Properties Of Progesterone Receptor Research Articles

Progesterone receptors in reproduction: functional impact of the A and B isoforms

Progesterone (P) is a key regulator of female reproductive activity. The effects of P are mediated by two progesterone receptor (PR) proteins, termed A and B, that arise from a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes. Null mutation of the PR gene in mice (PRKO) leads to pleiotropic reproductive abnormalities. This paper will review the reproductive functions of PRs delineated using the PRKO mouse. Further, we will summarize the structure and functional properties of PRs and discuss how functional differences between the PR-A and PR-B isoforms are likely to impact on the overall physiological role of the receptor in reproductive systems.

An N-terminally truncated third progesterone receptor protein, PR C, forms heterodimers with PR B but interferes in PR B-DNA binding

Multiple progesterone receptor (PR) isoforms may explain in part the complex and diverse biological actions of progestins. Recent studies demonstrate that the human 116 kDa B-receptor (PR B) and the 94 kDa A-receptor (PR A) can have very different transcriptional functions that are cell- and promoter-specific. Additionally, we have shown the existence of a smaller N-terminally truncated 60 kDa progestin-specific binding protein, called the C-receptor (PR C), that has unique transcriptional potentiating properties. In the presence of the other two PR isoforms, PR C enhances the transcriptional activities of the larger PR proteins. In order to determine the mechanism of action for the transcriptional promoting abilities of PR C, the structural and functional properties of PR C were analysed and compared to those of PR A and PR B. PR C consistently displayed a dissociation constant (K d) ∼5 times higher than that for PR B and PR A, suggesting that the N-terminal truncation of PR C results in a conformation different from the two larger PR isoforms, that affects the hormone-binding region and its interaction with hormone. Despite this change, PR C is still capable of forming heterodimers with the larger PR B in solution, as determined by co-immunoprecipitation studies, but PR C interferes in tight PR B binding to DNA in gel-shift assays. Surprisingly, progestin and anti-progestin autoregulation of PR C protein levels parallel those for PR B and PR A.

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 properties of PR from T47D cells that are similar to RU486.

ZK98299—A new antiprogesterone: Biochemical characterization of steroid binding parameters in the calf uterine cytosol

We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11β-(4-dimethylaminophenyl)-17α-hydroxy-17β-(3-hydroxypropyl)-13α-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [ 3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity ( K d = 2.5 nM) interaction between [ 3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2–3 h at 0 °C, and was facilitated by 20 m m Na 2MoO 4. Under nontransforming conditions, [ 3H]ZK98299-receptor complexes sedimented as 8 S species in 8–30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5–7%) binding to DNA-cellulose. In contrast, transformed [ 3H]R5020-receptor complexes exhibited a greater extent of binding (25–55%) to DNA-cellulose. [ 3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 °C. [ 3H]ZK98299 binding was sensitive to sulfhydryl group modification as β-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [ 3H]R5020 binding, there was a significant (nearly twofold) increase in the [ 3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.

Mammalian Progesterone Receptor Shows Differential Sensitivity to Sulfhydryl Group Modifying Agents When Bound to Agonist and Antagonist Ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.

Monoclonal antibody characterization of progesterone receptors, estrogen receptors and the stress-responsive protein of 27 kDa (SRP27) in human uterine leiomyoma.

Uterine leiomyoma occurs in one of every four or five women during their reproductive life. Its origin is unknown but it is accepted that estrogens play a significant role in its development. In order to learn more about the estrogen dependency of leiomyoma, the biochemical and immunological properties of two markers of estrogen response in target cells (the progesterone receptor (PR) and the stress-responsive protein of 27 kDa (SRP27)) were studied in leiomyoma. The ER (estrogen receptor) and PR content were determined by conventional DCC exchange assays. Specific anti-ER, anti-PR and anti-SRP27 monoclonal antibodies were used in immunoblots and immunohistochemical (IHC) studies. The binding properties of PR from cytosol of leiomyoma showed a Kd of 0.8-1.3 nM, which is in the range described for other human tissues. 80% of all studied leiomyoma contained PR, in a range of 805-2000 fmol/mg protein. The Kd for leiomyoma ER was 0.1-0.9 nM, and 84% of the samples were positive for ER. The PR of leiomyoma has the two A and B forms of 120 and 94 kDa, as shown in the immunoblot using the AB52 anti-PR monoclonal antibody. The IHC study revealed that the PR is concentrated in the cell nuclei, in the form of perinuclear bodies, with a homogeneous staining pattern from cell to cell. The leiomyoma fibres contain SRP27 in a higher concentration than the healthy myometrium. The leiomyoma SRP27 shows a typical doublet of 24 kDa and 27 kDa in immunoblot, the same as in MCF-7 cells. The IHC study revealed a high degree of organization of SRP27 in leiomyoma cells, suggesting that this protein may be part of the cytoskeleton. The results obtained show that human leiomyomas contain ER, PR and RSP27 with similar immunological and biochemical properties to those of other human tissues, including the MCF-7 breast cancer cell line.

Unspecific and Sequence-Specific Deoxyribonucleic Acid Binding of the Partially Purified Human Progesterone Receptor

The progesterone receptor (PR) was partially purified from T47D human breast cancer cells by sequential chromatography on phosphocellulose, heparin-Sepharose, and DNA-cellulose. Heparin-Sepharose chromatography resulted in an efficient conversion of the receptor to a DNA-binding form (activation) since more than 85% of the 3H-R5020 labeled eluate from heparin-Sepharose was retained on DNA-cellulose and since the cytosolic 8S receptor was converted to a 4S moiety after chromatography on heparin-Sepharose. The 3H-R5020 labeled human PR eluted from DNA-cellulose as a single symmetrical peak at 0.2 M NaCl; after photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this species was shown to consist of about equal amounts of two proteins of Mr approximately equal to 96,000 and 120,000 (the so called A- and B-subunits, respectively). This partially purified receptor preparation (SA 490 pmol/mg protein) did not contain any glucocorticoid receptor (GR) as shown by immunoblotting with a monoclonal antirat GR antibody that cross-reacts with the human GR. Therefore, this preparation was used to compare the specific DNA-binding properties of the human PR with those of the purified rat GR. The human PR bound specifically to the promoter region of mouse mammary tumor virus (MMTV) at a molar ratio between receptor and DNA similar to the molar ratio between GR and DNA needed for binding of rat GR to MMTV, indicating that the PR was purified in a biologically active form.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486.

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Progestin receptors from tissues either exhibiting or lacking estrogen response mechanisms : Comparison of conventional and high-performance liquid chromatography methodology

Evidence from a variety of target organs has shown that progesterone receptor (PR) is induced by estrogen receptor (ER) in normal and neoplastic tissues. However, approximately 12% of the normal human uterine samples exhibit only PR with no measurable ER, suggesting the expression of both inducible and constitutive receptor isoforms. We investigated several molecular properties of PR from tissues either exhibiting or lacking ER. All studies were conducted in potassium phosphate buffer containing 10 m M sodium molybdate with a synthetic progestin, [ 3H]R5020 as the ligand. Radioinert R5020 was used as competitor to assess nonspecific association. Competition analysis showed that PR from both sources exhibited similar ligand specificities and affinities. Relative affinities were ORG 2058 > R5020 > medroxyprogesterone acetate > progesterone /2>-testosterone K d values ranged from 10 −9 to 10 −10 M; testosterone showed no specific competition). We utilized high-performance liquid chromatography in the size-exclusion (HPSEC) and ion-exchange (HPIC) modes to probe the size and ionic properties of PR. HPSEC profiles showed that the PR isoform from both sources was eluted as a single, sharp peak > 76 Å. HPIEC elution profiles indicated no differences in the surface ionic properties in that PR from both tissue types eluted with ca. 100 m M phosphate. These experiments show no difference between the inducible and the putative constitutive form of PR. Thus, some PR species may not require estrogen for their formation.

Effects of the hormonal agents on the steroid receptors and DNA synthesis in MCF-7 cells

We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11β-(4-dimethylaminophenyl)-17α-hydroxy-17β-(3-hydroxypropyl)-13α-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2–3 h at 0 °C, and was facilitated by 20 mm Na2MoO4. Under nontransforming conditions, [3H]ZK98299-receptor complexes sedimented as 8 S species in 8–30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5–7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25–55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 °C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as β-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.

Progesterone receptors in rat brain and uterus: dependence on the hormonal milieu.

Cytosols were incubated from the hypothalamus and mid-brain and from the uterus, and incubated with [3H]progesterone alone or in the presence of excess radioinert steroid to reveal saturable binding sites. Bound and free hormone were separated by gel filtration. Scatchard analysis of the binding sites yielded evidence for only one class of binding sites of high affinity and limited capacity. The binding components in the hypothalamus and uterus appeared to fluctuate during the oestrous cycle, attaining a nadir at metoestrus, while those in the mid-brain were apparently unchanged. During pregnancy hypothalamic [3H]progesterone-binding sites appeared to lose affinity for the steroid while in the uterus the affinity for the steroid was unchanged but the absolute numbers of binding sites were greatly increased at day 10. It is concluded, both from studies of the properties intrinsic to the binding reaction and from endocrine correlates, that the macromolecular progesterone-binding components in the brain may be receptors for the hormone and that there may be differences between the properties of progesterone receptors in different tissues. Furthermore, during pregnancy there may be qualitative changes in the neural progesterone receptors which are not mediated by oestradiol.

Progesterone receptors in human breast cancer. Stoichiometric translocation and nuclear receptor processing.

In a subline of T47D human breast cancer cells, progesterone receptors (PR) are synthesized at very high levels, but their synthesis is not estrogen-dependent. Despite the unusual control of synthesis, the physicochemical properties of PR are normal. These are, therefore, ideal cells to study PR regulation by progesterone, free of estrogen effects. In this paper, we show that nuclear translocation of PR is stoichiometric, and that an unusual and very rapid nuclear turnover, or processing step, characterizes receptor-DNA interactions. In intact T47D cells, PR are translocated to the nucleus only by progestins; 70-90% of cytoplasmic receptors are depleted at 37 degrees C within 5 min of progestin addition. After PR are translocated by 0.1 muM progesterone, they can be quantitatively recovered from nuclei only in the first 5 min; thereafter, a rapid nuclear processing step results in loss of 50-80% of the newly translocated sites. Rapid processing may be inherent to PR; it also occurs in PR of MCF-7 cells. The extent of receptor translocation and of nuclear receptor processing is dependent on the progesterone concentration and on the treatment time, and can be masked by endogenous hormones. Proteolytic enzyme inhibitors (leupeptin, antipain) do not prevent nuclear PR loss. G-C specific DNA intercalators that prevent nuclear estrogen receptor processing (actinomycin D, chromomycin A3) also fail to prevent PR loss, but some A-T specific DNA-binding dyes (chloroquine, primaquine, quinacrine) protect 50-75% of nuclear PR. We conclude that translocated nuclear PR can be quantitatively measured only at early time points because the nuclear receptors are rapidly processed. Furthermore, the processing step may involve an interaction of receptors with DNA since it can be partially blocked by DNA-binding agents.

The effect of progesterone in vivo on the subcellular distribution and properties of progesterone receptors in guinea pig uterus

The distribution of progesterone receptors between cytosol and nuclear fractions of guinea-pig uterus was determined after the injection of 1 mg/kg and 10 mg/kg progesterone. After the lower dose, the concentration of cytoplasmic receptors decreased to approx. 2 3 of the preinjection level within 30–60 min and then gradually increased to almost the control value by 24 h. The nuclear receptors increased by an equivalent amount within 30–60 min and were retained at a slightly lower concentration for 4 h. Nuclear receptors decreased to the control level within 8 h and were not changed further. After 10 mg/kg, the cytoplasmic receptors decreased to approx. 1 2 the control value within 30 min and thereafter continued to decrease gradually. The nuclear receptors increased correspondingly to a maximum after 30 min and remained at somewhat less than the maximum concentration for 4 h. By 8 h the concentration of nuclear receptors had decreased to the control level. The properties of the cytoplasmic receptor (sedimentation coefficient of 7S (unactivated) and 5.5S (activated); K D 6–9 nM) were not changed after the administration of progesterone. However, the properties of the nuclear receptor changed in some respects. When exchange with [ 3H]-R5020 was carried out on nuclei suspended in 5 mM phosphate buffer, at least 80% of the specifically bound 3H was found in the supernatant. The tritium was not absorbed by dextran-coated charcoal and on sucrose density gradient centrifugation, a binding peak with a sedimentation coefficient of 3.5S was seen. Binding was temperature sensitive and was suppressed by unlabeled steroids in the following order: progesterone > 5α-pregnanedione testosterone cortisol. The nuclear receptor, extracted by 0.3–0.5 M KCl, also had a sedimentation coefficient of 3.5S on sucrose gradients in 5 mM phosphate. Competition by unlabeled steroids was similar to that seen with the receptor soluble in 5 mM phosphate.

Purification and Properties of Progesterone Receptors from Chick Oviduct*

Purification and Properties of Progesterone Receptors from Chick Oviduct^*