Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

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.     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: biochemical characterization

Polyclonal antiserum was generated in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR). The PR antigen was partially purified by DEAE-cellulose chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and the 116,000 Mr band excised and injected into guinea pigs. The antiserum recognized on protein blots rabbit uterine PR of Mr 116,000 and 81,000. The antiserum was judged to be specific for PR from normal and malignant human tissues as determined by sedimentation shift on sucrose gradients, immunoprecipitation studies, protein blotting, and fluorographic analysis using photolabelled samples. Comparison of protein blots probed with this polyclonal antiserum or with a recently obtained monoclonal antibody to human PR indicated that similar PR structures were recognized in rabbit and human samples by both antisera. Characterization of the polyclonal antiserum has demonstrated its suitability for investigating the immunolocalization or PR in normal and malignant human tissues as well as the receptor structure detected on protein blots.     

Steroid production by dispersed cells from fetal membranes and intrauterine tissues of sheep

The hypothesis was examined that the fetal membranes and the endometrium and myometrium of pregnant sheep have the ability to produce oestrogens and progesterone from exogenous precursors, and that this capacity might change during the course of pregnancy, and in relation to the onset of parturition. Cells were dispersed from samples of myometrium, endometrium, allantois, chorion and amnion from sheep at Day 50, Days 130-135 of pregnancy, and at term, in labour, and were incubated in the presence of pregnenolone and 20 alpha-dihydroprogesterone as potential precursors for progesterone production, and oestrone sulphate and androstenedione as potential precursors for oestrogen production. In addition, the metabolism of radioactive progesterone and oestrone sulphate by the dispersed cells was examined. Pregnenolone was converted to progesterone in significant amounts by dispersed cells from chorion and endometrium only. At Day 130 and at term this conversion was blocked by the addition of trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity. There was no significant change in the net production of progesterone from exogenous pregnenolone with gestation. 20 alpha-Dihydroprogesterone was converted to progesterone by all tissues, and at each stage of gestation. Formation of progesterone from 20 alpha-dihydroprogesterone was invariably greater than that from pregnenolone, but did not change with pregnancy. Oestrone sulphate was converted to oestrone and oestradiol by all tissues. In the myometrium and chorion this conversion was lower at term than at Day 50 of pregnancy. In contrast, there was very little conversion of androstenedione to unconjugated oestrogen, minimal activity being demonstrable only in dispersed cells from the chorion in some preparations. Radioactive progesterone was converted to radiochemically pure 17 alpha-hydroxyprogesterone by chorion, and to radiochemically pure 20 alpha-dihydroprogesterone by amnion, chorion, allantois and endometrium obtained at term pregnancy. At term [3H]oestrone sulphate was converted to radiochemically pure oestrone by all tissues. We conclude that there is a tissue-specific distribution of different steroid metabolizing enzyme activities in the fetal membranes and intrauterine tissues of pregnant sheep. Of the substrates examined, 20 alpha-dihydroprogesterone and oestrone sulphate were preferred for progesterone and oestrogen production, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel monoclonal anti-rabbit hsp90 antibody: Usefulness for studies on hsp90-steroid receptor interaction

The M_r 90,000 protein associated with steroid receptors in their non-transformed state has been identified as a heat shock protein (hsp90) but the relationship between hsp90 binding and receptor function is still poorly understood. In this work, we have obtained and characterized one monoclonal anti-rabbit hsp90 antibody (7C10), among more than 2000 wells plated. This antibody was able to complex both free and rabbit uterine progesterone receptor-associated hsp90 as demonstrated by sedimentation analysis on sucrose gradients. As assessed by ELISA, 7C10 displayed a high binding affinity for hsp90 ( 4 nM). A standardized and specific competitive binding assay was developed for accurate quantification of hsp90 in rabbit tissues including reticulocyte lysate. 7C10 also permitted immunolocalization of hsp90 in various rabbit tissues. In Western blot, the monoclonal antibody recognized a single polypeptide band of M_r 90,000 in crude or purified rabbit preparations but failed to cross-react with any other mammalian or avian hsp90. These findings suggest that hsp90, a highly conserved protein, is a weak immunogen and elicits a strict species specific immunological response. Owing to its high affinity and specificity for rabbit hsp90, the monoclonal antibody 7C10 was used for purification and total depletion of hsp90 from the reticulocyte lysate, an efficient system for in vitro receptor translation and reconstitution studies. Thus, 7C10 represents a new powerful tool to further investigate the importance of hsp90 in steroid hormone receptor function.     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

The ubiquitous nature of the progesterone receptor binding factor-1 (RBF-1) in avian tissues

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by ～ 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley-Liss, Inc.     

Characteristics of progestin binder in hypertrophic human prostate

The human prostate contains a protein which binds with progesterone in a high affinity and low capacity fashion. Characteristics of the progestin-binding protein in the prostate have been disputable; whether it is progesterone receptor or not. Therefore, the characteristics of the progestin binder in the benign hypertrophic human prostate was examined in the present study. After photoaffinity labeling with 3H-R 5020, the binder in the prostate migrated to the site of 42K on polyacrylamide gel electrophoresis under denaturing conditions, and the mobility was apparently different from that of the progesterone receptor in the human uterine endometrium. There was no protein in the prostate immunoreacted with a monoclonal antibody raised against the human progesterone receptor. It was concluded that the progestin-binding protein in the human prostate was different from the progesterone receptor observed in the female human organs.     

Purification and properties of an oestrogen-stimulated mouse uterine glycoprotein (approx. 70 kDa).

An oestrogen-induced secretory protein from mouse uterine luminal fluid was purified by CM-Affi-Gel Blue chromatography and reverse-phase h.p.l.c. This protein has an apparent molecular mass of approx. 70 kDa both by SDS/polyacrylamide-gel electrophoresis (with or without 2-mercaptoethanol) and by gel-filtration column chromatography, indicating that it exists as a single-chain polypeptide. Further analysis of the protein revealed that it is highly basic (pI greater than or equal to 10) and is a glycoprotein. The N-terminus appears to be blocked to Edman degradation. The partial amino acid sequence of a fragment was obtained by cleavage with CNBr; no sequence homology was apparent between the analysed fragment and other known sequences. The incorporation of [35S]methionine into uterine proteins in vitro revealed that oestrogen treatment of immature mice stimulates both synthesis and secretion of the 70 kDa protein. An enzyme-linked immunosorbent assay with polyclonal antibody was used to determine the tissue distribution of the protein. Tissues such as lung, brain, spleen, muscle, intestine, liver, kidney and ovary of oestrogen-treated mice did not have detectable amounts of the 70 kDa protein. Immunoreactivity was present in uterine and vaginal tissues from oestrogen-treated animals. The 70 kDa protein was not induced by testosterone or progesterone. Although the function of this protein is unknown, it is useful as a marker for the study of oestrogen action in the mammalian uterus as well as regulation of gene expression at the molecular level.     

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

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

Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies

Previous studies have shown that the molybdate-stabilized progesterone receptor from the chick oviduct contains a nonhormone binding component with a molecular weight of 90 000. This protein has also been shown to be associated with some other molybdate-stabilized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, we have developed an isolation procedure based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and one-dimensional peptide mapping. Four immunoglobulin G secreting hybridoma cell lines were generated against the 90 000-dalton antigen. All of the antibodies recognize the 90 000-dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, two of the antibodies complex the molybdate-stabilized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90 000-dalton component in molybdate-stabilized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.     

Interaction of murine progesterone receptors with specific monoclonal antibodies to the avian progesterone receptor

Monoclonal antibodies raised against purified chicken progesterone receptor (PgR) have been described and characterized recently. In this study we have screened these antibodies for cross-reactivity with murine PgR. Of the six anti-PgR antibodies tested, one (alpha PR6) precipitates murine PgR in an assay using protein A-sepharose as an absorbent for the antibody. The antibody is specific for PgR and does not react with the estrogen receptor or the glucocorticoid receptor in the same cytosol. In immunoblot experiments, both alpha PR6 and alpha PR11 recognize a 115,000 Da protein, however, alpha PR11 gives a weaker signal than alpha PR6. In photoaffinity labeling experiments, a 115,000 Da and an 83,000 Da protein covalently bind tritiated R5020 in a receptor-specific way. We conclude that the alpha PR6 antibody can be used as a tool to study the structure and function of the murine PgR.     

Rapid actions of progesterone on granulosa cells

Ovarian granulosa cells are responsive to progesterone but do not express the nuclear progesterone receptor. In an attempt to identify a receptor for progesterone (P4) in granulosa cells (GCs), an antibody built against the ligand binding site of the P4 receptor (i.e. C-262) was used. This antibody detected a 60 kDa protein in GCs as well as spontaneously immortalized granulosa cells (SIGCs). This C-262 detectable protein localizes to the plasma membrane and binds P4. Importantly, this C-262 detectable protein appears to be involved in mediating P4's biological actions. This is based on the findings that C-262 1) blocks P4's ability to inhibit mitogen-induced mitosis and apoptosis and 2) FITC-BSA-conjugated P4 binding to granulosa cells. A C-262 detectable protein was isolated using a C-262 affinity column and sequenced. This analysis identified an unnamed protein referred to as RDA288 that was found in the rat genome (Accession number: XM216160). A nearly identical unnamed protein was found in a cDNA library of mouse lung (Accession number: AK004678). Whether RDA288 functions as a membrane receptor for P4 remains to be determined.     

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.     

Characterization of the chromatin acceptor sites for the avian oviduct progesterone receptor using monoclonal antibodies

Monoclonal antibodies (MAb) against the chromatin acceptor sites for the avian oviduct progesterone receptor were prepared with highly purified hen oviduct acceptor proteins reconstituted to hen DNA. Addition of the MAbs to a cell-free assay blocked progesterone receptor from chick oviduct (PRov) binding to native-like acceptor sites on nucleoacidic protein (NAP) representing a partially deproteinized chromatin, which has been shown to be enriched in these binding sites. However, the antibodies do not block PRov binding to pure DNA, nor do they affect the receptor itself. Estrogen receptor binding to NAP was not inhibited, supporting a receptor specificity of the PRov acceptor sites as reported previously from direct competition studies. These data support earlier studies showing that (1) the reconstituted PRov acceptor sites resemble the native sites, (2) the acceptor sites are receptor specific, and (3) the PRov binding sites of NAP are different from those of pure DNA. While some animal-species specificity in the PRov binding inhibition was observed, no tissue specificity was seen. Direct binding of the antibodies to native acceptor sites was demonstrated in an enzyme-linked immunosorbent assay (ELISA) system. The antibodies showed little recognition of free acceptor protein or DNA alone, indicating specificity for the protein-DNA complex. A partial evolutionary conservation of the nuclear acceptor sites for PRov was shown by the fact that about 50% of the inhibition seen with hen NAP was obtained with NAPs from several other species, and this partial cross-reactivity of the MAbs with the same NAPs from other animal species was also seen in the ELISA.     

Composition, assembly and activation of the avian progesterone receptor

When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30°C), (3) presence of ATP, and (4) presence of Mg²⁺. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl₂ and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.     

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.