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Endocrine Sciences Research Group, Faculty of Medicine (A.S., H.G., A.B., C.W., A.W., D.R.) and School of Biological Sciences (C.W., A.W.), Stopford Building, University of Manchester, Manchester M13 9PT, United Kingdom
Address all correspondence and requests for reprints to: Dr. Adam Stevens, Endocrine Sciences Research Group, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: fras{at}fs1.ser.man.ac.uk.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Modified glucocorticoid ligands, which retain either transactivation or transrepression activity (dissociating ligands), provide an interesting insight on receptor function (9, 10, 11). The GR is capable of binding these ligands with high affinity, and the activated receptor is capable of nuclear translocation. It is likely that there are signals encoded within the ligand that act through recognition motifs within the ligand binding pocket to generate a new protein interacting surface on the receptor to regulate macromolecule complex formation. Understanding the nature of this switch has major implications for steroid biology and glucocorticoid therapy of inflammatory disease.
Nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT) are structurally related proteins originally identified as important mediators of gene repression by unliganded heterodimeric nuclear receptors (12, 13). More recently it was shown that steroid hormone antagonists recruit the two corepressors (12, 14, 15). The amount of residual, partial agonist activity of the antagonists was shown to be dependent on the expression level of cofactors (15, 16, 17, 18, 19). The best characterized GR antagonist is RU486, and RU486 promotes interaction between the GR and the receptor interacting domains of NCoR (15). These authors identified ligand-independent interaction between the GR N terminal and NCoR and also found that both dexamethasone and RU486 recruited NCoR to the GR C terminus. This latter finding is surprising as it might be expected that RU486 binding would generate an RU486-specific binding signature. The authors also identified multiple NCoR motifs, N1, N2, and N3, as capable of interacting with the GR, with evidence of cooperative binding (15). Interestingly, overexpression of SMRT has been shown to alter the dexamethasone dose response in transfected cells (17), suggesting a degree of functional interaction between the corepressor and the agonist-liganded GR.
By model building we have previously identified Tyr735 within the human GR as a residue contributing to the ligand binding cleft and forming a hydrophobic interaction with the C16 group of the steroid D ring of dexamethasone (20), confirmed by the subsequently published crystal structure (21). Mutation of tyrosine735, especially to serine, results in selective loss of transactivation with a relative preservation of ligand binding affinity and RelA repression (20).
We now use a panel of Tyr735-mutated GR molecules to dissect the interaction between ligand and receptor, and also post ligand-binding association with coregulatory proteins. Loss of transactivation by mutation to Tyr735 is accompanied by loss of p160 coactivator recruitment. However, none of the Tyr735 mutations allow recruitment of NCoR-N1 in response to dexamethasone. Those mutations that impair p160 recruitment, Tyr735phe, Tyr735val, and Tyr735 ser, result in enhanced NCoR-N1 recruitment in response to RU486.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In fact, loss of the terminal hydroxyl group of the tyrosine, by mutation to phenylalanine, did not alter the relative potency of hydrocortisone compared with dexamethasone (Fig. 1, A and B). Loss of the 735 side-chain aromatic ring, by change to valine, did reduce the relative potency of hydrocortisone compared with dexamethasone (Fig. 1, A and B) and when the small, polar amino acid serine was introduced at GR735, hydrocortisone had barely detectable activity up to 1000 nM (Fig. 1B). RU24858 has been reported to reduce transactivation activity compared with dexamethasone (11), but we found it caused significant, GR-dependent transactivation on wild-type GR (wtGR) (Fig. 1C). RU24858, with a 16-methyl group, had the same rank order of potency across the Tyr735-mutated GR molecules as dexamethasone, in contrast to the effect seen with hydrocortisone (Fig. 1B).
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RU486 Antagonism and Partial Agonist Activity Through Tyr735-Substituted GR
RU486 is a high-affinity GR antagonist, and we found that it efficiently antagonized dexamethasone transactivation on all the GR molecules examined (Fig. 2A). Furthermore, RU486 had significant, partial agonist activity on all the GR molecules analyzed (Fig. 2B), up to 5% of dexamethasone on the wtGR.
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). There was no ligand-independent interaction, and no interaction seen with 10 µM dexamethasone (Fig. 3A). The strength of this interaction was further quantitated using liquid cultures (Fig. 3B). We also found a strong dexamethasone-dependent interaction between the same GR construct and the receptor interacting domain (RID) of human SRC1 (SRC1-RID; amino acids 569-821) in the same assays (data not shown). There was no RU486-dependent interaction between the GR and SRC1 (data not shown).
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). Dexamethasone did not allow the hNCoR-N1/GR interaction (Fig. 4A). In contrast, RU486 did not promote recruitment of SRC-RID to the wtGR LBD, but SRC1-RID was recruited by 10 nM dexamethasone (Fig. 4B). These effects were specific as there was no interaction seen with the empty Gal4 vector (Fig. 4C).
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). GST-SRC1-RID pulled down little more GR than the GST alone, but 10 µM dexamethasone caused a 23-fold induction in the amount of GR pulled down by GST-SRC1-RID (P < 0.001; Fig. 5B), whereas RU486 caused no increase in GR/SRC1-RID interaction (Fig. 5, A and B). In contrast, there was a significant basal interaction between GST-hNCoR-N1 and the GR, compared with the GST control (Fig. 5A). RU486 caused no further increase, but dexamethasone inhibited the GST-NCoR-N1/GR interaction by more than 50% (P = 0.035) (Fig. 5B).
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).
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). Therefore, we examined RU486-dependent recruitment of SRC1-RID to the mutated GR LBDs. There was no ligand-dependent interaction seen with any of the mutated GR LBD constructs or with the wtGR LBD (Fig. 6B). We also examined the effects of progesterone, but again found no recruitment of SRC1-RID to the GR LBDs up to 500 nM concentration (data not shown).
There was reduced ligand-independent interaction with SRC1-RID for both the GR735phe (0.75-fold) and GR735val (0.67-fold) mutants (P = 0.035). The GR735ser had the lowest basal interaction (0.64-fold; P = 0.005; Fig. 6C).
Analysis of Ligand-Dependent Recruitment of SRC1-RID to the Tyr735-Mutated Full-Length GR Molecules: GST Pull-Down Assay
We also used our validated GST pull-down assay to measure changes in GR/SRC1 interaction induced by ligand binding. As shown above, the in vitro interaction between these two proteins is specific to agonist ligands.
We examined the absolute amounts of recruited full-length GR to the GST-SRC1-RID, by quantitating the radioactivity pulled down relative to input. Using this approach, we found there was no significant difference in basal SRC1-RID recruitment between the wtGR and the mutated GR molecules (n = 4; P = 0.412; Fig. 7B). Dexamethasone caused a significant increase in SRC1-RID recruitment to the wild type (TYR; P = 0.005), Tyr735phe (PHE) GR (P = 0.024), and Tyr735val (VAL) GR (P = 0.017). The Tyr735 ser (SER) showed no significant increase over basal interaction. RU486 did not induce SRC1 recruitment to any of the GR molecules examined (data not presented). All four GR molecules showed greater binding to the GST-SRC1 than to the GST-only control in the absence of dexamethasone, but this barely reached significance (TYR, P = 0.045; PHE, P = 0.048; VAL, P = 0.021; SER, P = 0.05).
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Effects of GR Tyr735 Mutations on Coactivation by SRC1
The mammalian two-hybrid analysis suggests that mutation of Tyr735 reduces recruitment of SRC1. In-vitroanalysis also shows a reduced dexamethasone dependent recruitment of SRC1 to the mutant Tyr735 molecules. Therefore, we measured the effect of over expressing full-length SRC1 on transactivation of the MMTV-luc reporter by the various GR molecules. wtGR (TYR) shows a marked enhancement of dexamethasone transactivation when SRC1 is expressed (Fig. 8), and the effect is also seen with the Tyr735phe (PHE). In contrast the effect on transactivation by Tyr735val (VAL) is more modest, and there was minimal impact on transactivation by Tyr735 ser (SER; Fig. 8).
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). In contrast to the agonist-directed recruitment of SRC1, RU486 promoted more efficient recruitment of hNCoR-N1 peptide to the mutated GR LBDs than to the wtGR LBD (Fig. 9A). Dexamethasone did not promote hNCoR-N1 recruitment to any of the GR mutants examined (Fig. 9B). There was significant interaction between hNCoR-N1 and GR735phe, -val, and -ser under ligand-free conditions compared with the wtGR (Fig. 9, A and B), in contrast to the reduced interaction seen between the mutated GR LBDs and SRC1-RID (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(PPAR), the LBD of the GR is likely to be responsible for interactions both with p160 family coactivators (SRC1) and the NCoR/SMRT corepressors (Fig. 10). There may be significant overlap between these GR surface domains (14, 21, 22).
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B (NFB) RelA (20). Therefore, we concluded that Tyr735 was important for interpretation of ligand.
Our model of dexamethasone binding to the LBD of the GR suggested an interaction with the 16-methyl group on the steroid D ring (Fig. 1A), and the crystal structure confirms this (21). Therefore, we compared the relative activities of an agonist steroid with the methyl group, dexamethasone, with one that lacked this group, hydrocortisone (Fig. 1, A and B). We also examined the activity of the partial agonist RU24858 (Fig. 1C), the low-affinity GR antagonist progesterone, and the high-affinity antagonist RU486 (Fig. 2). Tyr735phe did not alter the relative potency of hydrocortisone compared with dexamethasone, but Tyr735-val and Tyr735ser showed disproportionate reductions in hydrocortisone potency, indicating the importance of the Tyr735 interaction with the steroid D ring. Previously Lind et al. have noted that substitution of methionine at 560, which lies close to the Tyr735 within the GR ligand-binding pocket (21), has a differential impact on transactivation by synthetic glucocorticoids (23), again confirming the functional interaction between this portion of the ligand binding pocket and the steroid D ring. RU24858, which also has a 16-methyl group, had significant agonist activity on all the four constructs examined and showed the same rank order of potency as dexamethasone. This again suggests the functional interaction between Tyr735 and the 16-methyl group of some synthetic glucocorticoids. RU486 had weak agonist activity on all the four GR molecules, and progesterone had no agonist activity on any of the GR constructs tested.
To investigate the consequences of modification to the ligand/receptor interface for cofactor recruitment, we established a series of protein interaction assays. RU486, in common with other steroid receptor antagonists, leads to recruitment of corepressor proteins (15, 16, 24, 25, 26) and, in our assays, is both an efficient antagonist of dexamethasone transactivation and a partial agonist. Interestingly, NCoR appears to interact with multiple domains of the GR (15). We initially screened the NCoR-N1 domain using a yeast two-hybrid approach for steroid-dependent interaction with the GR C terminus (Fig. 3). In contrast to the in vitro analysis of Schulz et al. (15), we found significant recruitment of NCoR-N1 to the GR C terminus in an RU486-specific manner. It was previously found that including multiple copies of the NCoR N domains enhanced the interaction with the GR C terminus using a GST pull-down approach, but that this effect was seen using both dexamethasone and RU486 as ligands (15). This suggests that there is a loss of ligand specificity in protein interaction, possibly because of cooperative binding of multiple GR molecules to the NCoR peptide in a manner similar to that between retinoic acid receptor/retinoic X receptor and NCoR (13). However, we found that the NCoR-N1 peptide can act as a probe for the RU486-specific conformation of the GR C terminus. This was further examined using a mammalian two-hybrid approach, and the same pattern of RU486-specific interaction between GR LBD and NCoR-N1 was seen.
Because the studies in yeast and mammalian cells had shown a distinct pattern of ligand-dependent hNCoR-N1/GR and SRC1-RID/GR interaction, the proteins were also analyzed in vitro. There was a significant interaction between the GR and hNCoR-N1 in the absence of ligand. RU486 did not further enhance this interaction, but as the proportion of input GR pulled down by hNCoR-N1 was approximately 50% of that pulled down by SRC1 in the presence of dexamethasone, the interaction appears significant. Studies using dexamethasone show greater than 50% reduction in hNCoR-N1/GR interactions. This pattern of ligand-dependent dissociation of corepressors (NCoR) from nuclear receptors has been well defined for the heterodimeric nuclear receptor family (12, 13) and, under cell-free conditions, appears to be preserved for the GR.
The two-hybrid analysis suggests that antagonist is required for NCoR recruitment to GR, whereas the GST pull-down assay suggests that interaction is possible in the absence of ligand, and that antagonist adds little to the interaction. In intact cells the unliganded GR is sequestered in the cytoplasm, and is, therefore, in a separate compartment from the nuclear located corepressors. The GR expression vector used for the mammalian two-hybrid analysis directs proteins to the nucleus by virtue of a powerful nuclear localization signal (27). Indeed, we have shown by immunohistochemistry that all the GR-VP16 constructs are expressed and are located in the nucleus in the absence of ligand (data not shown). Other investigators have also described discrepant results when they compared mammalian two-hybrid against GST pull-down assays when examining steroid receptor interactions with NCoR proteins (16); as a result, additional controls on steroid receptor interactions with corepressors in living cells have been proposed (16).
Using both mammalian two-hybrid and GST pull-down approaches the recruitment of SRC1 to the GR was seen to be reduced by the Tyr735phe variant and reduced further by the Tyr735val; there was no significant induction seen when the Tyr735ser variant was examined. These results are concordant with those presented previously in which the rank order of potency for transactivation was wild type > Tyr735phe > Tyr735val > Tyr735 ser (Fig. 1; Ref. 20).
If the Tyr735 mutations reduce SRC1 recruitment to the GR, then coactivation by SRC1 would be expected to be impaired also. In fact there was no observed difference in SRC1 coactivation between the wtGR (Tyr) and the Tyr735phe (PHE). But both Tyr735val (VAL) and Tyr735 ser (SER) showed markedly reduced SRC1 coactivation (Fig. 8). This coactivation assay did not distinguish between the wtGR and the Tyr735phe, probably because the full-length SRC1 construct used synergistically interacted with both GR activation function 1 and 2 (AF1 and AF2) domains (28). More severe disruption of Tyr735 (to Val or Ser) presumably reduced the AF1/AF2 recruitment of full-length SRC1. This provides additional evidence for a Tyr735 effect on SRC family coactivator recruitment to the agonist-liganded human GR.
RU486 did not promote SRC-1 recruitment to the wtGR, nor to any of the three Tyr735-substituted GR variants, either in vitro or in vivo. Therefore, it does not appear that the partial agonist activity of RU486 could result from aberrant recruitment of SRC proteins. Others have shown that RU486 does not lead to recruitment of SRC1 to the GR in mammalian cells and have suggested that the partial agonist activity of RU486 observed in some cell types results from interaction of the GR with chromatin remodeling proteins (18).
There was basal interaction between the wtGR C terminus and SRC1 seen in the mammalian two-hybrid, but not in the GST pull-down, assays. This was made clear by the reduction in interaction seen when the Tyr735 was mutated. This implies that there are functional interactions between the unliganded GR and SRC1 in living cells, although the compartmentalization of GR in the cytoplasm in the absence of ligand may reduce the physiological importance of this phenomenon. However, there is evidence of nuclear trafficking of the unliganded GR, and even transient interactions with coactivators may be sufficient to promote changes in gene expression (19, 29).
As RU486 is capable of promoting interaction between the GR and NCoR, and as NCoR and SRC family coactivators are predicted to recognize a conserved hydrophobic cleft made up of helices 3, 4, and 5, we sought to define the effect of changes at Tyr735 on corepressor recruitment (13, 14, 21). We used the NCoR-N1 motif both because we found that it interacted with the GR C terminus in an antagonist-specific manner, and also because the interactions of the closely related SMRT-N1 with PPAR have been analyzed in detail (Fig. 10; Ref. 14). There is conservation of the critical charge clamp residues that in PPAR anchor the corepressor helix (Fig. 10). Strikingly, mutations to Tyr735 that impair SRC1 recruitment in response to dexamethasone enhance recruitment of NCoR-N1 in response to RU486 (Fig. 9). We have shown that the RU486-dependent interaction between GR and NCoR-N1 only occurs in living cells, either yeast or mammalian, and that GST pull-down approaches show the basal interaction between the two molecules only. This may indicate an important role for other proteins or factors in mediating or stabilizing protein interaction. Such discrepancies between in vivo and in vitro analyses have been reported before (16). It is also relevant that the unliganded GR interaction with NCoR may not readily occur in living cells due to compartmental segregation. Therefore we did not extend these studies into GST pull-down assays.
It is clear that mutation of Tyr735 increases the basal, unliganded interaction between NCoR-N1 and the GR C terminus. This is in contrast to the reduction in SRC1 interaction seen under the same conditions, with the same mutated GR molecules. This supports the existence of a functional overlap between the protein motifs responsible for interacting with coactivators in the presence of agonists, and corepressors in the presence of antagonists, as has been proposed for the heterodimeric nuclear receptors (14). By homology with PPAR, we have mapped candidate residues within the GR interacting with the NCoR peptide, and these overlie the motif found to bind to the transcriptional intermediary factor 2 (TIF2) LxxLL peptide (21) (Fig. 10). We also show that mutation to Tyr735 sufficient to reduce transactivation does not allow aberrant recruitment of corepressor to the GR in response to dexamethasone binding, but rather enhances binding to the NCoR-N1 repressor peptide in response to RU486 binding.
Glucocorticoid transactivation is served by recruitment of coactivators to both AF1 and AF2 of the ligand-bound, DNA-anchored GR. It is interesting that the GR C terminus has a cryptic surface capable of interacting with the potent corepressor NCoR either when unmasked by binding to RU486 or under basal, unliganded conditions. Coactivator recruitment to the GR can be selectively reduced by mutations to the LBD that retain high-affinity binding of agonists. These transactivation-defective GR molecules have enhanced recruitment of corepressors both basally and in response to antagonists, but not agonists. Such enhanced recruitment of NCoR might contribute to reduced transactivation (17). Therefore, Tyr735 appears to transmit a signal from the ligand to the surface of the GR, as measured by alterations in cofactor recruitment. However, the most conservative change to the tyrosine (Tyr735phe) did not show the predicted reduction in SRC1 coactivation, which suggests that recruitment of SRC1 is not the controlling factor for GR transactivation. The more severe disruptions to the tyrosine at 735 (Tyr735val, and Tyr735 ser), which result in loss of the aromatic ring and introduction of a small polar side chain, respectively, do result in concordant reductions in SRC1 recruitment and the predicted loss of SRC1 coactivation. Therefore, our data suggest that Tyr735 is an important amino acid for determining the final GR conformation after ligand binding, and contributes to final GR activity, but is not the sole determining element of cofactor recruitment. As both the SRC coactivators and NCoR/SMRT are predicted to recognize a common hydrophobic cleft in the GR, it seems likely that changes favorable to one interaction are detrimental to the other, i.e. a molecular switch.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gal-4 fusion constructs were made using the pBIND vector (Promega Corp.). The HF2 PCR kit (CLONTECH Laboratories, Inc., Oxford, UK) was used to amplify DNA coding for amino acid positions 569-821 of the human SRC1 gene, which includes the receptor interacting domain, RID), and 2262–2440 of the human NCoR gene, including the N1 motif, to give GAL4 SRC1 and GAL4 hNCoR. The specific primers had BamHI and NotI restriction enzyme sites added for ligation into pBIND (SRC1: forward primer, 5'-ggatcccacctagcagatta-aatatacaaccag-3'; reverse primer, 5'-gcggccgcagcgtggg-cagtaactgatc-3'; hNCoR: forward primer, 5'-ggatccttgggctggaagacattatc-3'; reverse primer, 5'-gcggccgctcagtcatca-ctatccgac-3').
The B42 activation domain fusion protein expression plasmids were made by inserting the NCoR and SRC1 gene fragments described above into pB42AD (CLONTECH Laboratories, Inc.) to give hNCoR-AD, and SRC1-AD, respectively.
The human GR LBD between codons 523 and 777 was amplified using the HF2 PCR kit and inserted into the pACT plasmid (Promega Corp.) to give VP16wtGRLBD. The VP16GR735phe, VP16GR735val, and VP16GR35ser plasmids were made by amplifying the GR LBD from the appropriate pcDNA3GR constructs detailed above. The specific primers had BamHI and NotI restriction enzyme sites added for ligation into the vector (forward primer for LBD, 5'-ggatcccaacgttaccacaactcacc-3'; forward primer for full-length, 5'-ggatcctggactccaaagaatcattaactc-3'; reverse primer, 5'-gcggccgccttttgatgaaacagaagttttttg-3').
The same GR LBD gene fragment described above was inserted into pLexA (CLONTECH Laboratories, Inc.) to give LexAGR.
A GSThNCoR-N1 fusion protein expression plasmid was made by inserting DNA coding for amino acid positions 2262–2440 of the hNCoR, including the N1 motif, gene into the pGEX-5X-3 vector (Amersham Pharmacia Biotech, Buckinghamshire, UK). This fragment was amplified using the HF2 PCR kit (CLONTECH Laboratories, Inc.). The specific primers had SalI and NotI restriction enzyme sites added for ligation into the vector (NCoR: forward primer, 5'-gtcgaccttgggctggaagacattatc-3'; reverse primer, 5'-gcggccgctcagtcatcactatccgac-3').
The GST/SRC1-RID plasmid (encoding 569-821) was a kind gift from Dr. Malcolm Parker (33). The pG5luc contains five copies of an upstream activator sequence upstream of luciferase (Promega Corp.). The yeast reporter gene was p8OP-lacZ (CLONTECH Laboratories, Inc.).
All plasmid constructs were sequenced to confirm the presence of the predicted changes and to exclude introduction of errors.
Transfection
COS 7 cells (obtained from ECACC:87021302), were cultured in DMEM with Glutamax (Life Technologies, Inc., Paisley, UK), and 10% fetal calf serum. All transfections were performed using lipofectamine plus (Life Technologies, Inc.), according to the manufacturers’ instructions, as previously described (20). Cells were divided into 24-well plates after transfection, and were treated with steroid for 16 h before harvest. All experiments were performed in triplicate and on at least three separate occasions with similar results.
Transactivation Assay
COS7 cells were seeded at 5 x 105 per 10-cm tissue culture plate and were transfected with 2 µg MMTV-luc, and 1 µg pcDNA3-GR (either wild-type or the three Tyr735-mutated GR cDNAs), and 1 µg CMV-ßGal, to control for transfection efficiency (20). Cell lysates were subjected to both luciferase assay and ß-galactosidase assay as previously described (20). Luciferase was corrected for ß-galactosidase activity as previously described (34).
Coactivation Assay
COS7 cells were seeded at 5 x 105 per 10-cm tissue culture plate and were transfected with 3 µg MMTV-luc, 0.12 µg pcDNA3-GR (wild-type or the three Tyr735-mutated GR cDNAs), either 0.6 µg pSG5-SRC-1 or 0.6 µg of the empty pSG5 vector, and 1 µg CMV-Renilla. Firefly luciferase and Renilla luciferase assays were performed on cell extracts from the same experiment using the dual-luciferase reporter assay system (Promega Corp.) according to the manufacturer’s instructions. All firefly luciferase results were normalized using the Renilla luciferase to control for differences in transfection efficiency.
Yeast Two-Hybrid Analysis
Saccharomyces cerevisae (EGY48) were sequentially transformed with reporter, bait, and then prey. The bait was LexAGR, and the prey was SRC1-AD or hNCoR-AD. Transformations were performed using a small-scale lithium acetate procedure (35).
The transfected yeast cells were replica plated onto synthetic dropout/Gal/Raf plates with X-gal, containing the appropriate treatment (ligand at 50 µM and dimethylsulfoxide vehicle as the no-ligand control).
Suspension cultures were used to quantitate the interaction. Briefly, overnight cultures were used to inoculate liquid SD/Gal/Raf with or without ligands and incubated until midlog phase (OD600 0.5–0.8). The cells were then washed and resuspended in buffer 1 [100 mM HEPES, 155 mM NaCl, BSA 1% (wt/vol), Tween-20, 0.05%]. This mixture was subjected to three freeze-thaw cycles using liquid nitrogen. Buffer 2 (2.23 mM chloro phenol red-ß-D-galactopyranoside in buffer 1) was added to the mixture, the color was allowed to develop, and the OD578 was recorded. The ß-Gal units were then calculated using the following equation: ß-Gal units = 1000 x OD578/(t x V x OD600) [t = elapsed time (in min) of incubation, v = 0.1 x concentration factor, OD600 = A600 of 1 ml of culture]. One ß-Gal unit was defined as the amount that hydrolyses 1 µmol of chlorophenol red-ß-D-galactopyranoside to chlorophenol red and D-galactose per min per cell (36).
Mammalian Two-Hybrid Analysis
COS 7 cells (5 x 105 per 10-cm plate) were transfected with 5 µg of each of the VP16/GR LBD, either Gal4/SRC1, or Gal4/NCoR and the pG5luc reporter plasmid. Firefly luciferase and Renilla luciferase assays were performed on cell extracts from the same experiment using the dual-luciferase reporter assay system following the manufacturer’s instructions. All firefly luciferase results were normalized using the Renilla luciferase to control for differences in transfection efficiency.
Interaction of SRC-1 and GRs in Vitro
GST and GST-SRC1 or GST-NCoR-N1 fusion proteins were expressed in Escherichia coli strain DH5 (Promega Corp.) and were purified as described (37). Briefly, a 50 ml culture of the expression vector was stimulated with 0.5 mM isopropyl-ß-D-thiogalactopyranoside (Sigma, Poole, UK) and grown on for 3 h. The bacteria were then pelleted and resuspended in NETN buffer (20 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.01% Nonidet P-40) supplemented with protease inhibitors (Complete, Roche Molecular Biochemicals, Mannheim, Germany). The bacteria were treated with lysozyme (100 µg/ml) for 15 min at 4 C and then disrupted using a 50 W, 20 kHz Sonicator (Jencons, Leighton Buzzard, UK) on full power for 5 x 30 sec. The sample was then centrifuged to remove the debris and glutathione-sepharose beads (Amersham Pharmacia Biotech) were added to the supernatant. The fusion proteins were allowed to bind to the beads for 1 h at 4 C. The beads were then washed three times in NETN plus protease inhibitors and stored at 4 C.
35S-labeled GR was synthesized using the TNT kit (Promega Corp.) following the manufacturer’s instructions with the full-length, human GR cDNA in pcDNA3 as template, [35S]methionine (Amersham Pharmacia Biotech), and T7 polymerase. Labeled GR was incubated with the GST proteins bound to glutathione-sepharose beads for 1 h at 4 C. The beads were then washed three times in NETN, and boiling in 2x SDS-PAGE sample buffer eluted the bound proteins. Proteins were analyzed by SDS-PAGE, and the gel was stained in Sypro red to visualize the GST fusion proteins. The 35S-labeled GR was then visualized by exposure of the gel to a phosphoimaging plate (BasIII, Fuji Photo Film Co., Ltd.).
The Sypro red-stained acrylamide gels were analyzed under UV light (Alphaimager 2000, Flowgen, Lichfield, UK). The amount of radiolabeled protein present on the gel was quantitated using a phosphoimager (FujiFilm, London, UK; BAS1800) and Aida 2.0 analysis software (Raytest, GMBH, Straubenhardt, Germany).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF, Activation function; CMV, Cytomegalovirus; GR, glucocorticoid receptor; GST, glutathione-S-transferase; LBD, ligand-binding domain; MMTV-luc, mouse mammary tumor virus-luciferase; NCoR, nuclear receptor corepressor; PPAR, peroxisomal proliferator-activated receptor-; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; TIF2, transcriptional intermediary factor; wtGR, wild-type GR.
Received for publication September 12, 2002. Accepted for publication January 21, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B -independent downregulation of NF-B activity by glucocorticoid receptor. EMBO J 16:4698–4707[CrossRef][Medline]
B-dependent mechanism. Mol Pharmacol 56:797–806. Nature 415:813–817[Medline]
NURSA Molecule Pages Link:
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 A. McMaster and D. W. Ray Modelling the glucocorticoid receptor and producing therapeutic agents with anti-inflammatory effects but reduced side-effects Exp Physiol, March 1, 2007; 92(2): 299 - 309. [Abstract] [Full Text] [PDF]
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 R. Donn, A. Berry, A. Stevens, S. Farrow, J. Betts, R. Stevens, C. Clayton, J. Wang, L. Warnock, J. Worthington, et al. Use of gene expression profiling to identify a novel glucocorticoid sensitivity determining gene, BMPRII FASEB J, February 1, 2007; 21(2): 402 - 414. [Abstract] [Full Text] [PDF]
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T. C. Voss, S. John, and G. L. Hager Single-Cell Analysis of Glucocorticoid Receptor Action Reveals that Stochastic Post-Chromatin Association Mechanisms Regulate Ligand-Specific Transcription Mol. Endocrinol., November 1, 2006; 20(11): 2641 - 2655. [Abstract] [Full Text] [PDF]
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S. Sola, J. D. Amaral, P. M. Borralho, R. M. Ramalho, R. E. Castro, M. M. Aranha, C. J. Steer, and C. M. P. Rodrigues Functional Modulation of Nuclear Steroid Receptors by Tauroursodeoxycholic Acid Reduces Amyloid {beta}-Peptide-Induced Apoptosis Mol. Endocrinol., October 1, 2006; 20(10): 2292 - 2303. [Abstract] [Full Text] [PDF]
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H. Garside, C. Waters, A. Berry, L. Rice, H. C Ardley, A. White, P. A Robinson, and D. Ray UbcH7 interacts with the glucocorticoid receptor and mediates receptor autoregulation. J. Endocrinol., September 1, 2006; 190(3): 621 - 629. [Abstract] [Full Text] [PDF]
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 T. Ichijo, A. Voutetakis, A. P. Cotrim, N. Bhattachryya, M. Fujii, G. P. Chrousos, and T. Kino The Smad6-Histone Deacetylase 3 Complex Silences the Transcriptional Activity of the Glucocorticoid Receptor: POTENTIAL CLINICAL IMPLICATIONS J. Biol. Chem., December 23, 2005; 280(51): 42067 - 42077. [Abstract] [Full Text] [PDF]
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 Z Alourfi, R P Donn, A Stevens, A Berry, A McMaster, and D W Ray Glucocorticoids suppress macrophage migration inhibitory factor (MIF) expression in a cell-type-specific manner J. Mol. Endocrinol., April 1, 2005; 34(2): 583 - 595. [Abstract] [Full Text] [PDF]
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H. Garside, A. Stevens, S. Farrow, C. Normand, B. Houle, A. Berry, B. Maschera, and D. Ray Glucocorticoid Ligands Specify Different Interactions with NF-{kappa}B by Allosteric Effects on the Glucocorticoid Receptor DNA Binding Domain J. Biol. Chem., November 26, 2004; 279(48): 50050 - 50059. [Abstract] [Full Text] [PDF]
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C E Waters, A Stevens, A White, and D W Ray Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line J. Endocrinol., November 1, 2004; 183(2): 375 - 383. [Abstract] [Full Text] [PDF]
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 G. Hochhaus New Developments in Corticosteroids Proceedings of the ATS, November 1, 2004; 1(3): 269 - 274. [Abstract] [Full Text] [PDF]
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 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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