The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Sex hormone binding globulin expression and colocalization with estrogen receptor in the human Fallopian tube.

The detection of sex hormone binding globulin (SHBG) or SHBG mRNA in several sex steroid target tissues, has raised the possibility that SHBG modulates the action of sex steroids outside the vascular compartment. The presence of SHBG mRNA was investigated by RT-PCR in the poly (A+) RNA fraction of the human Fallopian tube. Human and rat liver were used as positive and negative control tissues, respectively. The electrophoretic analysis of the amplified PCR products showed bands at 219 bp, corresponding to the expected size of the SHBG cDNA, in the Fallopian tube and human liver but not in rat liver, indicating that SHBG might be synthesized by the Fallopian tube. The cellular localization of SHBG and of estrogen receptor (ER) was examined by immunohistochemistry in consecutive sections of Fallopian tube tissues for individual staining or double immunostaining in the same section. Specific immunostaining of SHBG was present in the epithelial, vascular and muscle cells of the ampullary and isthmic region. In epithelial cells, immunoreactive SHBG was present in the apical end with the highest concentration close to the luminal membrane. The ER was localized in the nuclei of epithelial, stromal and muscle cells of the ampulla and isthmus. Double immunostaining showed that SHBG and ER are colocalized principally in epithelial cells of the ampulla and in muscle cells of the isthmus. In conclusion, the detection of SHBG and SHBG mRNA and the localization of SHBG in estrogen target cells was shown. These findings support the hypothesis that SHBG might regulate sex steroid action at the tissue level.     

The effects of various serine protease inhibitors on estrogen receptor steroid binding

Two serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP), were utilized to investigate the possible involvement of serine hydroxyl groups on 17 beta-estradiol binding to the rat estrogen receptor (ER). Single point saturation analysis and Scatchard analysis demonstrated that both 5 mM PMSF and 5 mM DFP were able to inhibit steroid binding to the ER after incubation at 37 degrees C, but neither were able to inhibit steroid binding of the nonactivated ER (0-4 degrees C). The reducing agent dithiothreitol (DTT) was used to differentiate between the interaction of PMSF with serine groups or with sulfhydryl groups of the receptor. When incubated in the presence of 5 mM PMSF, various concentrations of DTT up to 25 mM were not able to overcome the inhibition of this agent, indicating that there was no interaction of PMSF with sulfhydryl groups. Thus, these findings indicate that serine hydroxyl groups are involved in steroid binding of the rat ER.     

Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts

Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.     

Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.     

HPLC separation of anti-estrogen and estrogen receptor binding components of mouse plasma

Proestrous mouse plasma and urine were subjected to diethyl ether extraction, enzyme hydrolysis and HPLC separation of estrogen components. Radioimmunoassay of the treated proestrous samples with a broad spectrum anti-estrogen serum failed to detect estradiol-17 beta, estrone or estriol. HPLC chromatograms contained two peaks of immunoreactive and estrogen receptor binding material with polarities between those of estriol and estradiol-17 beta. Similar peaks were detected in HPLC chromatograms of urinary extracts from ovariectomized and ovariectomized-adrenalectomized mice. The least polar of the two peaks produced a mass spectrum identical to that of authentic equol [7-hydroxy-3-(4'-hydroxyphenyl)chroman], a phytoestrogen metabolite. The presence of significant quantities of circulating equol in all strains studied, combined with apparently low plasma levels of endogenous classical estrogens during proestrus, confound attempts to study estrogen secretion in the mouse.     

Characterization of estrogen binding in the developing rat testis. Ontogeny of the testicular cytoplasmic estrogen receptor.

The properties and physical characteristics of a steroid binding component present in the immature (7 to 35 day) rat were investigated and found to be different from those of the 17 beta-estradiol receptor in the mature rat testis. These properties include a binding capacity of 483 fmol estradiol/mg protein, a Ka at equilibrium of 4.23 x 10(7)M-1, and broad steroid specificity as shown by interaction with several steroids; no binding was observed with diethylstilbestrol. The component, found in blood and several tissues including the testis, migrated as a 4.6S peak on sucrose gradients. This 4.6S component, which interacted with an anti-alphafetoprotein antiserum, decreased with age and was not detectable in the testis after day 21 or in the serum after day 25. These data suggest that this component is alphafetoprotein. Ontogenic appearance of the testicular cytoplasmic 17 beta-estradiol receptor in the developing rat was further elucidated. Sucrose gradient sedimentation analysis of cytosols revealed an 8S binding component that was first detectable at 23 days. Specific binding (fmol [3H]-estradiol/testis) was relatively low in neonates, rose to 59 fmol during the third week, and increased dramatically to 333 fmol at 7 weeks; binding rose only gradually after maturity. The receptor was tissue specific and steroid specificity studies demonstrated that only diethylstilbestrol and other estrogens were effective in competing with 17 beta-estradiol for binding sites. The Ka at equilibrium was determined as 3 x 10(10)M-1 and the binding sites were saturable in an in vitro system. The receptor did not interact with anti-alphafetoprotein antiserum as indicated by sucrose gradient studies. These data demonstrate the developmental appearance of the testicular cytoplasmic estradiol receptor in the immature rat.     

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.     

The binding of estrogen and estrogen antagonists to the estrogen receptor.

The model of the estrogen receptor as a dimer of identical, interacting subunits and data obtained by Sasson and Notides (1988, Mol. Endocrinol. 2, 307-312) were used to find the standard free energy changes that describe the binding of estradiol and 4-hydroxytamoxifen to the estrogen receptor. For the binding of estradiol or 4-hydroxytamoxifen to the estrogen receptor the data do not deviate systematically from the best fit to the model. The standard free energy change for binding of one molecule of estradiol at one site and one molecule of 4-hydroxytamoxifen at the second site of estrogen receptor indicates that 4-hydroxytamoxifen antagonizes the binding of estradiol to the estrogen receptor.     

Preferential oxidation of zinc finger 2 in estrogen receptor DNA-binding domain prevents dimerization and, hence, DNA binding

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.     

Characterization of estrogen binding proteins in mouse liver cytosol: absence of sexual dimorphism

Estrogen binding proteins in mouse liver cytosol were characterized by separation on Sephadex G-75 columns, by Scatchard plot analysis, and by hormonal competition studies. A high affinity receptor (56-70 fmol/mg cytosolic protein) with a mol. wt greater than 75,000, Kd of 5.7-8.4 X 10(-10) M was identified in male and female C3H liver. A second high capacity low affinity (HCLA) binder (200-300 fmol/mg cytosolic protein) with a mol. wt of about 50,000, Kd of 1.7-7.2 X 10(-8) was also identified. Following partial purification of the estrogen binders by ammonium sulfate precipitation, Scatchard plot analysis revealed selective removal of HCLA. On Sephadex G-75 filtration, the purification also resulted in selective removal of the 17 beta-estradiol binding component with a mol. wt of 50,000. Comparison with rat cytosol separations show that the sexual dimorphism in HCLA binding proteins (5 times higher in male than female rat liver) was absent in the mouse liver. These studies document the presence of a specific high affinity estrogen binding protein in mouse liver and indicate that the sexual dimorphism in HCLA proteins is not a universal feature of all rodent species.     

Effects of estrogen and growth hormone on steroid sulfatase activity and estrogen binding in rat liver

It is now well established that the activity of certain liver enzymes displays sex differences and that administration of human growth hormone to male rats alters the liver metabolism in a "female" direction. In this work we studied steroid sulfatase activity and binding of estradiol-17 beta in livers from intact rats and found a sex difference, with considerably higher enzyme activity in male as compared to female liver tissue. Continuous infusion of native and recombinant human growth hormone and estradiol-17 beta to male rats reduced sulfatase activity to "female" levels. A specific binding of estradiol-17 beta with receptor properties was found in the rat livers, but the concentration of binding sites did not change after administration of growth hormone or estradiol in this group of intact animals. Our data confirm previous reports that continuous administration of human growth hormone "feminize" liver metabolism, and since estradiol was found to have an identical effect on sulfatase activity it is suggested that the effect of estradiol-17 beta in this respect may be indirect, mediated via an altered secretory pattern of rat growth hormone.