High level expression of a truncated chicken progesterone receptor in Escherichia coli

Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains. The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin. This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E. coli. Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols. We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays. Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element. This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies.     

Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.     

Identification of protein contact sites within the glucocorticoid/progestin response element

The glucocorticoid receptor (GR) and the progestin receptor (PR) bind specifically to a variety of DNA sequences, glucocorticoid/progestin response elements (GRE/PRE), located in the proximity of responsive gene promoters. Using the isolated recombinant GR DNA-binding domain (DBD), it has recently been shown that GR interacts with the GRE/PRE, a 15-basepair partially palindromic consensus sequence, as a dimer. In this study an investigation into the GR-GRE/PRE and PR-GRE/PRE interaction has been performed using missing base contact analysis with the tyrosine aminotransferase GREII (TATII) and recombinant GR DBD as well as a fusion protein consisting of the PR DBD fused to Staph. aureus protein-A. GR and PR had identical base contact points, localized within two consecutive major grooves, binding to the same face of the DNA. Ethylation interference was also performed on the GR DBD-TATII interaction. The contact points with the backbone phosphate groups flank the contacts within the major groove for each of the two half-sites. Knowledge of the contact points within the DNA sequence together with the three-dimensional structure of the protein enables modelling of the protein-DNA interaction.     

Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements

We demonstrated previously that two molecules of steroid hormone receptor bound efficiently to a single hormone response element (GRE/PRE) of the tyrosine aminotransferase gene (Tsai et al., 1988). Here, we show that two tandemly linked GRE/PREs conferred progesterone inducibility synergistically to a heterologous TK-CAT fusion gene. Binding studies demonstrated that occupation of one GRE/PRE site by a progesterone receptor dimer increased the binding affinity of receptors for the second GRE/PRE site 100-fold. Thus, the observed synergistic induction of TK-CAT may result from cooperative binding of receptor dimers to the two GRE/PRE sites.     

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.     

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.     

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.     

In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene.

The chicken progesterone receptor (cPR) gene and the ovalbumin (OA) gene, a target of cPR regulation, have been mapped via fluorescent in situ hybridization to the two largest chromosomes of the chicken karyotype. cPR is subtelomeric on the long arm of chromosome 1 and OA is on the long arm of chromosome 2, close to the centromere. A 35-kb cosmid probe for the cPR gene and two genomic fragments of 9.2 and 15 kb for the OA gene were biotin-labeled for nonradioactive localization of the two chicken loci.     

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB–CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB–CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.     

Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.