Regulation of progesterone receptor-mediated transcription by phosphorylation.

The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

Molecular cloning of the chicken progesterone receptor.

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Steroid receptor family: structure and functions.

Steroid receptors are a class of molecules that function as both signal transducers and transcription factors. From cloned sequences it is apparent that steroid receptors and other transcription factors belong to a superfamily of proteins that appear to function by similar mechanisms. Functional domains for hormone and DNA binding, and for transcriptional activation, have been defined for several receptors. In some cases, specific amino acids required for function have been identified. The multi-functional steroid receptor molecules are modular in nature in that domains function independently of structural position in receptor molecules and can even function after insertion into unrelated transactivation proteins. The mechanism of receptor action is complex and multistage and a number of unanswered questions remain to be defined. Receptors are inactive in the absence of hormone in vivo; the proposed components of this inactive complex include several proteins and RNA. Theories on the physiological role of HSP 90 in this complex range from an artifactual interaction to an absolute conformational requirement for hormone binding. Although its function has not been demonstrated clearly yet, there is a consensus that one major function is to inactivate receptor by blocking DNA binding. Most of the steroid receptors appear to be nuclear in the absence of hormone. The transformation process produces a receptor molecule that is capable of specific DNA binding and transcriptional activation. The specificity of DNA binding is conferred by as few as three amino acids in the first finger of the C1 region. Receptors appear to bind to DNA as dimers although whether dimers are preformed in cytoplasm remains unknown. Although the DNA binding domain is required for gene activation, other regions of the molecule in the carboxyl and amino terminus enhance activation function. Important interactions of steroid receptors with other receptors and unrelated transcription factors has been proposed and most certainly occurs. Finally, posttranslational modifications such as phosphorylation have been postulated to modulate several functional properties of steroid receptors.

Structure of chick progesterone receptors.

Oviduct progesterone receptors have been purified from both immature chicks and laying hens. The subunits, progestophilins A and B, have different molecular weights, but each is the same from either tissue source. Immunological data show that the subunits are cross-reactive. The hen B protein as isolated to homogeneity contains nonradioactive progesterone in stoichiometric amounts as shown by gas chromatography-mass spectrometry. These studies further confirm our earlier determinations on these proteins.

Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody.

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor.

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.

Dimerization of the chicken progesterone receptor in vitro can occur in the absence of hormone and DNA.

We have analyzed the dimerization of two forms of the chicken progesterone receptor (cPRA and cPRB) by nondenaturing gradient gel electrophoresis and chemical cross-linking with dimethylpimelimidate (DMP). We demonstrate by these two methods that the PRs assemble in vitro into dimers in the absence of DNA, and that dimerization does not require hormone. The cPRA homodimer binds quantitatively to its cognate DNA response element in our nondenaturing gradient gel assay. DMP cross-linking confirms that both forms of the receptor (cPRA and cPRB) assemble into dimers in solution. Finally, in a standard mobility shift assay, chemically cross-linked receptors bind to the progesterone DNA response element with high affinity. We conclude that the PR contains a dimerization motif, which can promote stable subunit-subunit contacts without the presence of hormone in vitro. The complex thus formed expresses sequence-specific DNA-binding activity indistinguishable from that observed in the presence of hormone.

Chicken progesterone receptor is phosphorylated by a DNA-dependent protein kinase during in vitro transcription assays.

We have reported previously that chicken progesterone receptor (PR) is phosphorylated in vivo in response to progesterone administration. Three phosphorylation sites have been reported, two of which show increased phosphorylation in response to hormone and one which is phosphorylated only in response to hormone administration. We found previously that PR lacking the hormone-dependent phosphorylation is active in an in vitro transcription assay. Since the source of general transcription factors is a HeLa nuclear extract which contains many kinases, we have analyzed the receptor for phosphorylation during the in vitro transcription assay. We report here that the receptor is rapidly and efficiently phosphorylated on new sites, causing a change in receptor mobility on sodium dodecyl sulfate-gels. This phosphorylation is strictly dependent upon the presence of double stranded DNA. A DNA-activated protein kinase with similar properties has been isolated previously from HeLa cell nuclei. We find that phosphorylation of PR with this purified enzyme mimics the phosphorylation observed in the transcription assay. These data suggest that a previously undetected additional series of DNA-dependent phosphorylations may be required for activation of the PR.

Chemical and antigenic properties of pure 108,000 molecular weight chick progesterone receptor.

Previous purifications of the progesterone receptor have yielded inadequate amounts of pure protein along with significant amounts of a nonreceptor contaminant. We have taken advantage of the high yield provided by an affinity chromatography method for partial purification and after the incorporation of additional steps, we obtained purified progesterone receptor devoid of detectable contaminants and suitable for chemical analysis. A polyclonal antibody was obtained using the pure receptor as the antigen. The antibody was specific for progesterone-binding receptor. Tissue distribution of cross-reacting material, analyzed by immunoblotting, confirmed the presence of the receptor protein only in the two tissues where progesterone binding has been described in the chick: the oviduct and the bursa of Fabricius. It was absent in receptor-negative tissues such as liver and lung. The receptor was cleaved with cyanogen bromide and trypsin to obtain fragments that were partially sequenced.

Structure-function properties of the chicken progesterone receptor A synthesized from complementary deoxyribonucleic acid.

The chicken progesterone receptor (PR) cDNA has been cloned and sequenced in our laboratory. Functional receptor A was synthesized from cDNA in two independent systems, by transient transfection of receptor-negative COS M6 cells and by in vitro transcription and translation. These receptors exhibited DNA and hormone binding properties similar to the native PR from oviduct. The ability of receptor to induce target gene transcription was measured by cotransfection of receptor-negative CV-1 cells with expression vectors containing the receptor A cDNA and a progesterone-inducible promotor linked to the chloramphenicol acetyl transferase (CAT) gene. In these assays, receptor A produced hormone-dependent induction of CAT activity. In order to define the functional domains of receptor A, expression constructs coding for C-terminal deletion proteins were prepared. Deletion of the C-terminus resulted in loss of hormone binding activity as well as a loss of CAT induction. However, when 290 amino acids were removed from the C-terminus, this severely truncated receptor protein produced hormone-independent target gene activation. Mutant receptor proteins which retained the highly conserved cysteine-rich (C1) region were able to bind to DNA-cellulose, although removal of 290 amino acids from the C-terminus resulted in reduced affinity for DNA. Deletion of part or all of the C1 region resulted in loss of both DNA-binding and transcriptional activation capacities. These results confirm that C1 functions in DNA binding and transcriptional activation and that hormone binding activity can be localized to the C-terminal half of the protein.

Sequence and expression of a functional chicken progesterone receptor.

We have cloned and sequenced 4.5 kilobases (Kb) of cDNA encoding the chicken progesterone receptor. The complete cDNA contains an open reading frame of 2361 nucleotides in length and encodes a polypeptide of 787 amino acids with a mol wt of 85.9 K. At least four mRNA species have been detected in chick oviduct cells. Direct sequencing of variant cDNAs has suggested that two of the mRNAs (4.5 Kb and 3.6 Kb) differ only in the length of their 3'-untranslated regions. A third mRNA (1.8 Kb) produces a truncated polypeptide which encodes the immunoreactive NH2 terminal sequence of the receptor but lacks the hormone binding regional and half of the DNA-binding domain. The polypeptide expressed from the receptor cDNA in progesterone receptor negative Cos M-6 cells is indistinguishable from oviduct progesterone receptor in terms of hormone binding and antibody reactivity. Furthermore, the cloned receptor is capable of activating transcription of a target gene. This activation is progesterone dependent (with half-maximal stimulation at approximately 3.3 x 10(-10) M) and specific for the target gene.

Antibodies to chicken progesterone receptor peptide 523-536 recognize a site exposed in receptor-deoxyribonucleic acid complexes but not in receptor-heat shock protein-90 complexes.

We have prepared monospecific polyclonal rabbit antibodies to the peptide sequence 523-536 of the chicken progesterone receptor. This region, located between the DNA-binding and hormone-binding domains, is predicted by hydropathic analyses to be on the surface of the protein. The synthetic peptide was coupled to keyhole limpet hemocyanin and injected into rabbits. Three rabbits produced antibodies; all three are specific for progesterone receptors, recognize both native and denatured receptor, and do not interfere with either hormone binding or receptor recognition of its DNA response element in gel retardation assays. However, the antibodies do not interact with cytosolic 8S receptor complexes which contain the heat shock protein hsp90, suggesting that this site is occluded in the 8S complex. In contrast, the antibodies recognize a type of receptor dimer which forms on the DNA response element. Thus, these antibodies are a unique tool for studying receptor protein-protein interactions.

Hormone-dependent regulation of chicken progesterone receptor deoxyribonucleic acid binding and phosphorylation.

To further understand the structure-function relationships of the chicken oviduct progesterone receptor, the effects of in vivo and in situ progesterone treatment were studied. Immunoprecipitated receptors isolated from oviduct slices incubated in the presence of H(3)32PO4 exhibited hormone-dependent phosphorylation. This was correlated with an increase in the apparent mol wt of receptors when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased DNA binding of total cytosolic receptors. Further, in vivo progesterone treatment resulted in dissociation of both the A and B receptor forms from nonhormone-binding proteins (such as heat shock protein-90) in vitro when analyzed by sucrose gradient ultracentrifugation. The 4S and 8S receptors were separated by phosphocellulose column chromatography, treated with ammonium sulfate to convert all receptors to DNA-binding forms, and analyzed for binding to DNA cellulose. The 4S receptor produced as a consequence of in vivo hormone treatment had a 3.35-fold higher affinity for DNA and bound to about a 3-fold greater extent than receptor that did not show altered interaction with other proteins. Thus, in vivo progesterone treatment results in increased receptor phosphorylation, altered interaction with heat shock protein-90, and increased DNA binding.

A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae.

A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation, resolution, and biological evaluation of 5-aryl-1, 2-dihydro-5H-chromeno[3,4-f]quinolines: potent, orally active, nonsteroidal progesterone receptor agonists.

Two potent nonsteroidal progestins from the 5-aryl-1, 2-dihydro-5H-chromeno[3,4-f]quinoline class (LG120746 and LG120747) were selected for scale-up, resolution, and biological evaluation of the purified enantiomers. For each quinoline, the levorotatory enantiomer was determined to be the more potent agonist of the human progesterone receptor isoform B (hPR-B) (EC50 < 3 nM), but the dextrorotatory enantiomers retained significant PR modulatory activity (EC50 < 200 nM). In two in vivo rodent models of progestational activity, a pregnancy maintenance assay and a uterine wet weight assay, the two eutomers displayed potent progesterone-like effects. In a third model for progestational activity, the mammary end bud assay, these compounds were significantly less active. These studies demonstrate that certain members of this class of selective progesterone receptor modulators display encouraging and potentially useful tissue-selective progestational effects.

Oviduct progesterone receptor: physical and chemical studies.

Chicken oviduct progesterone receptor has been purified to homogeneity. The protein consists of two dissimilar hormone-binding subunits, A and B, present in equal amounts in the complex. They have molecular weights of 79,000 and 108,000, respectively, as shown by both SDS-gel electrophoresis of the purified proteins and photoaffinity labeling of both with a labeled synthetic progestin. The two subunits show considerable homology (or identity) of structure at the hormone-binding domain, located at the N-terminus of the proteins. Considerable divergence of sequence must exist elsewhere in A and B, as shown by tryptic peptide mapping and by the fact that subunit A has a strong DNA-binding site lacking in B. Both are phosphorylated in vitro by cAMP-dependent protein kinase; this phosphorylation appears to be responsible for creation of a second, weaker progesterone-binding site on each subunit.

Progesterone receptors of chick oviduct.

The chick oviduct progesterone receptor has been purified to homogeneity by affinity chromatography and its molecular action studied in vitro. The native receptor is a 200,000 MW dimer of two dissimilar 4S subunits with different intranuclear function. The receptors directly regulate RNA chain initiation sites in oviduct chromatin by interactions involving target tissue nuclear acceptor sites. There is a 1:1 correspondence between receptor "acceptor" sites and RNA sites. Only the dimer form of the receptor is active in vitro on chromatin templates. The study suggests a novel model for hormone action which can be tested directly in this system.

Structural relationships between the chick oviduct progesterone receptor A and B proteins.

The chick oviduct contains two distinct forms of the progesterone receptor, termed progestophilins A (Mr = 79,000) and B (Mr = 117,000). Although these two hormone-binding proteins differ significantly in physico-chemical characteristics, a good deal of similarity exists between these two molecules. Thus, both proteins display identical hormone-binding kinetics and steroid specificity. The hormone-binding fragments obtained after the action of an endogenous oviduct Ca2+-activated protease are indistinguishable as analyzed by gel filtration chromatography. Both the A and B proteins are capable of binding to DNA-cellulose, although they elute at very different salt concentrations when subjected to gradient elution. In spite of these similarities all attempts to demonstrate the conversion of progestophilin B to A or to show a common precursor to both have been unsuccessful. Therefore, either the A and B receptor proteins are separate products of closely related genes or the conversion of one form to another occurs very rapidly either in vivo or in vitro.

Reversible dissociation of chick oviduct progesterone receptor subunits.

We report here the reversible dissociation of chick oviduct progesterone receptor subunits by pyridoxal 5'-phosphate. This agent has been reported to inhibit binding of steroid hormone receptors to DNA and nuclei (Cake et al., 1978). We have found that pyridoxal 5'-phosphate inhibits binding of chick oviduct progesterone receptors to DNA-cellulose, and also dissociates 6-8S cytosolic receptor aggregates to 4S monomers. Both of these effects are shown to be reversible if pyridoxal phosphate is removed, allowing in vitro reconstitution of receptor aggregates. Fidelity of reconstitution has been assessed by testing the reconstituted aggregate for binding to DNA-cellulose, phosphocellulose, and by studies using sedimentation velocity measurements. By these three criteria, the reconstituted product is indistinguishable from the native cytosol complex from which the monomers were derived. The reconstitution reaction shows an absolute requirement for the presence of both receptor monomers A and B. Titration experiments show a molar ratio of 1:1 for A and B in the reconstituted aggregates. These reconstitution studies confirm our hypothesis (originally based upon dissociation experiments) that native receptor aggregates are composed of the A and B proteins as subunits.

Progesterone receptor interaction in the 5'-flanking regulatory region of the ovalbumin gene.

Highly purified hen oviduct progesterone receptor A subunit has been found to interact preferentially with the 5'-flanking region of the chicken ovalbumin gene. Saturation kinetics showed that 2-fold less receptor was required for half-maximal binding to ovalbumin gene flanking sequences than to plasmid pBR322 sequences. Studies of selective binding were facilitated by a preferential adsorption assay involving gel electrophoretic analysis of [32P]DNA restriction fragments eluted from receptor-DNA complexes isolated on nitrocellulose filters. Approximately 10-fold difference in binding was observed at 37 degrees C for a 1.7 Kb ovalbumin DNA insert compared to binding of receptor to a 3.6 Kb plasmid pBR322 fragment. Selective receptor A binding was dramatically reduced when assayed at 4 degrees C. In addition to temperature dependence, preferential binding required the presence of glycerol or dimethyl sulfoxide during binding. Divalent cations and ionic strength had no apparent effect on the relative sequence selectivity but did alter the total DNA binding observed. Binding data from a variety of different restriction digests suggest receptor A interacts between -135 and -249 bp (upstream) from the start of ovalbumin transcription. Preferential binding to a DNA fragment containing the sequence -16 to -247 compared to an adjacent upstream fragment or plasmid DNA fragment confirm this assignment. This binding site lies within the regulatory sequence required for progesterone induction of a transfected fusion gene in primary chicken oviduct cells.