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.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

Hepatic glucocorticoid receptor behaves differently when its hormone binding site is occupied by agonist (triamcinolone acetonide) or antagonist (RU486) steroid ligands

We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [3H]TA or [3H]RU486 bound to macromolecule(s) which sediment as 8-9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2-10 mM dithiothreitol (DTT), beta-mercaptoethanol (beta-MER), and monothioglycerol (MTG) caused a 2-3 fold increase in the [3H]TA and [3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [3H]TA-receptor complex to DNA-cellulose by 70-90 percent whereas DNA binding of [3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites.     

Identification of the functional domain of glucocorticoid receptor involved in RU486 antagonism

Mifepristone, also known as RU486, is a potent glucocorticoid receptor (GR) antagonist that inhibits GR-mediated transactivation. As an alternative to existing antidepressants, RU486 has been shown to rapidly reverse psychotic depression, most likely by blocking GR. Although a number of studies have demonstrated RU486-induced GR antagonism, the precise mechanism of action still remains unclear. To identify the GR domain involved in RU486-induced suppression, GR transactivation and nuclear translocation were examined using cells transfected with human GR (hGR), Guyanese squirrel monkey GR (gsmGR), and GR chimeras into COS-1 cells. RU486 showed a much more potent suppressive effect in gsmGR-expressing cells versus hGR-expressing cells, without significant cortisol- or RU486-induced changes in nuclear translocation. A GR chimera containing the gsmGR AF1 domain (amino acids 132–428) showed a marked decrease in luciferase activity, suggesting that this domain plays an important role in RU486-induced GR antagonism. Furthermore, fluorescence recovery after photobleaching (FRAP) analysis indicated that, in the presence of RU486, gsmGR AF1 domain contributes to GR mobility in living COS-1 cells. Taken together, these results demonstrate, for the first time, that the antagonistic effects of RU486 on GR transactivation involve a specific GR domain.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Corepressor binding to progesterone and glucocorticoid receptors involves the activation function-1 domain and is inhibited by molybdate

Corepressors are known to interact via their receptor interaction domains (RIDs) with the ligand binding domain in the carboxyl terminal half of steroid/nuclear receptors. We now report that a portion of the activation function-1 domain of glucocorticoid receptors (GRs) and progesterone receptors (PRs), which is the major transactivation sequence, is necessary but not sufficient for corepressor [nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT)] RID binding to GRs and PRs in both mammalian two-hybrid and coimmunoprecipitation assays. Importantly, these two receptor sequences are functionally interchangeable in the context of GR for transactivation, corepressor binding, and corepressor modulatory activity assays. This suggests that corepressors may act in part by physically blocking portions of receptor activation function-1 domains. However, differences exist in corepressor binding to GRs and PRs. The C-terminal domain of PRs has a higher affinity for corepressor than that of GRs. The ability of some segments of the coactivator TIF2 to competitively inhibit corepressor binding to receptors is different for GRs and PRs. With each receptor, the cell-free binding of corepressors to ligand-free receptor is prevented by sodium molybdate, which is a well-known inhibitor of receptor activation to the DNA-binding state. This suggests that receptor activation precedes binding to corepressors. Collectively, these results indicate that corepressor binding to GRs and PRs involve both N- and C-terminal sequences of activated receptors but differ in ways that may contribute to the unique biological responses of each receptor in intact cells.