Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., O?ate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.     

The X-ray Structure of RU486 Bound to the Progesterone Receptor in a Destabilized Agonistic Conformation

Here we describe the 1.95 Å structure of the clinically used antiprogestin RU486 (mifepristone) in complex with the progesterone receptor (PR). The structure was obtained by taking a crystal of the PR ligand binding domain containing the agonist norethindrone and soaking it in a solution containing the antagonist RU486 for extended times. Clear ligand exchange could be observed in one copy of the PR ligand binding domain dimer in the crystal. RU486 binds while PR is in an agonistic conformation without displacing helix 12. Although this is probably because of the constraints of the crystal lattice, it demonstrates that helix 12 displacement is not a prerequisite for RU486 binding. Interestingly, B-factor analysis clearly shows that helix 12 becomes more flexible after RU486 binding, suggesting that RU486, being a model antagonist, does not induce one fixed conformation of helix 12 but changes its positional equilibrium. This conclusion is confirmed by comparing the structures of RU486 bound to PR and RU486 bound to the glucocorticoid receptor.The drug RU486, also known as mifepristone, is the only clinically approved antiprogestin (trade name Mifegyne® or Mifeprex®). It is applied to terminate pregnancy and has been clinically tested in many more indications (1, 2). Recently, it was shown that RU486 can prevent mammary tumorigenesis in Brca1/p53-deficient mice, implying a use for RU486 in breast cancer therapy (3).RU486 exerts its clinical effect by binding to the ligand binding domain of the progesterone receptor, although RU486 can also bind to the glucocorticoid receptor (GR)² and weakly to the androgen receptor (4). All these nuclear receptors are close sequence homologs (5). Because the anti-GR activity of RU486 might be problematic in chronic administration (1), past research has focused on finding RU486 variants with more selectivity (4, 6–10) (Open in a separate windowDespite the clinical importance of RU486, there is currently no three-dimensional structure of it bound to PR, its principal target. However, other complexes have been informative, such as that between the PR ligand binding domain and asoprisnil, which is biochemically a full antagonist and is chemically related to RU486 (11). Also informative is the crystal structure of RU486 bound to the GR LBD (12). In all these structures the antagonists bind to a receptor conformation in which the C-terminal helix (called helix 12) is displaced compared with structures of bound agonists. This so-called helix 12 displacement was first seen in the structure of raloxifene bound to the estrogen receptor α, and it is commonly thought to be a general nuclear receptor mechanism (5, 13).The indications from x-ray structures that in PR, RU486 can induce displacement of helix 12 are supported by biochemical data. For instance the truncation of the PR C terminus induces RU486 to act as agonist (14). Also, the C terminus of PR becomes prone to proteolysis when RU486 binds (15). Finally, from modeling RU486 into the structure of bound progesterone, it has been concluded that the 11β substitution of RU486 (16).Despite the evidence that RU486 can induce displacement of helix 12, it is still unclear if RU486 obligately dissociates helix 12 through steric repulsion or if RU486 allows multiple positions of helix 12 but changes their dynamic equilibrium. In this latter theory, called the dynamic model, RU486 would also be able to bind when helix 12 is in an agonist conformation. Indeed, under rare conditions RU486 can function as an agonist (7), and the compound asoprisnil, in vivo, is known as a partial agonist (11). The use of corepressor peptides in crystal complexes, such as in the PR-asoprisnil complex (11), might lock helix 12 into its final antagonist position, whereas the compound alone would have more subtle effects. The dynamic model controversy has also arisen with other nuclear receptors, indicating its wider scope (13, 17).To further elucidate the mechanism of RU486, we have determined the three-dimensional structure of RU486 bound to PR. For this, we developed a novel protocol in which ligands are exchanged in an existing PR crystal in which norethindrone is bound. The crystal lattice restricts the position of helix 12 to the agonist position, but RU486 is still able to bind, proving that it is sterically compatible with an agonist position of helix 12 and suggesting that RU486 works through changing the dynamic equilibrium of helix 12. Comparing our structure with the asoprisnil complex gives insight into the mechanism of helix 12 destabilization, which is confirmed by comparing our structure to that of RU486 bound to GR.     

Mammalian progesterone receptor shows differential sensitivity to sulfhydryl group modifying agents when bound to agonist and antagonist ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.     

Dissociation of steroid receptor coactivator 1 and nuclear receptor corepressor recruitment to the human glucocorticoid receptor by modification of the ligand-receptor interface: the role of tyrosine 735

Within the human glucocorticoid receptor (GR) steroid binding pocket, tyrosine 735 makes hydrophobic contact with the steroid D ring. Substitution of tyrosine735 selectively impairs glucocorticoid transactivation but not transrepression. We now show, using both mammalian two-hybrid and glutathione-S-transferase pull downs, that such substitutions reduce interaction with steroid receptor coactivator 1, both basally and in response to agonist binding. Using a yeast two-hybrid screen we identified one of the three nuclear receptor interacting domains (NCoR-N1) of nuclear receptor corepressor (NCoR) as interacting with the GR C terminus in an RU486-specific manner. This was confirmed in mammalian two-hybrid experiments, and so we used the NCoR-N1 peptide to probe the GR C-terminal conformation. Substitution of Tyr735phe, Tyr735val, and Tyr735 ser, which impaired steroid receptor coactivator 1 (SRC1) interaction, enhanced NCoR-N1 recruitment, basally and after RU486. RU486 did not direct SRC1 recruitment to any of the GR constructs, and dexamethasone did not allow NCoR-N1 recruitment. Using a glutathione-S-transferase pull-down approach, the NCoR-N1 peptide was found to bind the full-length GR constitutively, and no further induction was seen with RU486, but it was reduced by dexamethasone. As both SRC1 and NCoR are predicted to recognize a common hydrophobic cleft in the GR, it seems that changes favorable to one interaction are detrimental to the other, thus identifying a molecular switch.