Nuclear origin of progesterone receptor of the chick oviduct cytosol. An immunoelectron microscopic study

Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two forms of progesterone receptor from chick oviduct cytosol using non-denaturing gel electrophoresis

Non-denaturing polyacrylamide gel electrophoresis and non-denaturing agarose gel electrophoresis have been used to resolve [3H]R5020-binding components from chick oviduct cytosol. From both gel systems 2 peaks of bound radioactivity are resolved which display these properties of authentic progesterone receptor: binding of R5020: steroid specificity, saturability, and restriction to target tissues. The two peaks are approximately equal in magnitude, and there is no evidence for interconversion of the 2 peaks. The presence or absence of 10-20 mM sodium molybdate during cytosol preparation had no effect on the magnitude or mobility of either peak. Neither peak contains salt-dissociable components which affect its electrophoretic properties, suggesting a possible alteration of native receptor forms during electrophoresis.     

Evidence for two structurally related progesterone receptors in chick oviduct cytosol

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [3H]R5020. On SDS-polyacrylamide gels these two forms exhibit app. Mr-values of 79000 and 109000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity-labelled steroid receptors have been established by limited proteolysis with alpha-chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the Mr-value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with Mr-values of 6000 and 10000 from the hormone-binding domain of progesterone and glucocorticoid receptors, respectively.     

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.     

Immunoelectron microscopic localization of progesterone receptor in the chick oviduct

A peroxidase-anti-peroxidase (PAP) method using polyclonal anti-PR antibodies was used to localize progesterone receptor (PR) electron microscopically in the chick oviduct. The immunoreaction precipitate indicating PR was localized inside the nuclei of epithelial, glandular and stromal cells. In the estrogen withdrawn oviduct cytoplasmic immunoreaction precipitate was not seen. Inside the nucleus unoccupied PR was localized mainly like the heterochromatin. As visualized by the PAP technique, the localization of PR was not systematically changed after progesterone administration. In conclusion, we suggest that progesterone receptor in the chick oviduct is an intranuclear protein.     

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.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Antibodies against highly purified B-subunit of the chick oviduct progesterone receptor

A rabbit was immunized with the highly purified B-subunit (110kDa) (20 to 50 micrograms per injection) of the chick oviduct progesterone receptor (PR). Specific antibodies (IgG-RB) were observed 2 weeks after the first booster injection and high antibody titers in the serum were found after the second and third booster injections (with Kdeq of interaction integral of 2 nM). IgG-RB were tested by immunoprecipitation, immunoblotting, density gradient ultracentrifugation and protein A-sepharose assay methods. They recognized not only the B-subunit but also the A-subunit (79K), the nuclear PR, the mero-receptor (proteolytic cleavage product) and the "non-activated" molybdate-stabilized "8S" PR. However, IgG-RB did not interact with the 90K non hormone-binding component of this 8S-PR. IgG-RB did not affect the binding of the hormone to PR, whether incubated with the receptor before or after labelling with tritiated progesterone. They did not cross-react with glucocorticosteroid receptor of the chick oviduct. Weak interaction was observed with estrogen receptor of the chick oviduct and with KC1 activated "4S" forms of the rabbit and human uterus PR.     

Interaction of chick oviduct progesterone receptor with immobilized aurintricarboxylic acid

Aurintricarboxylic acid (ATA) was immobilized on Sepharose 4B via a carbodiimide coupling mechanism. A majority of the chick oviduct progesterone receptor was retained on the affinity resin and could be recovered upon washing the column with buffer containing free ligand or 3 M guanidine-HCl. The [3H]progesterone-receptor complex retained its integrity following the chromatography on ATA-Sepharose as judged by sedimentation analysis. The procedure allowed significant purification of progesterone receptor: SDS-polyacrylamide gel electrophoresis of the purified preparation revealed elimination of many peptide bands present in the cytosol prior to ATA-Sepharose chromatography. The technique thus has a clear potential in characterization and purification of progesterone receptor.     

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.     

Transformation of the chick oviduct progesterone receptor in the absence of hormone

Chicken oviduct progesterone receptor in cytosol was found to be transformed from the 8S to 4S form by incubation at 25 degrees C as well as by 0.3 M KCl in the absence of hormone. Heat transformation of ligand-free receptor took place at a much slower rate than that of ligand-bound receptor. The eventual percentage of transformation, however, was almost the same. The 4S form of the receptor transformed by KCl in the absence of hormone could bind to DNA-cellulose, but not to nuclei. However, upon exposure it acquired the ability to bind to nuclei. It was shown that the transformed ligand-free receptor could bind to progesterone to form the normal activated steroid-receptor complex. Conversely, when activated 4S progesterone-receptor complex was treated with DCC to peel off the hormone, a resulting ligand-free receptor was formed which behaved just like the KCl-transformed receptor in the absence of hormone.     

Immunological similarity between the chick oviduct progesterone receptor forms A and B

A large-scale purification of the progesterone receptor from laying hens is described which yields apparently homogeneous form A and form B receptor in denatured form. The purification procedure is based initially on differential DNA affinity chromatography of both form A and B receptors. Under the conditions of preparation and activation described, progesterone receptor form B binds to DNA-cellulose even in the presence of 100 mM salt. This binding cannot be observed after thermal activation. Receptors obtained at 5% purity using conventional chromatographic purification steps were covalently cross-linked with radioactive ligand by photoaffinity labeling and purified to homogeneity using preparative gel electrophoresis systems under denaturing conditions. This material has been successfully used to generate polyclonal antibodies in rabbits. Immunoblots demonstrated a high degree of cross-reaction between anti-A antibodies and progesterone receptor form B, as well as between anti-B antibodies and progesterone receptor form A, using homogeneous as well as 5% pure receptors as probes. Implications of the immunological data and the novel DNA-binding characteristics of form B are discussed with respect to topological conformation of the progesterone receptor and the structural similarity between forms A and B.     

Analysis of the molecular species of the chick oviduct progesterone receptor using isoelectric focusing.

Conditions are described for the preparative isoelectric focusing in flat beds of Sephadex of the progesterone receptor from the chick oviduct. The method allows the fractionation of the receptor into two molecular species, one focusing at pI 6 and the other at pI 7 with good purification and recovery. The pI 6 and pI 7 receptor species were purified 2- and 26-fold, respectively. The assaying of the focused fractions with the charcoal binding method provides an accurate identification and quantitation of the [3H]progesterone receptor. The method is reproducible in recovery, quantitation, and resolution of the two receptor species. The receptor with an apparent pI of 6 sediments at approximately 4 S on linear sucrose gradients, while the receptor with an apparent pI of 7 sediments at approximately 3.5 S. On the basis of the sedimentation values and elution patterns from diethylaminoethyl (DEAE) chromatography, the pI 6 component is equivalent to the "B" receptor species and the pI 7 component is equivalent to the "A" receptor species described previously [schrader, W, T., 7 O'Malley, B. W. )1972) J. Biol. Chem. 241, 51--59].     

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.