Purification of a progesterone receptor from rabbit uterus

A progesterone receptor has been purified to homogeneity from rabbit uterus by steroid affinity chromatography. The receptor was obtained in 5% yield, with a specific activity for [³H]progesterone binding of 14,580 pmol/mg protein. The pure receptor migrated as a single band on SDS-polyacrylamide electrophoresis, with a MW of 70,000. Progesterone binding to the receptor was heat labile and was displaced by an excess of R5020. Photoaffinity labeling of the pure receptor with [³H]R5020 corresponded to the major photoaffinity labeled species in crude cytosol.     

Rat uterine progesterone receptor: Stabilization of hormone-binding components for biochemical analyses

A method was developed for quantitative recovery of the labile rat uterine progesterone receptor hormone-binding components. Initial conditions were established by the sucrose gradient procedure. Upon centrifugation through low-salt 5–20% sucrose gradients prepared in 10% glycerol, the well-known 6–8 S progesterone receptor components were observed either when cytosol was prelabeled with [³H]17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione ([³H]R5020) or when prelabeled with [³H]progesterone followed by postlabeling the fractions collected after centrifugation with either [³H]progesterone or [³H]R5020. Recovery of progesterone receptor binding was improved by prelabeling with [³H]R5020, by adding 1.5 mm ethylene glycol bis(β-aminoethylether)N,N′-tetraacetic acid (EGTA) to all buffers, and at high tissue concentrations. Under these conditions quantitative conversion of the receptor to specific [³H]R5020-binding 4S components was achieved with 150 or 400 mm KCl. Similar conditions proved unsuitable for receptor analysis by gel filtration (Bio-Rad agarose A0.5M or A1.5M), apparently due to [³H]R5020 dissociation from the receptor in the large volume of elution buffer. However, excellent receptor recovery (97.2 ± 6.7%) was achieved by including 10 nm unlabeled progesterone in all preparation and elution buffers. Receptors were then detected by the addition of 5 nm [³H]R5020 to the column fractions, exchange incubation for 3–6 h at 4 °C, and subsequent separation of bound and free steroid by the hydroxyapatite assay. This method resulted in a consistent elution pattern suggestive of receptor heterogeneity. Identity of the peak(s) as progesterone receptor components(s) was confirmed by the lack of competition by 2 μm cortisol when added either to cytosol or during the post-labeling-exchange process. Neither the qualitative nor quantitative results of the column profiles were changed substantially in the presence of 20 mm molybdate. Although the receptor structure has yet to be established, both statistical analysis of the column profiles by computer curve-fitting procedures and rechromatography of peak fractions suggested that the rat uterine progesterone receptor may be composed of multiple components. This ligand-stabilization/postlabeling-exchange procedure provides a method for further studies of progesterone receptor biochemistry in mammalian systems. Additionally, similar procedures may stabilize other labile ligand-binding proteins for biochemical analyses and/or purification.     

Progestin binding sites in the rat hypothalamus pituitary and uterus

Specific, estrogen-inducible, nuclear radioactivity uptake has been demonstrated in the uterus, pituitary and hypothalamus of immature female rats after injection of a highly potent labelled progestin, R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione). A cytoplasmic progestin “receptor” has been characterized in these target tissues by an in vitro Dextran-coated charcoal adsorption method. R 5020 binds with the same intrinsic dissociation constant to the receptor present in these tissues ; it dissociates at the same rate from the uterine and pituitary receptor (k^?1 = 1×10^?2min^?1), but about 6 times slower than progesterone. This evidence, together with the similar hormone specificity in the uterus and pituitary, suggests that the progestin “receptor” is a similar entity in central and peripheral target tissues.     

Nuclear progesterone receptors and characterization of cytosol receptors in the rat hypothalamus and anterior hypophysis

Progesterone receptors in nuclei and cytosols from the hypothalamus and anterior hypophysis of oestrogen-primed immature and mature female rats were investigated. In the hypothalamic and hypophysial nuclei the binding and exchange of [³H]-R5020 with progesterone or R5020 was achieved after 2 h at 0–10°C, but rapidly degraded at 30°C. In addition, when unlabelled R5020 was added to the incubation tubes previously incubated with [³H]-R5020 at 0–10°C, unlabelled R5020 was found to exchange with [³H]-R5020 bound to nuclei, confirming that [³H]-R5020 binding is due to an exchange reaction. Scatchard analysis of the specific binding curves revealed high-affinity and low capacity binding. Progesterone receptor complexes extracted with 0.4 M KCl from purified and crude (800 g pellet) nuclei prepared from the hypothalamus and anterior hypophysis of the oestrogen-primed adult female rats incubated with [³H]-R5020 were identified in the vicinity of 5S by gradient centrifugation. From these results it is concluded that nuclear progesterone receptors exist in the hypothalamus and anterior hypophysis. Moreover, it is interesting to note that progestin binding sites resistant to extraction with 0.4 M KCl exist even in the purified hypothalamic and hypophysial nuclei.In the hypothalamic and anterior hypophysial cytosols an exchange reaction was observed at 0–10°C as in the nuclei. The 7S cytosol receptors at low ionic strength sedimented in the 4S region in a high salt medium (0.4 M KCl), both in the hypothalamus or hypophysis, suggesting a possible relationship between aggregate- and subunit receptors. Moreover, progesterone receptors in the hypothalamic and hypophysial cytosols were separated on polyacrylamide agarose gels electrophoretically from oestrogen- and androgen-receptors labelled with [³H]-R2858 and [³H]-R1881, respectively.The existence of nuclear progesterone receptors in the hypothalamus and anterior hypophysis, together with the cytosol receptors, provide further evidence for a possible role of the steroid-receptor interaction in the mechanism of the central action of progesterone.     

Uptake of three [3H]progestins by target tissues in vivo: Implications for the design of diagnostic imaging agents

We have investigated the tissue distribution of radioactivity for 0.5–4 h follwing the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [³H]progesterone shows minimal uterine uptake (<0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [³H]R 5020 (promegestrone) and [³H]ORG 2058 show highly selective uptake that reaches 4–5% ID/g by 1–3 h. The uterus to non-target tissue activity ratio at 2–4 h is approximately 12–20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.     

Characterization of a progestin-binding component in lactating rat mammary glands

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 · 10^?9 M under low-salt conditions and with that of 8.2 · 10^?10 M in the presence of 0.3 M KCl. Specificity studies showed a higher degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [³H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [³H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.     

Heterogeneity and ontogenesis of progestin receptors in rabbit lung

In order to evaluate the possible role of progesterone in fetal lung development, the presence of specific pulmonary progestin receptors and their ontogenesis were investigated in the rabbit fetus. Scatchard analysis of binding in lung cytosol from 29-day fetuses over a wide range of [³H]-R5020 concentrations indicates the presence of at least two binding sites. One of these sites, type I, is of very high affinity (K_D = 0.12 nM) and low capacity (26fmol per mg protein). The second binding site, type II, is of lower affinity (K_D = 36 nM) and higher capacity (240 fmol per mg protein). These two binding sites can be distinguished by sucrose density gradient centrifugation, the type I component sedimenting at 7.1 S and the type II component sedimenting at 4.5 S. Similar type I and type II sites are present in adult lung cytosol except that the type II binding component in adult lung sediments at 2.8 S rather than 4.5 S. Progesterone and R5020 compete well with [³H]-R5020 for binding to both sites while dexamethasone and cortisol do not compete. Thus the type I and type II binding sites appear to represent specific progestin receptors distinct from transcortin or the glucocorticoid receptor. The concentration of the type I sites increases significantly between the 20th and 29th day of gestation, with a further increase being observed in adult animals. The type II site is not measurable until 26 days of gestation and attains adult levels by day 29. Among a large number of fetal tissues examined, the lung contained the highest concentration of type I progestin receptor sites.Although cortisol and dexamethasone, even at very high concentrations, do not compete with [³H]-R5020 for binding to lung cytosol, the binding of [³H]-dexamethasone is inhibited significantly by nonlabeled progesterone or R5020 and this inhibition appears to be due to dissociation of [³H]-dexamethasone-receptor complexes. These results indicate that, in addition to type I and type II progestin receptor sites, fetal lung cytosol contains a third binding site, type III, which appears to be different from the glucocorticoid receptor site. Occupation of the type III site by progestins interferes with the binding of glucocorticoids to glucocorticoid receptors perhaps by increasing the rate of dissociation of glucoeortieoid-receptor complexes.     

Specific progesterone receptors in human breast cancer.

We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroid binding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by competition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextran-coated charcoal assays is approximately 2 times 10- minus 9 M.     

Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Target tissue uptake selectivity of three fluorine-substituted progestins: Potential imaging agents for receptor-positive breast tumors

We have studied three new fluorine-substituted progestins (1–3) as potential imaging agents for progesterone receptor (PgR)-positive human breast tumors. Two of these are fluorine-substituted analogs of the potent progestin R5020 (promegestone), derived from (21S)-hydroxy R 5020 (RU 27987) and (21R)-hydroxy R 5020 (RU 27988), known metabolites of R 5020, which have affinities for PgR that are 116 and 4%, respectively (relative to R 5020 = 100%). These precursors were protected as their 3,3-dioxolane derivatives and converted to the 21-trifluoromethanesulfonate derivatives. Fluoride ion displacement, followed by acid-catalyzed deprotection, furnished in good yield the epimeric fluoroanalogs, (21S)- and (21R)-fluoro R 5020 (1 and 2, affinities for PgR, 11 and 45%, respectively). These compounds were also prepared in ¹⁸F labeled form by the same route, in 14–32% overall radiochemical yield (decay corrected; synthesis time 90 min; sp. act. 370–1060 Ci/mmol). In tissue distribution studies in estrogen-primed immature rats, uterus-to-muscle ratios were 4.3 at 1 h for the 21S-epimer and 1.1 for the 21R-epimer, paralleling their relative binding affinities. Considerable metabolic defluorination was observed. The third fluorine-substituted progestin, DU 41165, has a novel retroprogesterone (9β, 10α) structure, substituted with fluorine at C-6; its binding affinity is 145% relative to R 5020, and it was prepared in tritium-labeled form by acetylation of DU 41231, the 17α-hydroxy precursor, with [³H]acetic anhydride. In estrogen-primed immature rats, this compound shows uterus-to-muscle ratios of 15 at 1 h, and 18–71 between 2 and 6 h, suggesting that compounds in this retroprogesterone series may be very promising candidates for selective imaging of PgR-positive tissues and tumors.     

Characterization of 7–8S progestin binding protein in human prostate using vertical tube rotor

The specific binding of the synthetic progestin, 17α-methyl [³H]promegestone (R5020), to the cytosol of human benign hyperplastic prostate has been studied in sucrose density gradients using a vertical tube rotor. The eytosol of human prostate was shown to contain substantial amounts of a 7–8S macromolecule with a high affinity (K_d = 0.5–1 nM) for R5020 which is saturated at low concentrations (10 nM). The conventional technique of sucrose density gradient analysis in a swinging bucket rotor was not suitable for reproducible optimal analysis of a 7–8S high affinity complex. The use of the salt, Na₂MoO₄, had a stabilizing effect on the complex. Comparison of saturation analysis assays using dextran charcoal assay and vertical tube rotor assay showed that the charcoal assay can give an over-estimation of the 7–8S saturable binding. Progestational steroids competed with R5020 for binding to 7–8S, whereas androgenic steroids, with the exception of 19-nor-testosterone, did not compete.Incubation of cytosol at elevated temperatures in the presence of DNA-cellulose resulted in the binding of the hormone-protein complex to DNA-cellulose. High ionic strength buffer was required to extract the complex which sedimented at 4.5S in sucrose gradients prepared in 0.4 M KCl. Based on the data presented, progestin binding in human prostate is clearly similar in physical chemical properties to progesterone receptors in “classical” target tissues. However, rapid sucrose gradient analysis with a vertical tube rotor is preferred over conventional techniques to evaluate progestin receptor binding in human prostate.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells

Phosphorylation of the tumor suppressor protein, retinoblastoma (pRb), regulates the progression of the cell cycle. Previous work from this laboratory had shown that estradiol (E(2)) regulates tumor suppressor proteins, p53 and retinoblastoma in breast cancer cells. In the present study, we have examined the phosphorylation of pRB in T47D breast cancer cells following treatments with R5020 and antiprogestins. In growth medium containing serum depleted of endogenous steroids by charcoal treatment, pRb appeared mainly in its hypophosphorylated form. Addition of 10 nM R5020 to the culture medium caused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated form of pRb began to accumulate after 72 h. Upon prolonged R5020 treatment (72-96 h), pRb was detected exclusively in its hypophosphorylated form. While treatment of cells with R5020 caused a transient increase in the level of cyclin D1, E(2) addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation. Antagonists of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E(2) and R5020-induced pRb phosphorylation, respectively. These results suggest that R5020 induces pRb phosphorylation via a transient increased expression of cyclin D1, whereas E(2) treatment results in sustained expression of cyclin D1 and increased pRb phosphorylation. Furthermore, R5020 effects on pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phosphorylation was observed: the R5020 effects were blocked by RU486 and ZK98299, but not by the pure ER antagonist, ICI 182, 780 (ICI).     

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., O?ate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.     

Properties of progestin-binding protein in benign hypertrophic human prostate

RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020. Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestin-binding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.     

Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.     

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.     

Progesterone receptor synthesis and degradation in MCF-7 human breast cancer cells as studied by dense amino acid incorporation. Evidence for a non-hormone binding receptor precursor

We have used the technique of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids to study the synthesis and degradation rates of the progesterone receptor in MCF-7 human breast cancer cells. In cells grown in the absence of progestin, sucrose gradient shift analyses reveal that it takes 17 h for the normal density progesterone receptor levels to be reduced to half the initial value, whereas in the presence of 10 nM of the synthetic progestin [3H]R5020, the receptor turns over more rapidly, such that the normal density R5020-occupied progesterone receptor complexes are reduced to half in 12 h. The accelerated progesterone receptor turnover in the presence of [3H]R5020 reflects increased turnover rates of both the A (Mr-85,000) and B (Mr-115,000) subunits, as determined by sodium dodecyl sulfate gel analyses of dense and light receptors photoaffinity labeled with [3H]R5020. In both control and progestin-exposed cells, the time course of progesterone receptor turnover shows a lag of approximately 6 h after dense (15N, 13C, 2H) amino acid exposure, before dense hormone binding receptor species are seen and before normal density progestin binding activity starts decreasing. Since our evaluations of progesterone receptor depend upon its binding of radiolabeled ligand ([3H]R5020), this lag in the density shift kinetics would be consistent with the presence of a non-hormone binding biosynthetic precursor, from which the hormone-binding form of progesterone receptor is derived. A kinetic model is used to analyze the lag-decay profiles and to determine the rate constants for progesterone receptor synthesis, activation to the hormone-binding form, and degradation.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.