Characterization of the Ah receptor from human placental tissue

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20°C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.     

Biochemical characterization of the progesterone receptor in the chick bursa of Fabricius

In a previous work we demonstrated estrogen-inducible progesterone binding sites in the bursa of Fabricius. In the present study these were characterized and compared to the progesterone receptor (PR) in the chick oviduct. When the size of the binding sites was analyzed with sucrose gradient centrifugation, 2 peaks of bound progesterone were obtained. The sedimentation coefficients of the peaks were 8-9 S and 3-4 S. In size exclusion HPLC only 1 peak was seen with a size corresponding to the 8-9 S in the sucrose gradient. The Stokes radius was 7.7 nm. When the ionic strength was elevated or CaCl2 was added, smaller steroid binding forms were detected. The sizes of these progesterone binding molecules at low and high ionic strength and in the presence of CaCl2 were equal in bursa and oviduct when analyzed with HPLC. The Stokes radii of these forms were 5.6 nm in high salt and 2.1 nm with CaCl2. The steroid binding components in the bursa cytosol eluated as 2 peaks from the DEAE column with KCl gradient. The peaks corresponded to the so-called A and B components in the chick oviduct. In the presence of molybdate, bound progesterone eluated as one peak from DEAE in both oviduct and bursa. The progesterone binding capacity was shown to be heat labile with equal half-lives in the bursa and the oviduct. Progesterone and ORG 2058 had a high affinity for the binding site and their binding was specific for progestins. It is concluded that the estrogen-inducible progesterone binding site in the bursa of Fabricius resembles the oviductal progesterone receptor in structural and binding properties.     

Heterogeneity of Estrogen Binding Sites in Mouse Mammary Cancer

Abstract

The recent demonstration in our laboratory of at least two specific estrogen binding sites in the rat uterus prompted us to investigate similar heterogeneity of binding sites in a trans-plantable ovarian dependent mouse mammary tumor (MXT-3590). Saturation analysis of cytoplasmic (protamine sulfate or hydroxylapatite exchange assay) or crude nuclear fractions (protamine sulfate precipitated nuclear exchange assay) revealed two binding components: type I which conforms to the classically described estrogen receptor and type II which has a lower affinity for estradiol but a greater capacity than type I sites. Exposure of cytosol to charcoal partially removes bound ³H-estradiol from type II sites but not from type I sites. Type II sites are specific for estrogens and do not translocate from the cytoplasmic to the nuclear compartment. Although Type II sites undergo dissociation on prelabeled sucrose density gradients, they are readily demonstrable by postlabeling sucrose density gradient fractions and hydroxylapatite adsorption. Since the presence of type II sites interferes with the measurement of the estrogen receptor (type I) which may also undergo dissociation on sucrose gradients, we recommended that the technique of postlabeling be used for the sucrose gradient analysis of type I and II sites. In addition, saturation assays should be performed over a wide range of ³H-es-tradiol concentrations (0.1–120 nM) for proper evaluation of both sites. These considerations may contribute to more accurate predictions about the response of breast cancers to endocrine therapies.     

A hydroxylapatite microassay for receptor binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene in various target tissues

A "batch" hydroxylapatite assay for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor that does not require detergents is described. The receptor could be assayed in rat target tissues using either of the cytochrome P1-450 inducers [3H]TCDD or [3H]3-methylcholanthrene as radioligands. A phosphate buffer washing procedure was developed on the basis of chromatographic data and optimized to separate nonspecifically and specifically bound ligand. The assay was characterized with respect to washing efficiency, binding specificity, competition, adsorption time, amount of hydroxylapatite required to bind receptor complexes, sensitivity, and effects of detergents. Equilibrium binding parameters were determined. Receptor extracted with phosphate from hydroxylapatite was analyzed on sucrose gradients and was found to exhibit the same sedimentation properties as the receptor in crude cytosol. Furthermore, the applicability of the assay has been demonstrated in cytosolic preparations from three different target tissues: liver, lung, and thymus.     

A specific progesterone receptor of myometrial cytosol from the rhesus monkey.

A specific progesterone receptor of myometrial cytosol from the rhesus monky is described. Characterization of the receptor by sucrose density gradient centrifugation revealed 2 peaks at 4s and 7.5s. The 4s peak seen in all groups (castrate; castrate plus estrogen treated; castrate plus estrogen and progesterone treated) contained little specific progesterone binding but the 7.5s peak, seen only in the estrogen-treated animal, was specific for progesterone. Competition studies revealed the reeceptor affinities to be: progesterone 100, 5alpha dehydroprogesterone 81.9 melengestrol acetate 72.5, norgestrel 53, desoxycorticosterone 25.9, 5beta-dihydroprogesterone 1.2, and 17 hydroxyprogesterone less than 1. Receptor levels measured from Scatchard plot analysis of of equilibrium data were 7 fM/mg cytosol protein (castrate), 45.2 fM/mg (p less than .01, estrogen treated), and 10.5 (estrogen plus progesterone treated). The association constant (approximately 5 x 10(-9)M) was similar in all 3 groups.     

Two binding proteins for progesterone in the bovine corpus luteum

The cytosolic supernatant of bovine corpus luteum contains two proteins which bind progesterone specifically. Bovine luteal cytosol was fractionated on hydroxylapatite and the peaks of protein obtained subjected to equilibrium dialysis against progesterone. Progesterone-binding activities (Ka approx. 10(6) 1/mol) was eluted at 40 mM (Binding Protein 1) and 100 mM phosphate (Binding Protein 2). They sedimented differently (3.95 and 4.65, respectively) on sucrose gradients. In contrast to Binding Protein 1, Binding Protein 2 bound R5020 better than progesterone on sucrose gradients. Purification of the binding activity eluted by 40 mM phosphate from the hydroxylapatite column showed that it resided in a single protein (molecular weight 65,000 daltons). The function of these proteins is presently unknown, but they may participate in the biosynthesis and/or secretion of progesterone from bovine luteal cells.     

Progesterone receptor in human endometrium of leiomyoma uteri

This study was designed to examine whether 8S protein as progesterone receptor exists in the human endometrium which has been primed with estrogen. The kinetic study showed that 8S-progesterone binding was specific with Kd of 2.0 X 10(-9) M. 5S-progesterone binding was inhibited competitively by cortisol. The study of ligand specificity also showed that progesterone and its related steroids had much stronger affinity for 8S component than for 5S component. Therefore, 5S protein may be CBG. Progesterone-8S protein binding was easily dissociated during the 5-20% sucrose gradient centrifugation, but such a protein from which progesterone had been dissociated could be sedimented at 8S region. Glycerol could stabilize progesterone-8S protein binding. These results indicate the existence of 8S protein as a progesterone receptor under the low salt medium in the estrogen primed human endometrium.     

Ovarian hormone receptors in human mammary stromal cells

Mesenchymal cells of the rodent breast express both estrogen and progesterone receptors. Searches for these molecules in the human breast have yielded conflicting results. Following immunohistochemical staining of samples of normal human breast tissue, the authors detected estrogen receptor alpha protein and progesterone receptor protein in extralobular (non-specialized) fibroblasts and estrogen receptor alpha protein in adipocytes. Tissues from young teenage girls and pregnant women contained the greatest number of receptor positive fibroblasts. These observations confirm prior reports of the presence of ovarian hormone receptors in mammary fibroblasts. The findings also illustrate similarities in the organization of the rodent and human breasts and thereby suggest that regulation of the gland by ovarian hormones involves similar mechanisms in both species.     

Recognition of two forms of the estrogen receptor in the guinea-pig uterus at different stages of development by a monoclonal antibody to the human estrogen receptor. Dynamics of the translocation of these two forms to the nucleus

Two forms of the estrogen receptor were recognised in the cytosol fraction of fetal uterus of guinea pig by a monoclonal antibody (D547Spγ) to the human estrogen receptor. It was observed that 60–65% of the total cytosol estrogen receptor (the α form) was bound to the antibody, increasing its sedimentation coefficient in a high ionic strength sucrose gradient (10–30% w/v sucrose, 0.4 M KCl) from 4.5 S to 7.4 S. The remaining fraction (the β form) has the classical sedimentation coefficient of 4.5 S. Dynamic studies of the translocation in vitro of the cytosol receptor to the nucleus as a function of time have shown that the a form decreases sharply while the β form is slightly affected when the cytosol was incubated with the nuclei. In contrast only one form, which is bound totally to the antibody, was found in the nuclear fraction. In addition, the presence of these two forms of the cytosol estrogen receptor was also demonstrated in newborn and immature animals.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Characterization of a Human Serum Inhibitor of Clostridium histolyticum Proteinase(s)

Normal animal sera inhibit at least one Clostridium histolyticum proteinase. An assay procedure based on immune hemolysis was developed for the estimation of this inhibition. This inhibitory activity occurs in various levels in the sera of different animal species. The highest titers have been obtained with rat sera. The inhibitory activity from human serum was isolated and purified 16- to 27-fold by Sephadex G-200 gel filtration and diethylaminoethyl cellulose or hydroxylapatite chromatography. The properties of the human serum inhibitor of the clostridial proteinase were compared with a trypsin inhibiting factor found in the partially purified preparations. Identical behavior of the two inhibitory factors was observed when measured by heat inactivation, beta-mercaptoethanol sensitivity, pH stability, and sucrose gradient centrifugation. The inhibitory factor has an approximate sedimentation coefficient (S(20,w)) of 17. Goat anti-alpha-2-macroglobulin specifically precipitated the clostridial proteinase inhibitor from a partially purified preparation.     

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.     

Steroid receptors in human endometrium. Comparison of data obtained by three different methods

To find a method for steroid receptor measurement in small endometrial tissue samples (less than 100 mg), an isoelectric focusing assay has been compared with a dextran-coated charcoal assay for oestradiol receptor. The results correlated well (r = 0.85) and this indicates that isoelectric focusing is a good technique for oestradiol receptor determination. Te isoelectric focusing of progesterone receptor has been compared with a dextran-coated charcoal assay and sucrose density gradient centrifugation. Isoelectric focusing gave recoveries of 0-26% compared to receptor values obtained with the two other methods, which correlated well (r = 0.97). The low recovery implies that the isoelectric focusing assay is not suitable for progesterone receptor determination.     

Characterization of estrogen-induced progestin binding in cytosol of the R3327 prostatic carcinoma of the rat

High affinity binding of the synthetic steroids methyltrienolone (R1881) and promegestone (R5020) to cytosol protein from the Dunning (R3327) experimental prostatic carcinoma of the rat was investigated. Animals bearing tumours of approx 1.5 cm mean diameter were either left untreated, or were administered diethylstilbestrol diphosphate (DESP) in the drinking water in doses close to those used clinically for the treatment of human prostatic carcinoma. Tumours were excised after 10-40 days, and binding of [3H]R1881 and [3H]R5020 to tumour cytosol was characterized using Scatchard analysis, sucrose density gradient centrifugation, and steroid competition, under conditions optimal for the conservation and assay of progesterone receptor. Both ligands were bound in much higher concentrations by cytosol from DESP-treated tumours than from untreated tumours. Binding was of high affinity (Kd congruent to 1 nM), was specific for progestins, and sedimented in peaks at approximately 8S and approximately 4S in sucrose density gradients. We conclude the DESP treatment of rats bearing the R3327 prostatic carcinoma induces synthesis of progesterone receptor in this tumour.     

11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.     

Characterization of two uterine proteases and their actions on the estrogen receptor

We have characterized two previously undetected proteases from the calf uterine cytosol and measured their actions on the estrogen receptor. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucylglycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant to exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity. These data indicate that a decrease in the sedimentation rate of the estrogen receptor in sucrose gradients with low salt or a change in the receptor's elution on DEAE-cellulose chromatography is not related to receptor activation but is produced by the receptor-active protease or other proteases.     

Isolation of untransformed bovine estrogen receptor without molybdate stabilization

A low concentration estrogen-derivatized affinity resin has been used in a rapid, single step purification of the untransformed estrogen receptor from calf uterine cytosols prepared without sodium molybdata. The procedure isolates the Mr 65,000 estrogen receptor in association with the bovine heat shock protein hsp90. Small amounts of proteolyzed receptor ranging in size from Mr 50,000 to 60,000 are also present in the purified extracts. Results from affinity chromatography of receptor cytosols either untreated or presaturated with estradiol suggest that two proteins of Mr 22,000 and 38,000 are co-purified with the untransformed receptor complex and may represent additional nonhormone-binding components of the native receptor form. Some indication of the stability of protein-protein interactions within the oligomeric complex has been derived from differential salt elution studies with heparin-sepharose and affinity gel-immobilized untransformed receptor. On size exclusion high performance liquid chromatography the untransformed complex eluted with a Stokes radius of 75 +/- 2 A (n = 18), but was shown to be sensitive to extended ultracentrifugal analysis dissociating to the receptor homodimer, sedimentation coefficient 5.3 +/- 0.3 s (n = 5). Preliminary data on urea- and heat-induced transformation of the isolated receptor to the DNA-binding state is presented.     

Effects of Temperature,Nucleotides and Sodium Molybdate on Activation and DNA Binding of Estrogen,Glucocorticoid, Progesterone and Androgen Receptors In MCF-7 Human Cancer Cells

Abstract

We studied the effects of temperature, ribonucleotides and sodium molybdate on the activation and DNA cellulose binding of estrogen, glucocorticoid, progesterone and androgen receptor complexes in MCF-7 cells. Using DNA cellulose binding as a measure of receptor activation, we found that ribonucleotides activated all four of these receptor complexes. Temperature also activated glucocorticoid receptor complexes efficiently but activated progesterone and androgen receptor complexes less well. Temperature did not activate estrogen receptor complexes. Sodium molybdate blocked either ATP or temperature induced activation of glucocorticoid, progesterone and androgen receptor complexes but only partially blocked estrogen activation. Sodium molybdate also prevented the formation of multiple forms of estrogen and glucocorticoid receptor complexes seen on DEAE cellulose and hydroxylapatite chromatography of crude cytosol. The mechanism by which ribonucleotide enhances and molybdate inhibits activation are discussed.     

Differential DNA binding by calf uterine estrogen and progesterone receptors results from differences in oligomeric states

The studies presented here provided evidence that the calf uterine estrogen and progesterone receptors exhibit different DNA-binding properties in vitro as a result of having different dimerization constants. The affinity of the estrogen and progesterone receptors for DNA was measured by using isocratic elution from DNA-Sepharose. The hormone-free estrogen receptor had a 10-fold higher affinity for DNA than did the hormone-free progesterone receptor when measured at receptor concentrations of 6-12 nM and 180 mM KCl. No effect on DNA binding by binding progesterone to its receptor was detected. This contrasts with the increased affinity for DNA and increased number of ions released upon DNA binding exhibited by the hormone-bound estrogen receptor. Between 2 and 3 ions were released when the progesterone receptor and the diluted estrogen receptor bound DNA. These observations suggested the progesterone receptor was in the monomeric state, whereas the estrogen receptor was in the dimeric state at receptor concentrations of 6-12 nM. When the dimerization constant of the progesterone receptor was measured, the value of approximately 7 nM obtained was 20-fold higher than the value of 0.3 nM reported for the estrogen receptor. This makes it likely the two receptors exist in different forms at the same concentration in vitro. It is also suggested the predominant form of the estrogen and progesterone receptors in vivo could differ.     

Steroid receptors analysis in human mammary tumors by isoelectric focusing in agarose

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.