Form Of Progesterone Receptor Research Articles

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201–215, 231–245, and 247–261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247–261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [ 3H]estradiol. The antibodies bound to ERs with high affinity ( K D = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1.000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens. (Steroids 55:196–208, 1990).

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo- p-dioxin: Use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by “3-methylcholanthrene-type” compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of ≈9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of ≈11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of ≈9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.

Biochemical characterization and subunit structure of quail oviduct progesterone receptor

The cytosolic quail oviduct progesterone receptor (PR) was studied under conditions that lead either to its stabilization or activation. Sedimentation coefficients and Stokes radii were respectively 7.8 ± 0.2 S and 7.6 ± 0.8 nm for the non transformed receptor (8S PR) and 3.9 ± 0.4S and 4.8 ± 0.6 nm for the transformed receptor (4S PR). The calculated molecular weight was 261 ± 29 KDa for the 8SPR and 83 ± 10KDa for the 4S PR. Density gradient centrifugation analyses showed that the monoclonal antibody BF4, directed against the 90 KDa hsp of the chick oviduct, cross-reacted with the quail 8S PR but not with the 4S PR. In contrast, polyclonal IgG-G6 antibodies, raised against the purified non transformed chick PR, cross-reacted with the non transformed as well as the transformed quail PR. The quail 8S PR was partially purified using NActac-Sepharose affinity chromatography and DEAE-Sephacel chromatography from cytosol prepared with protease inhibitors. The subunit structure of the purified quail and chick 8S PR were compared using SDS-PAGE, photoaffinity labeling and western immunoblotting. The quail PR was composed of two different proteins: a non-hormone binding protein (M r ∼ 90 KDa) which exhibited the same properties as the 90 KDa hsp protein of the chick oviduct and a single hormone binding subunit (M r ∼ 101 KDa). Based on its binding and immunological properties, this protein corresponded to the “B” form of the chick PR but was significantly smaller. In the quail cytosol or in purified PR preparations the “A” form of the PR was virtually absent; this observation is discussed.

Human Progesterone Receptor Binding to Mouse Mammary Tumor Virus Deoxyribonucleic Acid: Dependence on Hormone and Nonreceptor Nuclear Factor(s)

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.

Progesterone receptor is constitutively expressed in chicken intestinal mesothelium and smooth muscle.

We have previously shown that progesterone receptor (PR) is expressed in the mesothelium of the chick oviduct and ovary and in the smooth muscle cells of the oviduct and the bursa of Fabricius. Here, we investigated the presence of PR in different parts of the peritoneum and abdominal organs using an immunohistochemical staining based on monoclonal antibodies against chicken PR. In 4-week-old sexually immature chicks, PR expression was located in the mesothelial cells of different parts of the peritoneum, in a thin layer of muscle cells of the ileum and throughout the muscle tissue of the colon and cloaca. In chicks of the same age treated with estrogen, PR was demonstrated similarly in the peritoneum and in the smooth muscle cells of the ileum, colon and cloaca. Using 25-week-old mature chickens, PR was also detected in identical tissues. Immunoblotting of the cloacal cytosol revealed the B form, but no A form of PR, both of which were found in the oviduct samples. Muscle cells of the duodenum and jejunum were not found to contain PR. Estrogen treatment was not needed to stimulate the production of PR in any of the tissues examined. We therefore conclude that the B form of PR is constitutively expressed in the mesothelial cells in different parts of the peritoneum and also in the smooth muscle cells of the ileum, colon and cloaca.

Assay of activated form of progesterone receptor with ATP-sepharose column chromatography

Biological actions of progesterone are correlated with the ability of the progesterone receptor(PR) to bind to nuclear acceptor sites. Measurement of not only the presence of PR in a tissue but also the amount of that receptor which is able to bind to nuclear acceptor sites is important in predicting tissue response to progesterone. Activation of PR is required for effective binding to chromatin. Since the dextrancoated charcoal assay does not distinguish between an activated and a non-activated receptor, a rapid, relatively simple assay is needed which can account for an activated form of PR. Therefore, ATP-Sepharose column chromatography was tested to identify an activated form of PR. PR in crude uterine cytosol from estrogen-primed immature rabbits was labeled with 3H-progesterone at 0 degree C. The labeled PR was then incubated at 4 degrees C with uterine chromatin from ovariectomized mature rabbits. The freshly prepared PR had little capacity to bind to the chromatin. After activation manipulation at low temperature, low ionic strength and neutral pH, this PR was able to bind to chromatin approximately fourfold more than that activated by heating at 25 degrees C. The affinity of the activated and the non-activated PR for ATP was evaluated on ATP-Sepharose column chromatography. The activated PR was selectively adsorbed onto columns of ATP-Sepharose, and the binding ability of the activated PR to ATP paralleled that of the rabbit uterine chromatin. These results suggest that ATP-Sepharose column chromatography could be useful to identify an activated PR as a substitute for chromatin binding assay.

The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor

The salt-induced (0.3 M KCl) transformation of the non-transformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor (PR) was analyzed by density gradient ultracentrifugation (8S → 4S conversion) and DNA-cellulose chromatography (non-binding → binding forms). After 1 h treatment at 2 C, > 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist-and antagonist-bound non-transformed receptors were similar and the calculated Mr were ≈ 283,000 and ≈ 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.

Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E. coli.

Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively. The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.5 nM and was specifically photocross-linked with the synthetic progestin R5020 in crude bacterial lysates. Labelling of intact bacterial cells with [3H]R5020 revealed that the majority, if not all, of the bacterially produced hormone-binding domain was active. No differences in the binding to a synthetic palindromic glucocorticoid/progestin-responsive element (GRE/PRE) were found when the bacterially produced cPR DNA-binding domain was compared in methylation interference assays with the full-length chicken progesterone receptor form A expressed in eukaryotic cells. The study of dissociation kinetics, however, revealed differences in the half-life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA-binding domain. DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific 'footprints' on the mouse mammary tumour virus long terminal repeat (MMTV-LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor.

Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element.

We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.

Receptors bound to antiprogestin from abortive complexes with hormone responsive elements.

The mechanism of action of antisteroids is not understood and explanations of their antagonistic activity have been sought at all levels of hormone action. It has been proposed that antisteroids, after binding to receptor, trap it into a non-activated (non DNA-binding) form possibly through interaction with a heat-shock protein of relative molecular mass (Mr) 90,000 (90 K), or that the antisteroids provoke binding of receptor to nonspecific DNA sites but not to hormone responsive elements (HREs), or that the antisteroid-receptor complexes can bind to HREs but form abortive complexes that fail to regulate transcription. We have constructed a deleted cDNA encoding a mutant form of rabbit progesterone receptor which exhibits constitutive activity, that is, binds to HREs in the absence of hormone and thus bypasses the first two steps discussed above. Co-transfection experiments allowed the expression of both constitutive and wild-type receptors in the same recipient cells. Antiprogestin RU486-wild-type receptor complexes completely suppressed the activity of the constitutive receptor on a reporter gene, showing that the inhibition is at the level of their common responsive elements.

Purification of T47D Human Progesterone Receptor and Immunochemical Characterization with Monoclonal Antibodies

In order to obtain steroid-independent probes for human progesterone receptor (PR), the A [88-93 kilodalton (kDa)] and B (109-119 kDa) forms of PR from T47D human breast cancer cells were partially purified and used to generate a series of 14 monoclonal antibodies. Initially, unoccupied PR was isolated from cytosol extracts by steroid affinity chromatography, followed by chromatography on diethylaminoethyl Bio-Gel. The partially pure (3-15%) PR consisted of two steroid-binding components that migrated at 89 kDa and 109 kDa in reducing sodium dodecyl sulfate gels after being photoaffinity labeled with the synthetic progestin [3H]R5020. Two unique monoclonal antibodies to PR were derived from a male Lewis rat immunized with this material. One of these antibodies (JU601) was coupled to Sepharose 4B and used to purify T47D nuclear PR for additional immunizations. Highly purified (30-70%) PR migrated as 93 kDa and 119 kDa progestin-binding proteins in sodium dodecyl sulfate gels. In all, thirteen monoclonal antibodies were obtained that recognized epitopes shared by both receptor forms. One mouse immunoglobulin G (KC146) was completely specific for the larger B form. Interestingly, the epitope for this antibody was present on all PRs tested, including the B form of PR from chicken oviduct, whereas nine other antibodies recognized only human PR and the remaining four cross reacted with rabbit PR. With the exception of the JU145 and JU601 rat immunoglobulin Ms, all antibodies appeared to be completely specific for the A or B forms of PR. Each recognized the cytosol and nuclear forms of occupied as well as unoccupied PR. Although the relationship between B and A was not established, it is clear that an amino-terminal region of B is not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence.

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)

Peptide sequencing of the chick oviduct progesterone receptor form B

The progesterone receptor form B has been isolated to apparent homogeneity from large scale preparations of laying hen oviduct cytosol. The quantities obtained were sufficient to monitor the separation of tryptic peptides on HPLC columns. Using a multi-dimensional microbore HPLC peptide purification protocol, several peptides were isolated in homogeneous form and sequenced up to 34 steps at the sub-40 pmol level using a gas phase sequenator. One of the peptides showed a striking homology with sequences of the putative steroid binding domain of the human glucocorticoid receptor; this region is also conserved in the human and chick estrogen receptor.

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)

Characterization of chromatin binding sites for different forms of uterine progesterone receptor

We tested hamster uterine progesterone receptor (Rp) forms for binding to different chromatin preparations. Similar forms of chick oviduct Rp were used for comparison. Hamster Rp elutes from DEAE-Sephacel in the two peaks, peak I at 115 mM KC1 and peak II at 205 mM KC1. Chick Rp peaks I and II elute at 125 mM and 300 mM KC1, respectively. Both chick and hamster peak I displayed a higher level of binding to SDS-stripped chromatin (DNA) than to crude chromatin or 4 M guanidine hydrochloride (GuHCl)-extracted (nucleoacidic protein, NAP) chromatin while peak II bound 50% better to the NAP chromatin than to crude chromatin or DNA. 10 mM molybdate was used to stabilize Rp and to increase Rp recovery. Molybdate-stabilized hamster Rp elutes from DEAE at the peak II position and like peak II, binds poorly to DNA. Since molybdate prevents receptor activation, DNA-Rp interactions require activated Rp. Because molybdate did not prevent Rp binding to NAP chromatin, we conclude that both activated and unactivated Rp bind well to that matrix. Activated hamster Rp could be extracted from crude chromatin, NAP chromatin and DNA with 200 mM KC1. Unactivated Rp was extracted from NAP only with 6 M GuHCl or NaSCN, whereas KC1, glycerol or pyridoxal 5'-phosphate were not able to remove unactivated Rp from NAP. Various Rp forms did not compete with [ 3H]ORG 2058-Rp for binding to NAP but BSA did compete. Thus a large portion of Rp binding to NAP may represent nonspecific binding rather than binding to a finite number of Rp acceptor sites. These results suggest that the binding of activated Rp to crude chromatin may represent the actual acceptor sites in target cell nuclei. Since the high level of Rp binding sites in NAP chromatin may be an extraction artifact, the involvement of proposed masking proteins in regulating the availability of acceptor sites should be reconsidered. As an alternative to acceptor site regulation, changes in the Rp molecule itself may be important. Rp isolated from hamster uteri on days 1–4 of the estrous cycle was incubated with crude chromatin, NAP chromatin and DNA. The apparent level of Rp binding to chromatin and NAP chromatin increased 2.5-fold from day 1 to day 4, but Rp binding to DNA remained constant. This suggests that ovarian cycle-dependent changes occur in the unactivated Rp which affect its interactions with chromatin, and these changes disappear when receptor is activated. In summary, these studies indicate that the three matrices, crude chromatin-cellulose, NAP chromatincellulose, and DNA-cellulose, bind activated Rp in a KCl-extractable manner. Although NAP chromatin displayed increased salt-resistant Rp binding, such binding was not sensitive to competition with various forms of hormone-Rp complex and it was unaffected by Rp activation state. Our findings indicate that the previous concept of Rp acceptor sites in chromatin should be expanded to include another level of control which appears to involve the receptor protein itself.

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.

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.

Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor.

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)

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.