Chick oviduct glucocorticosteroid receptor. Specific binding of the synthetic steroid RU 486 and immunological studies with antibodies to chick oviduct progesterone receptor

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen-primed, 4-week-withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18 300 receptor sites/cell were measured assuming that all cells contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: cortisol less than deoxycorticosterone less than dexamethasone less than corticosterone less than triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was approximately equal to 10-fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species. The molybdate-stabilized, 8-S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand--8-S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG-G3) or monoclonal (BF4) antibodies generated against the molybdate-stabilized, 8-S form of the chick oviduct PR. Since neither IgG-G3 nor BF4 interacted with the steroid binding 4-S form of GR, it is suggested that these antibodies recognized a non-steroid binding protein common to molybdate-stabilized, 8-S forms of GR and PR.     

Kinetics of progesterone binding to the chick oviduct receptor protein

Kinetic parameters have been determined for the binding of progesterone to receptor proteins of the chick oviduct. Association and dissociation rate constants and the equilibrium constant have been determined as a function of temperature and ionic strength. Both the association and dissociation rate constants vary with temperature by about 2 orders of magnitude. However the equilibrium constant for the binding reaction does not change substantially over the temperature interval from 0 to 24°. The half-life of the complex under pseudo first-order conditions decreases from about 10 h at 0° to about 3 min at body temperature of the animal (42°). Arrhenius plots of the dissociation rate constant information show a linear plot over the temperature range studied indicating that the aggregation and the conformational changes accompanying this temperature range do not cause any apparent change in the hormone binding site.On the other hand glycerol has a considerable stabilizing effect on the receptors increasing the half-life at 24° from about 24 min to a matter of about 100 min as the glycerol concentration is increased to 40%. The hormone binding site can be disrupted irreversibly by high salt or urea concentrations in excess of one molar. Ionic strength over the range from 0–0.5 M has essentially no effect on any of the parameters measured. The half-life of the complexes decreases by only about 10–15% over this interval.The possible perturbation of the hormone binding sites accompanying binding of the proteins to ion exchange resins was studied. There is no detectable change in any of the kinetic parameters when the receptor protein is adsorbed to either DEAE cellulose or phosphocellulose. The hormone binding characteristics of the protein remain the same for up to 100 hours; only a single class of hormone binding site could be detected. Variations in half-life accompanying dissociation of hormone off the receptors was shown to be due merely to the presence of non specific binding in the preparation. The study concludes that the conformation of the hormone binding site is not strongly perturbed by the environment of the protein.     

Chick oviduct progesterone receptor phosphorylation: characterization of a copurified kinase and phosphorylation in primary cultures

A protein kinase activity was copurified with the chick oviduct progesterone receptor. The enzyme is magnesium dependent and can use the B subunit of progesterone receptor or histones as substrates. The physiochemical parameters of the kinase were determined [pI approximately 5.3; Stokes radius approximately 7.2 nm; sedimentation coefficient (S 20,w) approximately 5.6] and compared to those of the purified B subunit. The results were consistent with the presence of an unique enzyme distinct from the receptor itself. The physiological significance of receptor phosphorylation was investigated in oviduct cells grown in primary culture. Cells were labeled with [32P]orthophosphate in presence or absence of progesterone and the receptor components were immunoprecipitated with a specific polyclonal antibody. Although progesterone treatment lead to the attachment of most of the receptor (approximately 80%) to nuclear structures, the 32P-labeled B subunit was only recovered in the cytosol fraction. Different procedures to extract the nuclear receptor did not allow detection of any 32P-labeled form in the nuclear-soluble fractions, suggesting that the B subunit was not further phosphorylated upon the exposure of cells to progesterone.     

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor

A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified. This paper demonstrates that RBF-1 and also the PR nuclear binding sites are localized in the oviduct nuclear matrix. RBF-1 is found in abundance in the nuclear matrix of liver but only in traces in the nuclear matrix of spleen. Extraction of the nuclear matrix with 4.0 M Gdn-HCl results in the complete removal of RBF-1 as occurs with whole chromatin. Interestingly, a second class of specific PR binding, termed RBF-2, remains on the nuclear matrix after the removal of all RBF-1. Southern blot analysis indicates that the nuclear matrix DNA contains sequences homologous with the 5'-flanking domains of the rapidly steroid regulated c-myc and c-jun protooncogenes and the beta-actin gene, but not genomic sequences of the late sex steroid regulated gene, ovalbumin, or the alpha-actin gene. A specific, small region in the 5'-flanking domain of the c-myc gene appears to be associated with the nuclear matrix. Southwestern blot analysis using partially purified RBF-1 shows a marked affinity and specificity of the RBF-1 for the nuclear matrix DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological activity and ligand binding mode to the progesterone receptor of A-homo analogues of progesterone

The biological activity of two seven-membered A-ring (A-homo) analogues of progesterone was evaluated by transactivation assays in Cos-1 cells and by determination of Bcl-x(L) expression levels in T47D cells. The results show that both compounds act as selective progesterone receptor (PR) agonists but lack mineralocorticoid receptor (MR) activity. Molecular modelling using semiempirical AM1 and ab initio HF/6-31G** calculations, showed that the A-ring of the A-homo steroids may adopt five different conformations, although only three correspond to low energy conformers. The low energy conformers of each analogue were introduced into the ligand binding pocket of the PR ligand binding domain (LBD) obtained from the PR LBD-progesterone crystal structure. The steroid binding mode was then analyzed using 10 ns of molecular dynamics (MD) simulation. The PR LBD-progesterone complex was also simulated as a control system. The MD results showed that both A-homo steroids have one conformer that may be properly recognized by the PR, in agreement with the observed progestagen activity. Moreover, the simulation revealed the importance of a water molecule in the formation of a hydrogen bonding network among specific receptor residues and the steroid A-ring carbonyl.     

Novel phosphorus-containing 17beta-side chain mifepristone analogues as progesterone receptor antagonists

A novel series of steroidal compounds were designed and synthesized with various phosphorus-containing groups on the 17beta-side chain as progesterone receptor antagonists. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in an rat progesterone-sensitive assay. Some of these compounds are more potent than mifepristone, with a better selectivity profile in differentiating progesterone receptor from glucocorticoid receptor.     

Evidence of a progesterone receptor in the liver of the green frog Rana esculenta and its down-regulation by 17 beta estradiol and progesterone

Progesterone is a versatile hormone showing an ample variety of effects. One of the numerous functions attributed to progesterone is the modulation of vitellogenesis in oviparous vertebrates. As a prerequisite for the possible involvement of progesterone in vitellogenesis modulation, we investigated the presence of a progesterone receptor (PR) in the liver of the female green frog Rana esculenta. 3H-Progesterone (3H-P) binding activity was found in both cytosol and nuclear extract of the liver of Rana esculenta. The progesterone-binding moiety showed the typical characteristics of a true receptor, such as high affinity, low capacity, and specificity for progesterone. It also bound to DNA-cellulose and was eluted with a linear salt gradient at a concentration of 0.05 M of NaCl. The progesterone-binding moiety was down regulated by steroid hormones, in that ovariectomy resulted in a significant increase, in both cytosol and nuclear extract, of 3H-P binding activity with respect to intact females. On the contrary, 3H-P binding activity was almost undetectable after estradiol and/or progesterone treatment. The progesterone binding moiety of Rana esculenta was analyzed by Western blotting with the aid of a monoclonal antibody raised against the subunits A and B of the chicken PR. An immunoreactive band of about 67 kDa was observed in the liver of both intact and treated females. The 67 kDa band showed an increased intensity in ovariectomized animals, while it was faint following treatment with estradiol and/or progesterone. This is the first report on the presence of a progesterone receptor (PR) in the liver of an amphibian. PR of Rana esculenta is down regulated by estradiol and/or progesterone and shows peculiar immunological and biochemical characteristics, which make it rather different from the PR of other vertebrates.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Non-activated progesterone receptor extracted from nuclei of hen oviduct

When the progesterone receptor was extracted from nuclei of laying hen oviduct with 0.5 M sodium molybdate, a large, 7-8 S, form of the receptor was observed. This receptor form resembled non-activated cytoplasmic receptor not only in displaying the same sedimentation coefficient, but also in rapid dissociation rate of the hormone-receptor complex. This finding suggests that either activation may occur within the nuclear compartment, or that activation may be reversed under certain conditions.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

Augmentation of progesterone receptor concentration by progesterone and estrogen treatments in the chick oviduct

Cytosol receptors for progesterone in the chick oviduct were measured by charcoal-adsorbtion assay by using ORG 2058 as a ligand after long-term administration of progesterone and diethylstilbestrol (DES). Steroid administration was carried out by using daily injections or silastic capsules. DES treatment increased the progesterone receptor concentration (from 11500 to 21500 sites per cell, day 14). Progesterone also augmented the concentration of its own receptors (from 11500 to 29000 sites per cell, day 14). In the experiments with capsule administration the same trend was seen. This indicates that both diethylstilbestrol and progesterone are able to increase the concentration of progesterone specific cytosol receptors in the non-differentiated chick oviduct.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.