Novel mechanisms of progesterone antagonists and progesterone receptor.

The progesterone receptor (PR), as a member of the nuclear receptor superfamily of ligand-dependent transcription factors, activates gene transcription through binding to specific palindromic progesterone response elements (PRE) in the promoter region of progestin-responsive genes. The progesterone antagonists ZK98299 (Onapristone) and RU 486 (Mifepristone) inhibit the transcriptional activity of PR by complex mechanisms at concentrations much lower than the progestins. Altered conformation is central to antagonist inhibition of the transcriptional activity of PR. Antagonists also promote inappropriate association of PR with corepressors. We speculate that the different PR conformations induced by agonist and antagonists results in an asymmetric agonist/antagonist heterodimer that binds inefficiently to palindromic PREs. PR, under the same cellular conditions but with different promotor contexts, can repress (beta-casein) or enhance (3 beta-HSD) signal transducer and activator of transcription (Stat5)-mediated gene activation. The beta-casein promoter appears to contain a composite DNA-binding element for PR and Stat5 and that occupancy by PR in response to progestins or antagonists suppresses Stat5 transactivation function.

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay.

This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro and in vivo, but have distinct effects on receptor conformation.

Three types of transfection experiments were used to detect the abilities of different classes of antagonists to stimulate binding of progesterone receptor (PR) to progesterone response elements (PRE) in intact mammalian cells. These included a promoter interference assay, in which PR binding to PREs positioned between the TATA box and the start of transcription is detected as a reduction of expression of a constitutively active reporter gene, competition of PR antagonist and glucocorticoid receptor agonist for a common glucocorticoid response element/PRE-controlled reporter construct, and activation of a chimeric receptor (PR-VP16) containing the constitutive trans-activation domain derived from the VP16 protein of herpes simplex virus. By each approach, all antagonists tested were equally effective in stimulating PR binding to PREs in the cell. This included previously designated type I (ZK98299) and type II (RU486, ZK98734, and ZK112993) 11beta-aryl substituted steroid analogs. Stimulation of PR binding to PREs in the cell by ZK98299 was of interest because this antagonist has been reported to lack the ability to stimulate PR-DNA binding in vitro by electrophoretic gel mobility shift assay compared with RU486, which promotes efficient binding of PR to PREs. To clarify the apparent discrepancy between intact cell and in vitro results with ZK98299, we altered electrophoretic gel mobility shift assay conditions to allow detection of less stable DNA complexes. Under these conditions, ZK98299 induced the formation of specific PR-PRE complexes. Further analysis of the ZK98299-induced DNA complexes revealed that they exhibited an electrophoretic mobility different from that of the complexes induced by RU486, and the off-rate of PR from DNA was faster than that of the PR bound to agonist. This suggests that ZK98299 promotes a conformational change within PR distinct from that induced by RU486. The present results are consistent with the conclusions that ZK98299 stimulates PR binding to target DNA sequences and that ZK98299 and RU486 represent two mechanistic classes of antagonists based on inducing different conformational changes in PR.

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.