type II, this form could be detected in a nuclear exchange assay at both 37 and 4 degrees C, but at neither temperature in the presence of reducing agent. Additionally, the steroid specificity of the curvilinear form was identical to the hook-shaped form. The hook-shaped form was found in both immature and ovariectomized adult rats implanted for 6 days with an E2-releasing Silastic capsule to provide pharmacological E2 levels. When uteri from implanted animals displaying the hook-shaped form were mixed in various ratios with uteri lacking type II sites, the curvilinear form was produced. Animals given an E2 implant for 3 days, followed by a 3 day hormone-free period showed a curvilinear form. In vivo E2 dose-response experiments showed the curvilinear form at low E2 doses and the hook-shaped form at the high dose and in implanted animals. We conclude that curvilinear Scatchard plots result from the presence of authentic type II at lower concentrations than those giving rise to the hook-shaped form.     

Catecholestrogens are MCF-7 cell estrogen receptor agonists

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to effect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10⁻⁸ M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). Form these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.     

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.     

A male specific hepatic estrogen binding protein: characteristics and binding properties

Mammalian liver is a sex-steroid responsive tissue in that androgen and estrogen receptors are present and mediate differential hepatic hormonal effects. Further, we and others have found a sexual dimorphism in the hepatic cytosolic content of estrogen binding proteins. In addition to the estrogen receptor, the male has a high-capacity (12.0-15.0 pmol/mg protein) estrogen binding protein (MEB) which demonstrates a moderate affinity for estradiol (Kd = 31.0-43.2 nM) if estradiol metabolizing enzymes are first precipitated with protamine sulfate. This protein exhibits a unique specificity for steroidal estrogens: 2-methoxyestriol greater than estradiol greater than estriol = 2-methoxyestradiol greater than 2-hydroxyestradiol greater than estrone greater than 2-methoxyestrone greater than estriol 3-glucuronide greater than 2-hydroxyestrone = 3-methoxyestriol greater than androstanediol greater than dihydrotestosterone greater than testosterone. Other androgens such as androstenedione and methyltrienolone, nonsteroidal estrogens such as diethylstilbestrol, and the antiestrogens tamoxifen and 4-hydroxytamoxifen do not compete for [3H]estradiol ([3H]E2) binding. MEB is a relatively small-molecular-weight protein with a Sr of 20.4 A as determined by gel filtration on Sephadex G-100. The kinetics of [3H]E2 association and dissociation at 4 degrees C are very rapid, with t1/2 values of less than 5 s. Sodium molybdate, generally used to stabilize steroid receptors, inhibits MEB-[3H]estradiol binding activity in cytosol in a time- and dose-dependent manner, an effect not observed with partially purified MEB. Magnesium chloride inhibits binding activity of the Sephadex G-100 MEB pool, an effect reversed by EDTA. Other divalent cations also inhibit binding: Mn2+ greater than Mg2+ greater than Ca2+. Furthermore, EDTA complexes of these cations slightly enhance binding relative to EDTA alone: Ca2+ EDTA greater than Mg2+ EDTA greater than Mn2+ EDTA. These results demonstrate that MEB is a unique sex-steroid binding protein, albeit of unknown function, which is distinct from hepatic steroid receptors.     

Synthesis and estrogen receptor affinity of a 4-hydroxytamoxifen-labeled ligand for diagnostic imaging

A 10-step synthesis of a novel 4-hydroxytamoxifen-DTPA ligand (HOTam-DTPA) is reported. Tamoxifen and its primary metabolite 4-hydroxytamoxifen are common estrogen receptor ligands. Consequently, tamoxifen has found utility as the targeting component of various diagnostic agents for selective imaging of estrogen receptor-rich tissue, specifically breast cancer. An L-aspartic acid-derived DTPA analogue was attached to the ethyl side chain of 4-hydroxy-tamoxifen using N,N'-dimethylethylenediamine as a hydrophilic linker. A competitve estrogen receptor binding assay using [3H]-17beta-estradiol was performed to determine the effect of the ethyl side chain modification on estrogen receptor affinity. The results show that while the relative affinity of HOTam-DTPA for the estrogen receptor is approximately 10-fold lower than that of tamoxifen, it still remains a potent ligand at relatively low concentrations.