Glucocorticoid Receptor Ligand Binding Domain Is Sufficient for the ...

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Molecular Endocrinology 19(2):290–311 Copyright © 2005 by The Endocrine Society doi: 10.1210/me.2004-0134

Glucocorticoid Receptor Ligand Binding Domain Is Sufficient for the Modulation of Glucocorticoid Induction Properties by Homologous Receptors, Coactivator Transcription Intermediary Factor 2, and Ubc9 Sehyung Cho, Benjamin L. Kagan, John A. Blackford, Jr., Daniele Szapary, and S. Stoney Simons, Jr. Steroid Hormones Section, National Institute of Diabetes and Digestive and Kidney Diseases/Laboratory of Molecular and Cellular Biology, National Institutes of Health, Bethesda, Maryland 20892 Several factors modulate the position of the doseresponse curve of steroid receptor-agonist complexes and the partial agonist activity of antagonist complexes, thereby causing differential gene activation by circulating hormones and unequal gene repression during endocrine therapies with antisteroids. We now ask whether the modulatory activity of three factors (homologous receptor, coactivator transcription intermediary factor 2, and Ubc9) requires the same or different domains of glucocorticoid receptors (GRs). In all cases, we find that neither the amino terminal half of the receptor, which contains the activation function-1 activation domain, nor the DNA binding domain is required. This contrasts with the major role of activation function-1 in determining the amount of gene expression and partial agonist activity of antisteroids with most steroid receptors. However, the situation is more complicated with Ubc9, where GR N-terminal sequences prevent the actions of Ubc9, but not added GR or transcription intermediary factor 2, at low GR concentrations.

Inhibition is relieved by deletion of these sequences or by replacement with the comparable region of progesterone receptors but not by overexpression of the repressive sequences. These results plus the binding of C-terminal GR sequences to the suppressive N-terminal domain implicate an intramolecular mechanism for the inhibition of Ubc9 actions at low GR concentrations. A shift from noncooperative to cooperative steroid binding at high GR concentrations suggests that conformational changes reposition the inhibitory Nterminal sequence to allow Ubc9 interaction with elements of the ligand binding domain. Collectively, these results indicate a dominant role of GR C-terminal sequences in the modulation of the dose-response curve and partial agonist activity of GR complexes. They also reveal mechanistic differences both among individual modulators and between the ability of the same factors to regulate the total amount of gene expression. (Molecular Endocrinology 19: 290–311, 2005)

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development, differentiation, and homeostasis of multicellular organisms (1–3). This can be accomplished with steroid hormone receptors by three general methods: by causing the de novo expression of previous silent genes, by increasing the total levels of expression of basally transcribed genes, and by modulating the position of the dose-response curve of responsive genes so that they will have different EC50s and therefore will be unequally induced by the same subsaturating concentration of circulating hormone. Research of these general methods has focused on the first two. However, the third method can be equally effective in producing significantly different levels of gene products at physiological concentrations of steroid. Interestingly, changes in the dose-response curve to lower concentrations of agonist steroid have so far been inextricably associated with increases in

HE DIFFERENTIAL CONTROL of gene induction by steroid hormones is especially important for the preferential expression of selected genes during the

First Published Online November 11, 2004 Abbreviations: AF, Activation function; AR, androgen receptor; BAP, bacterial alkaline phosphatase; DBD, DNA binding domain; Dex, dexamethasone; ER, estrogen receptor; GME, glucocorticoid modulatory element; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GRIP, GRinteracting protein; HA, hemagglutinin; LBD, ligand binding domain; MES, 21-mesylate; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; PR, progesterone receptor; SRC, steroid receptor coactivator; SRM, selective receptor modulator; SUMO-1, small ubiquitin-like modifier-1; TIF, transcription intermediary factor; UAS, upstream activating sequence; wt, wild-type. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 290

Cho et al. • Modulation of Glucocorticoid Induction

the partial agonist activity of receptor-antagonist complexes (reviewed in Ref. 4). Such changes in partial agonist activity have attracted extensive recent investigations due to the realization that the blockage of all steroid responses during antisteroid therapies is unnecessary. Selective repression of the target gene will afford the same desired physiological outcome with many fewer of the adverse side effects that result from the indiscriminant repression of all responsive genes. For these reasons, antisteroids with significant amounts of partial agonist activity in at least one assay system (5–10) have considerable clinical potential and have been called selective receptor modulators (SRMs). Multiple factors have been described that modulate the dose-response curves and partial agonist activities of the classical steroid receptors (reviewed in Ref. 4). The first factor to be described was a cis-acting element, called a glucocorticoid modulatory element (GME), that exists 1 kb upstream of the glucocorticoid response elements of the rat tyrosine aminotransferase gene and 3.6 kb upstream from the start of transcription (11–13). Another factor that modulates GR properties is the homologous receptor itself. Elevating the concentration of GR causes a left-shift in the dose-response curve and increases partial agonist activity for antagonists in a manner that is independent of the cells, reporter, and promoter in both transiently transfected cells (6, 14) and transgenic mice (15). Similar results have been reported for progesterone receptors (PRs) (9), mineralocorticoid receptors (MRs) (8), and estrogen receptors (ERs) (8). Coactivators, such as the p160 coactivators transcription intermediary factor (TIF) 2/GR-interacting protein (GRIP) 1, steroid receptor coactivator (SRC)-1, and AIB1/pCIP/ACTR/RAC3/ TRAM1, and comodulators, such as cAMP response element binding protein-binding protein and PCAF, are a third group of factors for modifying GR transactivation properties. The EC50 is lowered, and the partial agonist activity of antiglucocorticoids is increased with exogenous coactivators or comodulators (6, 7). These three factors (GME, GR, and TIF2) appear to modulate GR induction properties by acting through a common rate-limiting intermediate or step (16). An additional, unrelated modulatory factor is Ubc9 (17), which is a human homolog of the E2 ubiquitin-conjugating enzymes of yeast and often conjugates an ubiquitin-like molecule, called small ubiquitin-like modifier-1 (SUMO-1), to proteins in vertebrate cells (18, 19). Greater mechanistic understanding of how the above factors modulate the dose-response curve of GR-agonist complexes, and the partial agonist activity of GR-antagonist complexes, is clearly desirable. Recent studies suggest that the molecular details for this modulation of GR properties are different from those by which the total level of GRregulated gene induction is achieved. For most of the classical steroid receptors, major determinants

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for the amount of gene induction are the activation function (AF)-1 domain in the amino terminal half of the receptor (20–22) and the p160 coactivators, which bind strongly to the C-terminal third of receptors that contains the ligand binding domain (LBD) and the AF-2 domain (23–25). The AF-1 domain of ER and PR also binds the coactivator SRC-1 (26, 27) and is required for the expression of the partial agonist activity of antisteroids bound to PR (28), androgen receptor (AR) (29), and ER (30–35). Likewise, SRC-1 binds to the GR AF-1 domain (36) and augments the activity of the GR AF-1 domain in HeLa cells (26). One study shows that the N terminus of GR is required for the partial agonist activity of the antagonist RU486 (37). In contrast to the above ability of the p160 coactivators to augment the total levels of receptor-mediated gene induction, a truncated form of the coactivator TIF2 has been reported to lose the majority of TIF2’s ability to increase the total amounts of GR-regulated transactivation but still retains most of the modulatory activity for the GR dose-response curve and partial agonist activity of antiglucocorticoids (7). Furthermore, Ubc9 increases the levels of induced gene product under conditions that have no effect on the EC50 or partial agonist activity (17). Changing the position of GME in the reporter gene has unequal effects on the total amounts of activation vs. the EC50 and partial agonist activity (13). Structure/activity relationships of the two proteins (GMEB-1 and -2) whose binding to the GME appears to be required for GME activity (11, 38) reveal that domains of GMEBs that modulate GR properties are separate from those possessing intrinsic transactivation activity (39, 40). Finally, several of the cofactors that cause changes in the EC50 and partial agonist activity do so independently of their ability to alter the levels of induced gene product (6, 41). On the basis of these results, it is reasonable to propose that the role of the AF-1 and AF-2 domains in modulating the GR dose-response curve and partial agonist activity may be different than those that have been implicated for regulating the maximal levels of gene activation. The purpose of this study is to determine the importance of the AF-1 and AF-2 domains for the modulatory activity of several factors: GRs, the coactivator TIF2, and Ubc9. These factors were selected because earlier results in HeLa cells (14) suggested that the full-length GR (and thus the AF-1 domain) is required for modulatory activity with GRs. TIF2 is known to bind to the AF-2 domain in the LBD of GRs (25, 42) and thus could act independently of the AF-1 domain. Ubc9 is of interest because it modulates GR activity via a different mechanism than is used when simply changing the concentrations of GR (17). Thus, the mechanisms of action of these three factors could have different requirements for the AF-1 and AF-2 domains. Nonetheless, we find that the modulatory activity of each factor requires only the GR LBD with the AF-2 domain. Unexpectedly, N-terminal GR sequences inhibit the modulatory activity of Ubc9.

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RESULTS Role of AF Domains in the Modulation of GR Properties by Increasing Concentrations of GR The AF-1 domain of the rat GR is contained within the region of amino acids 98–317 (43–45), whereas helices 3, 4, 5, and 12 in the region of residues 572–781 comprise the AF-2 domain (25, 42). The AF-1 domain contributes much more to the total transactivation activity of GRs than does the AF-2


domain (14, 20). To determine whether the AF-1 domain is similarly important for increasing GR concentrations to modulate the EC50 of agonists and the amount of partial agonist activity of antagonists, we compared the responses of full-length GR with those of the amino terminal-truncated GR, GR407C (Fig. 1A). The antiglucocorticoid dexamethasone (Dex)-21-mesylate (Mes) was used throughout because it usually displays appreciable partial agonist activity (6, 14, 41, 46), which makes it an ideal model SRM with which to examine the consequences of

Fig. 1. Ability of Truncated GRs to Modulate their Transactivation Properties A, Schematic of rat GR constructs used. DBD (amino acids 440–506) and LBD (amino acids 550–795) domains of GR are indicated by the cross-hatched and diagonally striped boxes. The regions of the AF-1 and AF-2 domains are indicated by the solid bars above the cartoon for GR. The GAL DBD is depicted by the shaded box, with the dimerization domain being the horizontally striped box. The amino acids present in each construct are indicated by the numbers after GR, with C representing the C-terminal amino acid. B, Effect of increasing concentrations of full-length GR on the EC50 or partial agonist activity. Triplicate samples of CV-1 cells in 60 mm dishes were transiently transfected with the indicated amounts of GR plasmid, GREtkLUC reporter, and Renilla control plasmid, and then induced with EtOH ⫾ the indicated concentrations of Dex or 1 ␮M Dex-Mes. The luciferase activities, normalized to the internal Renilla control values, were then expressed as percent of the maximal response with 1 ␮M Dex as described in Materials and Methods. Similar results were obtained in eight additional experiments. The total amount of transactivation with 1 ␮M Dex increases 3.0 ⫾ 0.1-fold (SEM) in going from 40–1000 ng of plasmid, which is consistent with an increase in functional GR protein (data not shown). Effect of increasing concentrations of GR407C (C) and GAL/GR525 (D) on the EC50 or partial agonist activity. Triplicate samples were prepared and analyzed as in Fig. 1B except that GAL/GR525C and the (UAS)5tkLuc reporter were used in Fig. 1D. Similar results were obtained in three additional experiments with GR407C and five additional experiments with GAL/GR525C. The total amount of transactivation by GR407C is about 20% of that for equivalent amounts of full-length GR plasmid (data not shown). The total amount of transactivation, and the fold induction, with 1 ␮M Dex increases by 2.1 ⫾ 0.5-fold (SEM) in going from 16–300 ng of GR407C plasmid (data not shown) and by 2.2 ⫾ 0.4-fold (SEM) for a 20- to 25-fold increase in GAL/GR525C plasmid (data not shown), which is consistent with an increase in functional GR407C and GAL/GR525C proteins.



various parameters. As shown in Fig. 1B, a higher concentration of the full-length GR causes a shift in the dose-response curve to lower steroid concentrations (average shift ⫽ 7.5 ⫾ 1.3-fold; SEM, n ⫽ 9) and an increase in the partial agonist activity of the antiglucocorticoid Dex-Mes, just as reported (14, 16). Importantly, the modulation of the dose-response curve and partial agonist activity with increasing concentrations of the truncated GR407C (Fig. 1C) mimics the behavior of the full-length GR. The average left-shift in the dose-response curves for 300 ng vs. 16 ng of GR407C is 6.2 ⫾ 1.1-fold (SEM, n ⫽ 4), which is not significantly different from the 7.5-fold shift seen with full-length GRs. Thus, the amino terminal domain, and AF-1, of GR is not required for this modulation. In both cases, the total amount of transactivation increases with additional GR plasmid (data not shown), indicating that the modulation of full-length GR and GR407C transcriptional properties occurs under conditions where the levels of receptor are not a limiting determinant for the transactivation process.

To assess the requirement of the GR DNA binding domain (DBD) for the modulation of GR transactivation properties, we removed the DBD from GR407C. The remaining C terminus (residues 525–795) containing the GR LBD (amino acids 550–795) (47) plus 25 additional amino acids of the hinge region was fused to the GAL4 DBD (amino acids 1–147) (Fig. 1A). This chimera is known to induce reporter genes under the control of the GAL4 upstream activating sequence (UAS) such as in the (UAS)5tkLuc reporter (17). Elevated concentrations of transfected GAL/GR525C plasmid gave a proportionally higher amount of induced gene product (data not shown), again indicating that the chimeric receptor is limiting under these conditions. At the same time, elevated amounts of the GAL/GR525C produce a progressive left-shift in the dose-response curve to lower Dex concentrations and more partial agonist activity with Dex-Mes (Fig. 1D). The average left-shift produced by a 20- to 25-fold increase in GAL/GR525C plasmid is 4.5 ⫾ 0.8-fold (SEM, n ⫽ 6), which is again similar to the 7.5-fold shift seen with full-length GRs (Fig. 1B). These data argue that neither

Fig. 1. Continued

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the N-terminal half of GR nor the DBD is required for the modulatory properties of added GR. However, the slightly lower values for GAL/GR525C vs. GR407C may indicate a minor contribution by the GR DBD. Role of AF Domains in the Modulation of GR Properties by Increasing Concentrations of Coactivator The p160 coactivators, along with cAMP response element binding protein-binding protein and PCAF, are able to modulate GR transactivation properties (6, 7). To determine whether this property of the p160 coactivators is mediated through the LBD, which is the known interaction site in GR for these coactivators to increase the absolute levels of gene transactivation (25, 42), we compared the ability of TIF2 to modulate the properties of the full-length and truncated GRs (Fig. 1A). At the same time, we compared these results to those obtained with the chimeric protein of the activation domain of the herpes simplex virus VP16 protein fused to the full-length GRIP1/TIF2 (Fig. 2A). This construct was chosen to examine whether the nature of the activation domain (the AD1 and AD2 domains of GRIP1 (48, 49) vs. that of the heterologous transactivator VP16) affects the ability to modulate GR transactivation properties. The ability of both GRIP1/TIF2 and VP16/GRIP1 to modulate the EC50 of agonists and the amount of partial agonist activity of antagonists is the same for full-length GR (Fig. 2B) (6, 7) and the truncated GR407C (Fig. 2C). The change in EC50 of full-length GRs with added GRIP1 and VP16 is 3.0 ⫾ 0.3-fold (SEM, n ⫽ 3). This value is very similar to both the 3.6-fold shift previously reported for just GRIP1/TIF2 with full-length GR (6) and the 3.1 ⫾ 0.6-fold change (SEM, n ⫽ 3) seen for GR407C with GRIP1 and VP16. GRIP1 and VP16/GRIP1 were equally effective in increasing the level of induced luciferase activity by full-length GR (about 4-fold) and by GR407C (about 11-fold). This suggests either that the VP16-AD is inactive in this system or that, when the VP16-AD is active, the net result is no more than that of the GRIP1 ADs alone. In either case, we can conclude that the amino terminal half of GR is not required for the modulation of GR properties by GRIP1/TIF2. These data also indicate that the lower activity of GR407C vs. full-length GR can be largely recovered with added GRIP1. GRIP1 and VP16/GRIP1 cause an even greater increase the total amount of transactivation by GAL/ GR525C (about 30-fold). Both GRIP1 and VP16/ GRIP1 modulate the position of the dose-response curve (7.4 ⫾ 3.1-fold [SEM, n ⫽ 2] for GRIP1 and VP16) and the amount of partial agonist activity (Fig. 2D), as seen for full-length GR (Fig. 2B) and GR407C (Fig. 2C), although VP16/GRIP1 is somewhat less active here. These results indicate that the GR LBD (plus 25 amino acids of the hinge region) is


sufficient for the induction properties of GR to be modulated by the coactivator GRIP1/TIF2. Amino Terminal Domain of GR Inhibits the Modulatory Activity of Ubc9 with Low Concentrations of GR We previously reported that Ubc9 is active as a modulator of the transcriptional properties of GR in the presence of saturating concentrations of GR (e.g. ⱖ900 ng of plasmid/60-mm dish), where the presence of additional GR has little further ability to modulate the position of the dose-response curve or the partial agonist activity of antisteroids. In contrast, Ubc9 has no modulatory activity at low concentrations of GR plasmid (e.g. 40 ng) (17). This unique response at saturating GR concentrations suggested the presence of a rate-limiting step or intermediate X (16), with Ubc9 acting downstream of X (17). At the same time, Ubc9 was found to modulate the properties of 40 ng of GR407C and GAL/ GR525C. What was not known was whether 40 ng represents a subsaturating concentration. The data of Fig. 1 establish that 40 ng of plasmid is a subsaturating concentration of truncated receptors, conditions where the full-length GR is not modulated by Ubc9 (17). This suggests that the amino terminal half of GR restricts the ability of low concentrations of full-length GR to respond to added Ubc9. To identify this putative inhibitory sequence, we prepared a series of amino terminal deletions of GR (Fig. 3A) and asked what has to be removed to enable the properties of low concentrations of GR to be modulated by Ubc9. To be sure that we are using subsaturating concentrations of each truncated GR, we first confirmed that more receptor plasmid is capable of further modifying the EC50 of each construct (Fig. 3B). This behavior also argues that the N-terminal deletions have not dramatically altered the properties of the resulting GRs. It should be noted that the larger change in EC50 for wild-type (wt) GRs is due to the fact that a greater difference in receptor plasmid concentration was used for wt GR (4 vs. 100 ng) than for any of the other constructs (15–30 vs. 145 ng). Smaller differences in wt GR concentrations cause less dramatic changes in EC50 (14). Using the lower, subsaturating concentration of receptors of Fig. 3B, we then asked whether the addition of Ubc9 could decrease the EC50 by shifting the dose-response curve to the left. As expected, Ubc9 affects GR407C but not wt GRs (Fig. 3C). Deletion of the core of AF-1 and the first 260 amino acids is not enough to relieve the suppressive effects of the wt GR. However, removal of the next 100 amino acids (261–360) renders low concentrations of GR responsive to Ubc9 (Fig. 3C). This suggests that amino acids 261–360 contain a domain that represses Ubc9 modulatory activity. Ubc9 is also able to modulate the properties of these same truncated GRs (GR361C and GR407C) at higher



Fig. 2. Modulatory Activity of GRIP1 and VP16/GRIP1 with Truncated GRs A, Schematic of GRIP1/TIF2 constructs used. RID, Receptor interaction domain; AD1 and AD2, activation domain 1 and 2. The black box indicates the position of the VP16 activation domain. Modulatory activity of GRIP1 and VP16/GRIP1 with subsaturating concentrations of (B) full-length GR (100 ng), (C) GR407C (100 ng), and (D) GAL/GR525C (50 ng). Triplicate samples of CV-1 cells were transfected in 60-mm dishes and analyzed as in Fig. 1B but with 400 ng of VP16 ⫾ 400 ng GRIP1 or 500 ng VP16/GRIP1. Similar results were obtained in one to three additional experiments.



Fig. 3. Localization of GR Sequence that Inhibits the Modulatory Activity of Ubc9 with Low GR Concentrations A, Schematic of rat GR constructs used. The designations of the various GR domains is the same as in Fig. 1A, with the letter S marking the positions of the known sumoylation sites. B, Ability of increasing concentrations of truncated GR construct to shift the dose-response curve of agonists to lower steroid concentrations. Dose-response curves were determined as in Fig. 1B, but in 24-well plates, for different concentrations of the indicated GR plasmids (4 and 100 ng for full-length GR, 15–30 and 145 ng for all other constructs). The EC50 value of each curve is determined and the average of the ratio of EC50(low GR)/EC50(high GR) (⫽ fold increase in EC50) ⫾ SEM from three to five separate experiments is plotted. C, Ubc9 modulation of EC50 value for truncated GRs. Triplicate samples of CV-1 cells were transiently transfected with low concentrations of GR constructs (4 ng for full-length GR, 15–30 ng for all other constructs), 125 ng of Ubc9 plasmid, GREtkLUC reporter, and Renilla control plasmid, and then analyzed as in Fig. 3B. The plotted values represent the averages (⫾SEM) of three to five separate experiments. The dashed line indicates no change in the position of the dose-response curve with added Ubc9.

concentrations of GR (data not shown), which again argues that the truncations have not drastically modified their biological properties. Modulation by Ubc9 Is Independent of Sumoylation of GR Ubc9 can sumoylate proteins by mediating the covalent attachment of SUMO-1 (18, 19). The above iden-

tified inhibitory sequence in GR contains two consensus sumoylation sequences (⌿KxE, where ⌿ is a hydrophobic group and x is any group) (50) that have recently been shown to be sumoylated (51) at residues K297 and K313 (52). We previously established that the modulatory effects of Ubc9 on GRs are independent of the sumoylation activity of Ubc9 because the C93S mutant of Ubc9 is incapable of sumoylating


proteins (19, 53) but still modifies GR transactivation properties (17). However, it is possible that GRs are sumoylated by other proteins and that the sumoylated GR now interacts with Ubc9. To investigate this in greater detail, we prepared the K297,313R double mutant. This K-to-R mutation preserves the local charge in the protein while blocking sumoylation because only lysines are sumoylated (50). The wt and K297,313R mutant GRs have indistinguishable properties. In both cases, Ubc9 causes the same left-shift in the Dex dose-response curve and increase in Dex-Mes partial agonist activity at high, but not low, GR concentrations (data not shown). This suggests that sumoylation of GR at K297 and/or K313 by any means is not required for the modulatory activity of Ubc9. It should be noted that this double mutation also has no effect on the ability of elevated concentrations of receptor to modulate GR EC50 and partial agonist activity. Whereas K297 and K313 are the only consensus sumoylation sites in GR, mutation of both sites does not eliminate all sumoylation of GRs (52). To determine the possible role of sumoylation of additional residues, we examined the effects of added SUMO-1. Cotransfection of SUMO-1 (80 ng) causes a decrease in the total amount of gene induction that is more dramatic at lower GR concentrations but does not alter the inability of Ubc9 to change the EC50 or partial agonist activity with low concentrations of GR (data not shown). Likewise, the modulatory effects of Ubc9 at elevated GR concentrations are unaffected (data not shown). Collectively, these data argue that sumoylation of GR is not required for the modulatory activity of Ubc9 with elevated concentrations of GR. GR Sequences Involved in Binding of Ubc9 It is known that Ubc9 binds to GR (17). To determine the region in GR to which Ubc9 binds, we turned to an in vitro pull-down assay. Three different [35S]methionine-labeled, in vitro-translated GR species (fulllength GR, GRN523, and GR486C) (Fig. 4A) were examined for their ability to bind to Flag-tagged Ubc9 that was overexpressed in Escherichia coli. Whereas all three GR species are initially present at about equal concentrations, only the full-length GR and GR486C bind strongly to Ubc9 in a steroid-independent manner. Much less of the available GRN523 binds to Ubc9 (Fig. 4B). These results indicate that Ubc9 preferentially associates with the C-terminal amino acids of 524–795, which consists mostly of the LBD. These data are consistent with GAL/GR525C being a target of Ubc9 (Fig. 1C). Inter- vs. Intramolecular Action for Inhibition of Ubc9 Activity by GR Amino Acids 261–360 The demonstration that Ubc9 binds to the C-terminal half of GR (Fig. 4B) suggests that GR residues 261–


Fig. 4. Effect of GR Sequences and Steroid on GR Cell-Free Binding of Ubc9 A, Schematic of rat GR constructs used. The designations of the various GR domains is the same as in Fig. 1A. B, Binding of Ubc9 to GRs in cell-free pull-down assays. Flagtagged Ubc9 or Flag-tagged BAP were overexpressed in E. coli, immobilized by matrix-bound anti-Flag antibody, and treated with in vitro-translated GRs that had been bound by agonist Dex, antiglucocorticoid (Dex-Mes [DM] or Dex-Ox [Dox]), or left unbound. GR complexes that were retained on the matrix by binding to the Flag-tagged proteins were separated on SDS-PAGE gels and visualized by autoradiography. Specific binding (left column) is seen as the GR binding to Ubc9 that is in excess of the nonspecific binding to BAP. The right column shows 10% of the amount of each GR construct that was loaded onto the matrices. Luciferase was used as an additional control for nonspecific binding. Similar results were obtained in three additional independent experiments.

360 may inhibit Ubc9 modulatory activity by preventing Ubc9 from interacting with residues 525–795 at low GR concentrations. This could occur by two mechanisms. GR residues 261–360 could recruit a molecule that binds to the GR LBD. Alternatively, GR residues 261–360 could directly interact with the GR LBD. To test the first hypothesis, we asked whether overexpression of a larger fragment [hemagglutinin (HA) tagged GR261–488] can titrate out the putative inhibitor and thus render low concentrations of GR


sensitive to the modulatory effects of Ubc9. Western blots show that equal levels of plasmid cause similar levels of expression of wt GR and HA/GR261–488 (Fig. 5A, inset). However, a 25-fold excess of HA/GR261– 488 has no effect on the ability of Ubc9 to alter the dose-response curve and partial agonist activity of low concentrations of GR (Fig. 5A). This inability to titrate out a putative factor is consistent with the inhibitory activity of GR261–360 being due to the sequence itself, which would interact intramolecularly with another region of GR and thus would be resistant to intermolecular competition by added GR fragments. Under the same conditions, HA/GR261–488 has no effect on the activity of Ubc9 with high concentrations of GR (Fig. 5B). Direct support for an intramolecular interaction was obtained from cell-free pull-down assays. [35S]methi-


onine-labeled GR and GR486C (Fig. 4A) are each retained by bacterially expressed GST/GR261–488 but not the GST control (lanes GR vs. C respectively in Fig. 6). This interaction is ligand independent and is limited to the C-terminal half of GR as very little binding of GRN523 is seen under the same conditions (Fig. 6). Thus, we conclude that the sequence of GR261–360 could inhibit the actions of Ubc9 by preventing Ubc9 from binding to sequences in the C-terminal half of GR (see Fig. 4B). Ubc9 Modulatory Activity with Chimeric Receptors ⴞ GR N-Terminal Domain To obtain additional support for the above hypothesis that residues 261–360 of GR block the actions of Ubc9 at low concentrations of GR by interacting with the GR LBD in an intramolecular interaction, we examined the behav-

Fig. 5. Effects of Overexpression of GST/GR261–488 A, Inset, Western blot of overexpressed wt GR and GST/GR261–488. Equal amounts cytosols from COS-7 cells that had been transiently transfected with the same amount of plasmid for full-length GR and GST/GR261–488 were separated on SDS-PAGE gels. After being transferred to nitrocellulose filters, the GRs were detected by Western blotting with BUGR-2 anti-GR antibody as described in Materials and Methods. The positions of the wt GR and GST/GR261–488 are indicated by the arrows on the left and right sides of the blot, respectively. Transactivation properties of 4 ng (A) and 100 ng (B) full-length GR ⫾ Ubc9 ⫾ GST/GR261–488. Triplicate samples of CV-1 cells were transiently transfected with the indicated amounts of GR ⫾ 125 ng Ubc9 ⫾ 100 ng GST/GR261–488 plasmids, treated with the indicated steroids, assayed as in Fig. 1B. Similar results were obtained in seven additional independent experiments.


Fig. 6. Interaction of N- and C-Terminal GR Sequences in Cell-Free Pull-Down Assays GST-tagged GR261–488 was overexpressed in E. coli, immobilized by matrix-bound glutathione, and treated with in vitro-translated GRs (full-length GR, GR486C, and GRN523) that had been bound by agonist Dex, antiglucocorticoid (DexMes or Dex-Ox), or left unbound. GR complexes that were retained on the matrix by binding to the GST chimeras were separated on SDS-PAGE gels and visualized by autoradiography. Specific binding is indicated by receptor binding (⫾ ligands) to GST/GR261–488 (column GR) that is in excess of the nonspecific binding to GST (column C). The right column shows 10% of the amount of each GR construct that was loaded onto the matrices. Luciferase was used as an additional control for nonspecific binding. Similar results were obtained in two additional independent experiments.

ior of two chimeras of GR and PR: PR/GR and GR/PR (Fig. 7A). Given the no more than 15% homology between the N-terminal domains of GR and PR along with the 55% homology between the GR and PR LBDs and the very close correlation between the x-ray structures of GR and PR LBDs (54, 55), we predicted that the PR N-terminal domain in PR/GR would not prevent Ubc9 actions because the PR N-terminal domain would not interact with the GR LBD. Conversely, the PR LBD could retain sufficient critical residues to enable the binding of the GR N-terminal domain in the GR/PR chimera and prevent modulation by Ubc9 at low GR/PR concentrations. As shown in Fig. 7, B and C, precisely this result was observed. PR/GR is limiting at receptor concentrations up to 0.5 ng/well, as determined by increasing levels of reporter activity (data not shown). Similarly, GR/PR is limiting at receptor concentrations up to 1 ng (data not shown). Whereas PR/GR properties are modulated at both low and high receptor concentrations by Ubc9 (Fig. 7B), the properties of GR (data not shown; see also Fig. 4A and Ref. 17) and GR/PR (Fig. 7C) are significantly altered only at high receptor concentrations. These results are consistent with the GR, but not the PR, N-terminal domain interacting with either the GR or PR C-terminal domain to prevent the modulatory activity of Ubc9 at low receptor concentrations. Effect of GR Concentration on Steroid Binding Cooperativity In an effort to find a biochemical property of an early step in GR action that varies with GR concentration


and could be responsible for the ability of high, but not low, GR concentrations to respond to added Ubc9, we examined the kinetics of steroid binding. Scatchard analysis of [3H]Dex binding to concentrated cytosolic solutions of wt GR from transiently transfected COS-7 cells gave a highly curvilinear plot (Fig. 8B), similar to that seen with ERs and indicative of cooperative binding of steroid to receptor dimers (56). In support of this interpretation, reanalyzing the data of Fig. 8B in the form of a Hill plot gave a Hill plot coefficient (nH) value of 1.479 (Fig. 8G). Values for nH ⬎ 1 are diagnostic of a high level of cooperativity in steroid binding (57). Dilution of the sample gives a linear Scatchard plot (Fig. 8C) and a Hill plot with a nH value of 0.995 (Fig. 8H), which argues that the cooperativity for steroid binding to GR is very concentration dependent and disappears at lower GR concentrations. As a further test of this interpretation, we examined the properties of the chimera GAL/GR, which contains the GAL DBD and full-length GR (Fig. 8A). The GAL DBD contains a strong dimerization domain at amino acids 67–94 (Fig. 1) (58). Thus, GAL/GR is predicted to display cooperative steroid binding even when diluted. In fact, high concentrations of GAL/GR give a curvilinear Scatchard plot (Fig. 8D) and a Hill plot nH value of 1.457 (Fig. 8I), just as is observed for GR. Western blots show that the same amount of GR and GAL/GR protein are present (Fig. 8F). More significantly though, as Western blots detect only protein and cannot determine the amount of functionally active protein present, the x-axis intercepts are about the same for the solutions of GR and GAL/GR, which indicates that the amount of receptor protein capable of binding steroid is similar in the solutions of GR vs. GAL/GR. In contrast to the behavior of low concentrations of GR, a comparably diluted solution of GAL/GR still shows a nonlinear Scatchard plot (Fig. 8E) and a Hill plot nH value much greater than 1 (i.e. 1.390; Fig. 8J). These data argue that conditions sufficient to dissociate the weakly bound dimer of GR do not cause the dissociation of a strongly dimerizing species such as GAL/GR. These results are consistent with the monomeric species of GR present at low concentrations of GR not being responsive to added Ubc9 whereas the dimerization that occurs at high GR concentrations permits Ubc9 to modulate GR activities. Proposed Model for Ubc9 Action and Tests of Model The above results support a model in which Ubc9 is inactive at low GR concentrations due to the intramolecular inhibitory effects of amino acids 261–360 in the N-terminal region of GR. However, Ubc9 becomes able to express its modulatory activity upon deletion or replacement of this GR N-terminal domain. Our model further proposes that the elevation of wt GR concentration causes an intermolecular dimerization that displaces the inhibitory N-terminal domain and uncovers

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Fig. 7. Ability of Ubc9 to Modulate the Transactivation Properties of Chimeras of PR and GR A, Schematic of composition of chimeric receptors. The linear structures of the wt rat GR and human PR-B are displayed on top. The individual domains of the receptors are labeled as A–E on top of the GR structure. The C and D domains, corresponding to the DNA binding domain and the hinge region respectively, are differentially shaded. The chimeric receptors are joined at the interface of the C and D domains, indicated by the vertical line. The precise amino acids from each receptor are shown above and below the two segments comprising each chimera. B and C, Triplicate samples of CV-1 cells were transiently transfected with low and high concentrations of the indicated wt GR and chimeric plasmids ⫾ 125 ng of Ubc9 plasmid and analyzed as in Fig. 1B, but in 24-well plates. Similar results were seen in one (GR/PR) and two (GR and PR/GR) additional independent experiments.



Fig. 8. Effect of Receptor Concentration on Binding Kinetics of GR and GAL/GR A, Schematic of rat GR constructs used. The designations of the various GR domains is the same as in Fig. 1A. B–E, Scatchard analysis of Dex binding to cell-free solutions of GR and GAL/GR. Wt GR and GAL/GR were overexpressed in COS-7 cells, the cytosolic preparations were diluted to concentrations ranging from 30% to 5.3% of the initial receptor solution, and analyzed for the specific binding of [3H]Dex by the method of Scatchard as described in Materials and Methods. F, Western blots of overexpressed wt GR and GAL/GR. Equal amounts COS-7 cell cytosols containing overexpressed wt GR and GAL/GR were separated on SDS-PAGE gels. The amount of receptor was determined by Western blotting with BUGR-2 anti-GR antibody. G–J, Hill plot analysis of Dex binding to GRs. The binding data of panels B–E were replotted by the method of Hill, as described in Materials and Methods, to give panels G–J, respectively. (Figure continued on next page.)

a strong Ubc9 binding domain in the C-terminal region of GR (Fig. 9). Recent x-ray structures of the GR LBD indicate several residues that are important for LBD dimerization in the crystals (25, 55). Thus, a mutation of one of these residues might thwart Ubc9 actions at high GR concentrations due to a reduced abundance of the intermolecular dimer of Fig. 9. We chose to look at the I646A mutant of rat GR, which appears to have normal

nuclear translocation, to cause a 10-fold reduced ability to dimerize compared with the wt GR LBD, and to reduce the fold transactivation of a mouse mammary tumor virus (MMTV) reporter in the presence of 100 nM Dex by 60% (25). With the GREtkLUC reporter, both low (4 ng) and high (100 ng) amounts of the GR(I646A) mutant plasmid afford very little difference in either fold transactivation or total activity with 10 ␮M Dex (0 to 20% reduction; data not shown), compared with the



Fig. 8. Continued

wt receptor. However, the GR(I646A) displays a doseresponse curve that is shifted about 10-fold to the right (Fig. 10; note the 10-fold difference in the x-axis values in panels A and B) and a greatly reduced partial agonist activity with Dex-Mes (from 45 ⫾ 2% for 100 ng wt GR to 12 ⫾ 2% for 100 ng of GR(I646A); n ⫽ 3, ⫾ SEM). Nevertheless, the GR(I646A) acts like the wt receptor in that increased functional receptor (as indicated by a ⱖ3-fold increase in total activity) causes a left-shift in the dose-response curve (Fig. 10) and increased partial agonist activity (from 19 ⫾ 1 to 45 ⫾ 2% for wt GR and from 2 ⫾ 0 to 12 ⫾ 2% for GR(I646A); n ⫽ 3, ⫾ SEM). Similarly, the addition of Ubc9 to high concentrations of either wt or I646A GR plasmids produces about a 6-fold left shift in the dose-response curve, and increased partial agonist activity of Dex-Mes (from 45 ⫾ 2 to 85 ⫾ 5% for wt GR and from 12 ⫾ 2% to 62 ⫾ 8% for GR(I646A)], although having no effect on the properties with low amounts of plasmid (Fig. 10 and data not shown). The magnitude of Ubc9-induced changes in Dex EC50 (Table 1A) and Dex-Mes partial agonist activity (Table 1B) increases in a similar manner with progressively higher concentrations of transfected wt and mutant receptor plasmids. These actions of Ubc9 therefore do not appear to be influenced by the reported 10-fold change in dimerization affinity

of the isolated GR LBD that is caused by the I646A mutation (25). Thus, whereas the I646A mutation dramatically affects the EC50 and partial agonist activity (Fig. 10 and Table 1), its inability to suppress the modulatory properties of Ubc9 suggests that residues other than I646 are involved in the dimerization of the full-length GR that is proposed to permit Ubc9 modulation (Fig. 9; see also Discussion). As an alternative test of the model in Fig. 9, we asked whether GAL/GR, which is present as a dimer even at low receptor concentrations (Fig. 8, D, E, I, and J), might be responsive to added Ubc9 at both low and high receptor concentrations. As is described elsewhere (59), GAL/GR is inactive on both the GAL-regulated reporter [(UAS)5tkLuc) and the GREtkLUC reporter but has very weak activity on a MMTVLuc reporter. Unfortunately, the narrow concentration range over which the GAL/GR plasmid gives some induction without evidence of squelching (from about 0.75 to 7.5 ng of plasmid) coupled with the very low fold induction of MMTVLuc by GAL/GR in the absence of Ubc9 (average ⫽ 3.6-fold for 0.75 ng of GAL/GR plasmid) prevented us from obtaining statistical significance for the slightly lower EC50 values with added Ubc9 (1.25 ⫾ 0.32-fold lower, n ⫽ 5, SEM). More significant is the ability of Ubc9 to increase the partial agonist activity of Dex-Mes both at low concen-



Fig. 9. Model of Involvement of AF-1 and AF-2 Domains in Modulation of GR Transactivation Properties by GR, TIF2, and Ubc9 An equilibrium between intramolecular and intermolecular association of sequences in the full-length GR is proposed to occur where high concentrations of GRs favors the intermolecular association (A). At low concentrations of full-length receptor, residues within the sequence of 261–360, which is downstream of the core of the AF-1 domain, are proposed to interact (dashed lines) with residues 525–795 in the C-terminal half of GR (blackened rectangle), which includes the LBD. This interaction prevents the modulatory activity of Ubc9 but not TIF2 or GR. At high concentrations of full-length GR, other interactions, such as those between the DBDs and/or LBDs (dashed lines), predominate and disrupt the intramolecular interactions, thereby making the LBD receptive to the modulatory actions of Ubc9 but not TIF2 or GR (stippled rectangle). This model accounts for the observations that the modulatory activity of added GR and TIF2 is seen with full-length GRs, but only at low GR concentrations (A), and with truncated receptors, which lack the AF-1 domain but retain the AF-2 domain, at all GR concentrations (B). It also explains why Ubc9 is able to modulate the properties of truncated GRs at all concentrations of GR but affects the properties of full-length GR only at high GR concentrations. See text for further details.

trations of GAL/GR (from 0.76 ⫾ 2.6% to 11.7 ⫾ 2.3%; n ⫽ 5, ⫾ SEM, P ⫽ 0.013) and at high GAL/GR concentrations (from 20.5 ⫾ 3.5% to 90.0 ⫾ 3.8%; n ⫽ 5, ⫾ SEM, P ⫽ ⬍0.0001) (Fig. 11). The fact that the activities ⫾ steroid with both concentrations of GAL/GR (middle and right panels) are higher than the control values with GAL (left panel) indicates that we are monitoring the responses to added GAL/GR. These changes in partial agonist activity of Dex-Mes support the prediction of the model in Fig. 9 that GAL/GR is responsive to Ubc9 even at low GAL/GR concentrations because the formation of GAL-GAL dimers disrupts the inhibitory actions of the GR N-terminal domain of amino acids 261–360. It is also very interesting to note that Ubc9 causes a 30–100-fold increase in the transactivation activity of GAL/GR (Fig. 11). Thus, whatever defect(s) is responsible for the low transcriptional activity of GAL/GR, it is effectively reversed by the addition of Ubc9.

DISCUSSION Several factors can modulate the EC50 of GR-agonist complexes and the partial agonist activity of GRantagonist complexes. The ability to modify the quan-

titative values of these properties are highly beneficial to cells with regard to both the differential control of gene induction by the single circulating concentration of steroid hormone and the possible limitation of full repression during endocrine antisteroid therapies to a subset of the regulated genes. Here, we find that the GR LBD is a necessary and sufficient target for the modulation of these GR transcriptional properties by changing concentrations of GR, of coactivator TIF2, and of Ubc9. The observations that the EC50 and partial agonist activity of the GAL/GR525C chimera, which contains only the GR LBD, are modulated by increased concentrations of the chimera, TIF2, and Ubc9 (Figs. 2 and 3 and Ref. 17) indicate that the mechanism of modulation does not require GR contacting DNA via its own DBD with a subsequent DNA-induced conformational change in GR being transmitted to the LBD. Whereas it is possible that an identical DNA-induced conformational change could be propagated from the GAL DBD to the GR LBD in the GAL/GR525 chimera, it seems unlikely because the structure-activity relationships of the two proteins have evolved independently. This therefore suggests that the transactivation properties of GRs noncovalently attached to DNA-bound pro-



Fig. 10. Effect of I646A Mutation of GR on the Modulation of Transactivation Properties by Ubc9 Triplicate samples of CV-1 cells were transiently transfected with low and high concentrations of (A) wt GR and (B) mutant GR(I646A) plasmids ⫾ 135 ng of Ubc9 plasmid and analyzed as in Fig. 1B, but in 24-well plates. Similar results were obtained in two additional independent experiments. Western blots with BUGR-2 anti-GR antibody show that equal levels of wt and I646A mutant receptors are expressed in Cos-7 cells (data not shown).

teins would also modulated by added receptor, coactivator, and Ubc9. GR represses many genes by mechanisms that involve GR binding to DNA-bound proteins, such as activator protein-1 (AP-1) and nuclear factor ␬-B (NF␬-B). It therefore will be of great interest to determine whether the parameters of GRmediated repression are modulated in a manner similar to that seen for GR-mediated induction. If so, this would greatly expand the applications of the present results (4). Whereas all three modulators (GR, TIF2, and Ubc9) require only the GR LBD (Fig. 9B), the precise molec-

ular mechanisms appear to be different because amino acids 261–360 in the GR N-terminal domain inhibit the actions of Ubc9 (Fig. 3C), but not GR or TIF2, at low GR concentrations. This inhibition appears to be intramolecular because it cannot be relieved by overexpressing a fragment of GR containing the inhibitory sequence of amino acids 261–360 (Fig. 5). The proposed intramolecular interaction is supported by the observation that a GR N-terminal fragment containing the inhibitory sequence binds to the C-terminal third of GR (Fig. 6). This intramolecular inhibition is sequence specific and is relieved by either removing

Table 1. Ability of I646A Mutation to Alter the Capacity of Ubc9 to Modulate GR Properties at Different Concentrations of GR 20 ng GR

⫺Ubc9

40 ng ⫹Ubc9

⫺Ubc9

100 ng ⫹Ubc9

A. Amount of Dex (nM) required for EC50 with different amounts of GR plasmid ⫾ Ubc9 Wild type 6.9 ⫾ 1.6 4.3 ⫾ 0.3 5.2 ⫾ 0.1 1.6 ⫾ 0.3 (1.6) (3.3) I646A 163 ⫾ 49 94 ⫾ 5 127 ⫾ 30 37 ⫾ 20 (1.7) (3.4)

⫺Ubc9

⫹Ubc9

2.8 ⫾ 0.3

0.55 ⫾ 0.18 (5.1)

71 ⫾ 29

B. Percent partial agonist activity of Dex-Mes with different amounts of GR plasmid ⫾ Ubc9 Wild type 31 ⫾ 1 57 ⫾ 1 40 ⫾ 0 96 ⫾ 1 46 ⫾ 1 (1.8) (2.4) I646A 4.8 ⫾ 0.5 6.1 ⫾ 0.9 7.3 ⫾ 0.3 17 ⫾ 1.7 11 ⫾ 1.2 (1.3) (2.3)

11 ⫾ 2 (6.5) 96 ⫾ 10 (2.1) 47 ⫾ 2 (4.3)

Experiments were performed as in Fig. 10 with the indicated amounts of wild-type or I646A mutant plasmids. The averages ⫾ SEM of two independent experiments are shown. The fold change caused by the presence of Ubc9 is indicated in parentheses below each set of data for ⫾ Ubc9.



Fig. 11. Ability of Ubc9 to Modulate the Transactivation Properties of GAL/GR Triplicate samples of CV-1 cells were transiently transfected with GAL/GR at low (0.75 ng, middle panel) and high (7.5 ng, right panel) concentrations of plasmid, or GAL plasmid at the same molar concentration as the high GAL/GR (left panel), ⫾ 135 ng of Ubc9 plasmid and analyzed as in Fig. 1B, but in 24-well plates. The relative luciferase values (normalized to Renilla to correct for transfection efficiency) of five independent experiments were averaged and plotted ⫾ SEM. The numbers in parentheses above the bars for Dex-Mes (DM) activity represent the percent partial agonist activity (⫽ activity above EtOH treatment as percent of activity with 1 ␮M Dex). Note the split y-axis scales for the data of GAL/GR with Ubc9.

the N-terminal half of GR or by replacing it with the N-terminal half of PR, which is less than 15% homologous. Furthermore, the inhibitory GR sequence is effective with the PR LBD, as was expected given the high degree of sequence homology and similar tertiary structure between the GR and PR LBDs (54, 55). However, the effects of the GR inhibitory sequence disappear at high concentrations of GR and GR/PR (Fig. 7C). Similarly, the binding of steroid to GR switches from noncooperative to cooperative at high GR concentrations. These observations are consistent with the proposed model (Fig. 9) in which an intramolecular interaction of GR N- and C-terminal sequences at low GR concentrations is replaced by an intermolecular interaction at high GR concentrations, which may involve the dimerization sequences of the DBD (60) and/or LBD (25, 55). This model is supported by the observation that Ubc9 modulates the partial agonist activity of antisteroids even at low concentrations of GAL/GR (Fig. 11), which exist as dimers under conditions where wt GRs are monomeric (Fig. 8). We conclude that dimeric but not monomeric GRs productively interact with Ubc9 in a manner that allows the modulation of GR transactivation properties. Our model (Fig. 9) also incorporates the observations that Ubc9, but not added GR or TIF2, displays modulatory activity at high concentrations of GR, whereas the

converse is true at low GR concentrations (this work and Refs. 16 and 17) (Fig. 9A). The present results are also consistent with our previous conclusion that Ubc9 acts downstream from a step or intermediate that is rate-limiting with added GR or TIF2 (4, 17) and suggest that the rate limiting step could be the formation of GR dimers in Fig. 9. The key GR residues for forming Ubc9-responsive dimeric GRs are not known. I646 of rat GR is important for GR-LBD dimerization (25), along with other residues (55). However, the I646A mutation does not reduce the modulatory activity of Ubc9 over a range of GR concentrations (Fig. 10 and Table 1). This absence of significant effects on Ubc9 actions with any concentration of the I646A mutant GR suggests that the formation of Ubc9-responsive full-length GR dimers depends more on the other participating residues of GR dimer formation that are found in the LBD (55) and DBD (60, 61). The intramolecular interactions between the N- and C-terminal domains of monomeric GR that are suggested by the data of Fig. 6 are similar to those seen for ER (62–64), PR (65), peroxisome proliferator-activated receptor (66), and ARs (21, 67–70). Thus, this type of interaction may be a common phenomenon among steroid/nuclear receptors. Further studies are

306


required to identify the precise region of GR to which residues 261–360 are thought to bind (Fig. 6). A strong Ubc9 binding site in GR is now identified as being within the C-terminal amino acids 486–795 (Fig. 4B). The comparatively weak binding to the amino terminal sequence of 1–523 (Fig. 4B) plus the similar responses of full-length GR and GAL/GR525C (17) suggest that the major Ubc9 binding site in GR is between residues 525 and 795. This region corresponds to sequences that are downstream of the Ubc9 binding site in the AR, which is amino acids 629–633 at the C terminus of the nuclear localization sequence (71). For this reason, it will be interesting to see if added Ubc9 modulates the EC50 and partial agonist activity of AR complexes. The interaction of Ubc9 with GR dimers, which are formed at high GR concentrations (Fig. 8), to modulate the EC50 and partial agonist activity of GR complexes appears separable from the binding of Ubc9 to GR, which can occur at low concentrations of GR (Fig. 4B). The ability of Ubc9 to increase the total amount of activation by both low and high concentrations of GR (this work and Ref. 17) suggests that Ubc9 binds to dilute solutions containing monomeric GRs; however, additional conformational changes produced by high GR concentrations are needed for Ubc9 to display its modulatory effects. Whereas the AF-1 domain does not appear to be involved in the modulation of the EC50 and partial agonist activity of GR complexes, it has long been known to be the major contributor to GR transactivation activity, as witnessed by the much lower activity of GR407C compared with full-length GR (this study and Refs. 14 and 20). This may be due to reduced interactions of the truncated GR with DRIP150 (72). Also, Hong et al. (42) reported that GRIP1 is required for AF-1 but not AF-2 activity of GR in yeast. Consistent with this is the fact that the AF-1 activity of several other receptors has been found to be accentuated by coactivators (21, 26, 27, 73, 74). However, under our conditions, GRIP1/TIF2 causes a much larger enhancement of the amount of gene product induced by the AF-2 domain of GR407C than when the AF-1 domain is present in the wt GR (11- vs. 4-fold). Similarly, GRIP1/TIF2 augments GAL/GR525C gene induction by 30-fold. Thus, the usually lower transcriptional activity of GRs lacking the AF-1 domain is mostly restored by elevated levels GRIP1/TIF2 due to the more dramatic increase of AF-2 activity than of AF-1/AF-2 activity. This ability of coactivators to rescue the transcriptional activity of N-terminal-deleted GRs may account for the often wt activity that was recently reported for the truncated GR that is produced in exon 2-disrupted GRs and is missing the AF-1 domain but still binds steroid (75, 76). Schulz et al. (37) reported that the GR AF-1 domain is required for the expression of the low amount of partial agonist activity of RU486. However, this is not necessarily in conflict with the present results. It is known that the amount of partial agonist activity of any


antagonist depends upon the concentration of receptor (4). Whether higher amounts of Schulz’s truncated receptor would give significant amounts of activity for RU486 was not investigated. Also, we have found that the partial agonist activity of a variety of antiglucocorticoids does not require the presence of the AF-1 domain (59). Therefore, the bulky 11␤ substituent of RU486 may have additional requirements not seen with the other less bulky antiglucocorticoids. The C-terminal end of the GR AF-1 domain, which is part of the region that inhibits Ubc9 modulatory activity, contains two consensus sumoylation sequences that were previously reported to constitute a synergy control motif. The mutation of both sites to yield GR/ K297,313R causes both a 6- to 12-fold increase in the total amount of gene activation from a reporter containing multiple glucocorticoid response elements (GREs), but not a single GRE, and shifts the doseresponse curve to the left (77). In our system, the same K297,313R mutant produced relatively little change in the amount of gene induction (2.0 ⫾ 0.3-fold increase [SEM, n ⫽ 4]) at both low and high concentrations of receptors (data not shown). Importantly, the presence of the double K/R mutation does not alter the ability of Ubc9 to modulate the EC50 and partial agonist activity (Fig. 4, B vs. A). These results suggest that sumoylation does not significantly affect GR transcriptional properties from our luciferase reporter with a tandem GRE. This is consistent with reports that the effect of these GR mutations on the magnitude of gene activation is promoter dependent (52, 78). The disparate importance of the AF-1 domain in several situations for determining the total amount of gene activation vs. the modulatory activity to both reposition the agonist dose-response curve and modify the amount of partial agonist activity of antisteroids is consistent with these two sets of processes being governed by different mechanisms. We have previously arrived at this same conclusion, albeit from numerous other directions. The modulatory activity of TIF2 and SRC-1 localizes to a different region than the transactivation domains of each coactivator (7). Ubc9 is adept at increasing the total amount of gene induction by both low and high concentrations of GR but displays modulatory activity only with high GR concentrations (this work and Ref. 17). The histone deacetylase inhibitor trichostatin A increases the amount of activation by GRs in the presence of the corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT) but has no effect on the modulatory properties of SMRT (79). With several other factors, there is no correlation between their ability to increase (or decrease) the levels of induction and the magnitude or direction of the modulatory changes (6, 7, 12–14, 16, 17). This accumulating evidence provides a compelling argument that the level of gene induction by GRs and the modulation of the position of the dose-response curve for agonists (and the amount


of partial agonist activity of antagonists) are determined by different mechanistic pathways.

MATERIALS AND METHODS Unless otherwise indicated, all operations were performed at 0 C. Chemicals and Antibodies [1,2,4,6,7-3H]Dex (91 Ci/mmol) and L-[35S]methionine (⬎ 1000 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Nonradioactive Dex is from Sigma (St. Louis, MO). Dex oxetanone (Dex-Ox) (80) and Dex-Mes (81) were prepared as described. Restriction enzymes and digestions were performed according to the manufacturer’s specifications (New England Biolabs, Beverly, MA). Anti-GR antibody BUGR-2 was purchased from Affinity Bioreagents (Golden, CO). Preparation of Plasmids Renilla null luciferase reporter was purchased from Promega (Madison, WI) and pM and pVP16 vectors were from CLONTECH (Palo Alto, CA). hPR-B was a gift from Hinrich Gronemeyer (IGBMC, Strasbourg, France). GREtkLUC (46), pVP16/ GR, GAL/GR (82), GAL/GR-525C, GAL/GR-407C, pSG5/ rUbc9, hSA/pSG5 (17), GR-407C (14), GR/PR and PR/GR chimera (41) have already been described. pSG5-GRIP1 and VP16/GRIP1 were obtained from Michael R. Stallcup (University of Southern California, Los Angeles, CA). pRBAL-GR and pSVL-GR (rat GR) were kindly provided by Keith Yamamoto (University of California, San Francisco, CA). To generate pSVL-GR⌬␶1, pSVL-GR was cut with AccI and BglII and self-ligated after filling-in the 3⬘-recessed termini with Klenow fragment. pSVL-GR201C, pSVL-GR261C, pSVL-GR361C, and pSVL-GR407C were prepared as follows. Upstream primers (5⬘-ATGGATCCATGTCAGTGTTTTCTAATGGGTAC for pSVL-GR407C; 5⬘-ATGGATCCATGCTTTCTCAGCAGGATCAG for pSVL-GR361C; 5⬘-ATGGATCCATGACGAATGAGGATTGTAAGCC for pSVL-GR261C; 5⬘-ATGGATCCATGAGTGTGAAATTGTATCCCAC for pSVLGR201C) and the common downstream primer (5⬘-ATCCCGGGTCTAGAAAGTTTTACCCAGC) were used for PCR amplification of the portions of GR. The resulting PCR products were digested with BamHI and XbaI and ligated into the 3.8-kb partial digestion product of BamHI/XbaI-cut pSVLGR. pSVL-GR261–488 was cloned by cutting pSVL-GR261C with BstB1, treating with alkaline phosphatase, and ligating the product with the double-stranded oligomer of 5⬘-CGATGATAATCGATCTAG to introduce stop codons. GST/GR261– 488 was constructed by inserting the BamHI fragment of pSVL-GR261–488 into pGEX-6P-1 vector (Amersham Pharmacia Biotech) at the BamHI site. pSVL-GR(K297,313R) double mutant was prepared by four rounds of PCR-based site-directed mutagenesis. Using pSVL-GR as a template, two separate sets of PCR primers (upstream PMUP, 5⬘-GGGCTGTATATGGGAGAGAC and downstream, 5⬘-GTTTTTCTGTTCGCACTTGGGG for one set and upstream, 5⬘-CCCCAAGTGCGAACAGAAAAAG and downstream PMDN, 5⬘-GGTAATTGTGCTGTCCTTCC for the other set) were used to generate two species of PCR products that share a 20-bp complimentary sequence. The two PCR products were mixed and subjected to a second round of PCR with PMUP and PMDN as primers. The resulting PCR product was cut with AccI and ApaI and inserted into pSVL-GR at the AccI/ApaI sites to provide the single mutant GR(K297R). The double mutant was generated in a similar manner using the pSVL-GR(K297R) plasmid as a template.


For the double mutant, PMUP, PMDN, and two more primers (5⬘-GTTTCTCTTGCCGAATTACCCG; 5⬘-CGGGGTAATTCGGCAAGAGAAAC) were used. pSVL-GR(I646A) was prepared using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA) and the pSVLGR construct as a template. Sense (5⬘-TGCTTTGCTCCTGATCTGGCTATTAATGAGCAGAGAATGTC-3⬘) and antisense (5⬘-ACATTCTCTGCTCATTAATAGCCAGATCAGGAGCAAAGCA-3⬘) primers containing base pair substitutions conforming to an amino acid substitution of isoleucine to alanine at position 646 in the rat GR DNA sequence, ATT to GCT respectively, were designed using Stratagene’s QuickChange Primer Design software (http://labtools.stratagene. com). The PCR products were digested with DpnI, incubated at 37 C to remove the wt original methylated template DNA, and then transformed into DH5␣ Library competent cells (Invitrogen Life Technologies, Carlsbad, CA). The correct sequence was verified by dideoxynucleotide chain termination sequencing using the rat GR-sequence-specific primer 5⬘-GGATGACCAAATGACCCTGCTAC-3⬘. To make pRBAL-GRN523, three fragments were ligated: the 0.5-kb fragment from BamHI/SalI digestion of pSVL-GR, the 1.2-kb fragment obtained by SalI/EcoRI digestion of the PCR amplified region of pSVL-GR (using upstream primer, 5⬘-AATAGGTCGACCAGCGTTCCAG and downstream primer, 5⬘-TAGAATTCGTCATGCAGTGGCTTGCTGAATC), and the 3.0-kb fragment from BamHI/EcoRI digestion of pRBAL-GR. For pRBAL-GR486C, the PCR amplified region of pSVL-GR using the upstream primer, 5⬘-ATGGATCCAATGATTGATAAAATTCGAAGG and downstream primer, 5⬘-TATCTAGAGTCATTTTTGATGAAACAG was cut with BamHI and ligated with the 3.0-kb fragment from BamHI/SmaI digestion of pRBAL-GR. pFLAG-MAC and pFLAG/BAP (bovine alkaline phosphatase) vectors were purchased from Sigma. The bacterial expression vector pFLAG/Ubc9 was cloned by inserting the HindIII/EcoRI fragment of Ubc9 from pFLAGCMV2/mUbc9 (kindly provided from Olli Ja¨nne, University of Helsinki, Finland) into the pFLAG-MAC vector at the HindIII/EcoRI site. Cell Culture and Transient Transfection COS-7 and CV-1 cells were maintained at 37 C with 5% CO2 in DMEM (Invitrogen Life Technologies) supplemented with 5 or 10% of fetal bovine serum (Biosource International, Camarillo, CA), respectively. For biological activity assays, CV-1 cells were seeded at a density of 2 ⫻ 104 cells/well in 24-well plates (Corning Inc., Corning, NY) or at 2 ⫻ 105 cells in 60-mm dishes (Corning Inc.). Unless specified otherwise, all transfections were in 24-well plates. After overnight culture in 24-well plates, a total of 0.3 ␮g plasmids containing 0.1 ␮g of reporter plasmid, 0.01 ␮g of Renilla internal control plasmid, and other expression plasmids as indicated in the figure legends were mixed with freshly diluted Fugene solution (0.7 ␮l of Fugene per 0.3 ␮g DNA), incubated at room temperature for 30 min, and added to the culture. For transfections in 60-mm dishes, 10-fold more DNA and Fugene were applied. One day later, transfected cells were induced with the appropriate steroid for 24 h. The cells were lysed and assayed for reporter gene activity using the luciferase assay reagent according to the manufacturer’s instructions (Promega). Luciferase activity was measured in an EG&G Berthold (Oak Ridge, TN) luminometer (Microlumat LB96P). The data were normalized for Renilla null luciferase activity to correct for differences in transfection efficiency. The partial agonist activity of a steroid A (expressed as percent) is defined as follows: 100⫻[(the activity with 1 ␮M steroid A) ⫺ (the basal level seen in the absence of hormone)]/[(the activity with 1 ␮M Dex) ⫺ (the basal level seen in the absence of hormone)].


In Vitro Transcription and Translation pRBAL-GR, pRBAL-GR486C, pRBAL-GR-N523, and luciferase control plasmid (Promega) are under the control of SP6 promoter. For each reaction, 1 ␮g of plasmid DNA was mixed with 2 ␮l of [35S]methionine plus 40 ␮l of TNT SP6 master mix (Promega), brought up to a total volume of 50 ␮l with H2O, and supplemented with steroid to give a final concentration of 1 ␮M. The reaction was conducted at 30 C for 90 min as recommended by the manufacturer (Promega). Bacterial Expression of Proteins FLAG/BAP, FLAG/Ubc9, GST, and GST/GR261–488-expressing constructs were transformed into BL21 bacterial cell line (Invitrogen Life Technologies) according to the manufacturer’s procedure. A single colony was selected, inoculated into 2 ml of Luria Bertani broth (Quality Biologicals Inc., Gaithersburg, MD) with 100 mM ampicillin (Sigma), and cultivated overnight at 37 C in a shaking incubator. One milliliter of the overnight culture was inoculated into 100 ml of LB broth containing 100 mM ampicillin, shaken at 37 C for 3–4 h and induced with 0.4 mM isoproply-1-thio-␤-D-galactopyranoside for 2.5 h. Cells were harvested by centrifugation and lysed for 15 min at room temperature in 5 ml of BugBuster Protein Extraction Reagent (Novagen, Madison, WI) containing Complete Mini protease inhibitor cocktail (one tablet per 10 ml solution; Roche Diagnostics GmbH, Mannheim, Germany), 25 U/ml Benzonase nuclease and 1 KU/ml rLysozyme solution (Novagen). After centrifugation at 20,000 ⫻ g for 20 min, the clear supernatants were used immediately for pull-down assays. Pull-Down Assay with Anti-FLAG M2 Agarose M2-agarose beads (anti-FLAG monoclonal antibody conjugated to agarose, Sigma) were prepared by treating them with 10 volumes of 100 mM glycine-HCl buffer (pH 3.5) for 2–3 min, which was then neutralized with 10 vol of 1 M Tris-HCl (pH 8.0). The beads were washed once with wash buffer (10% glycerol, 20 mM Tris-HCl, 0.4 mM EDTA, 0.2% Tween 20, 10 mM ␤-mercaptoethanol, and 500 mM KCl) and resuspended in 1 volume of wash buffer. Aliquots (200 ␮l) of bacterial extracts containing either FLAG/BAP or FLAG/Ubc9 were incubated with 20 ␮l of M2-agarose beads for 2 h at room temperature. The beads were then washed five times with wash buffer. The radioactive proteins (labeled with [35S]methionine during in vitro translation in rabbit reticulocyte lysates) were added separately to the M2 beads bound with FLAG/BAP or FLAG/Ubc9 and incubated at room temperature for 1 h and then at 4 C for 16 h. The beads were washed five times with wash buffer and finally resuspended in 20 ␮l of wash buffer. To extract the proteins from the beads, 20 ␮l of 2⫻ SDS-PAGE loading buffer was added and boiled for 5 min. After separating the extracted proteins by 10–12% SDS-PAGE gels, the gels were dried and exposed to x-ray films for 1 or 2 d. Pull-Down Assay with Glutathione-Sepharose 4B Beads To analyze the interactions between the GR261–488 fragment and GR constructs, Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) were loaded with bacterially expressed GST or GST/GR261–488 according to the manufacturer’s specifications. The subsequent binding of [35S]methionine-labeled GRs and washing of the beads are comparable to those with anti-FLAG M2 agarose. Western Blotting Sodium dodecyl sulfate-polyacrylamide gels were equilibrated in transfer buffer for 15 min before elctrophoretic


transfer of proteins to PROTRAN nitrocellulose membranes (Scheicher & Schuell GmbH, Dassel, Germany) using Xcell II transblot modules (110 mA overnight; Invitrogen Life Technologies). The membranes were stained in Ponceau S staining solution (0.2% PonseaUSAnd 0.04% glacial acetic acid in water) to localize the molecular weight markers, incubated with 5% Carnation nonfat dry milk in TBS (Quality Biologicals Inc.) containing 0.1% Tween 20 (Bio-Rad Laboratories, Hercules, CA) for 30 min, and washed three times with TBS containing 0.1% Tween 20 for 5 min each. Primary antibody were diluted in TBS containing 0.1% Tween 20 (1:5,000 for BUGR-2) and added to the membrane for 1 h at room temperature. Biotinylated secondary antibodies (antimouse IgG for BuGR2) and ABC reagents (Vector Laboratories, Burlingame, CA) were added sequentially for 30 min incubation each at room temperature. The nitrocellulose membranes were washed three times for 5 min each with TBS containing 0.1% Tween 20. The signals were detected by enhanced chemiluminescence using the recommended protocol of the supplier (Amersham Pharmacia Biotech). Steroid Binding Assays, Scatchard Plots, and Hill Plot Analysis GAL/GR and wt GR proteins were expressed in COS-7 cells by transfecting cells with pM/GR and pSVL-GR plasmids via calcium phosphate coprecipitation method as described previously (83). Cytosols of transfected cells containing the steroid-free receptors were obtained as reported previously (84). Thirty percent cytosol supplemented with 20 mM sodium molybdate was added to varying concentrations of [3H]Dex ⫾ 100-fold excess of nonradioactive Dex and incubated at 0 C for 18 h. Unbound [3H]Dex was removed by dextran-coated charcoal. The binding capacity and affinity were determined by Scatchard plot analysis by plotting [bound steroid]/[free steroid] vs. [bound steroid]. Receptor cooperativity was determined by Hill plot analysis by plotting Y/(1-Y) vs. [free dex], where Y is the fraction of [3H]Dex-binding sites occupied. The Hill plot coefficient (nH) was determined by the slope of the plot as previously reported (56). Statistical Analysis Unless otherwise indicated, all transient transfection experiments were performed in triplicate. KaleidaGraph 3.5 (Synergy Software, Reading, PA) determined a leastsquares best fit (R2 was almost always ⱖ 0.95) of the experimental data to the theoretical dose-response curve, which is given by the equation derived from MichaelisMenten kinetics of y ⫽ [free steroid]/([free steroid] ⫹ Kd) (where the concentration of total steroid is approximately equal to the concentration of free steroid because only a small portion is bound and Kd is the equilibrium dissociation constant), to yield a single EC50 value. The values of n independent experiments were then analyzed for statistical significance by the two-tailed Student t test using the program InStat 2.03 for Macintosh (GraphPad Software, San Diego, CA). When the difference between the SD of two populations is significantly different, then the Mann-Whitney test or the Alternate Welch t test is used.

Acknowledgments We thank Hinrich Gronemeyer, Olli Ja¨nne, Michael Stallcup, and Keith Yamamoto for generously providing plasmids, members of the Steroid Hormones Section for helpful comments, and Paul Mittelstadt (National Cancer Institute, National Institutes of Health) for critical review of the manuscript.


Received April 1, 2004. Accepted November 4, 2004. Address all correspondence and requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, National Institute of Diabetes and Digestive and Kidney Diseases/ LMCB, National Institutes of Health, Bethesda, Maryland 20892. E-mail: [email protected]. Present address for S.C.: I.G.B.M.C., B. P.10142, 1 rue Laurent Fries, 67404 Illkirch-Strasbourg, France.


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