Department of Medicine (D.W.R., J.S., A.W.) and School of Biological Sciences (C.-S.S., A.B., A.W.) University of Manchester Manchester, M13 9PT, United Kingdom
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ABSTRACT |
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Substitution to a nonaromatic hydrophobic amino acid, valine
(Tyr735Val), retained high-affinity ligand binding for dexamethasone
(Kd 6 nM compared with
4.6 nM) and did not alter transrepression of
NF-B. However, there was a 36% reduction in MMTV activity with a
right shift in EC50 (14.8
nM). The change to serine, a small polar amino
acid (Tyr735Ser), caused significantly lower affinity for dexamethasone
(10.4 nM). Maximal transrepression of NF-
B
was unaltered, but the IC50 for this effect was
increased. Tyr735Ser had a major shift in EC50
(118 nM) for transactivation of an MMTV
reporter.
Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.
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INTRODUCTION |
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The GR has a modular structure, with a central DNA binding motif, which
is well conserved among the nuclear receptor superfamily. The
N-terminal region contains a transactivation domain, and the C-terminal
region contains the ligand binding function as well as a further
transactivation domain and nuclear localization signal. Members of the
nuclear receptor superfamily whose crystal structures have been solved
have a ligand-binding domain (LBD) consisting of 12 -helices (7, 8, 9).
These 12 helices are thought to form a pocket with a hydrophobic lining
into which the ligand binds. The process whereby ligand binding results
in an activated receptor is uncertain. The crystal structure of the
estrogen receptor (ER) with agonist and antagonist has been revealing.
It appears that the C terminal helix moves across to enclose the ligand
and thereby generates a new composite surface on the receptor. The
crystal structure of the ER with antagonist bound, however, shows that
helix 12 is in a different position, and so the activated surface is
not formed. Presumably, this results in a receptor that is unable to
efficiently recruit the necessary coactivators to augment transcription
(8, 10).
Glucocorticoid effects on transcription may be mediated by both the
direct binding of activated GR dimers to target DNA, but also by
binding of receptor monomers to other transcription factors, including
AP-1, nuclear factor-B (NF-
B) and NUR77 (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). It has been
shown that these two modes of receptor activity are dissociable,
i.e. negative effects on NF-
B activity can be retained
when there is loss of transactivation.
Specific mutations in the GR cause selective loss of GR dimerization and so prevent dimer-dependent transactivation. Such dissociated receptors (22) retain the ability to oppose other transcription factor function. It is clear that the GR dimer-independent mechanism is capable of subserving more glucocorticoid actions than previously thought a total knockout of the GR prevents neonatal survival, but specific abrogation of receptor dimerization results in mice with no gross phenotype (23).
A number of synthetic GR ligands have been identified with similar
activity to dexamethasone in opposing NF-B signaling but reduced
ability to transactivate a GR dimer-dependent reporter gene (23). These
dissociating glucocorticoid ligands provide an interesting insight on
receptor function. The GR is capable of binding these ligands with high
affinity, and the activated receptor is capable of nuclear
translocation. However, the GR is presumably incapable of recruiting
the necessary cofactors for transactivation. This suggests that there
are signals encoded within the ligand, which, if detected by the
ligand-binding pocket, promote the receptor to undergo full
conformational change into a transactivating transcription factor.
Therefore, the partial agonist ligands would be capable of binding to
the receptor, but only of inducing a partial conformational change.
Further, true antagonists at the receptor would be predicted to bind
receptor but fail to induce the conformational changes needed for
either transactivation or interaction with other transcription factors.
The partial agonist RU24858 is the most specific yet characterized
(23). The molecule differs from dexamethasone or cortisol in the D
ring, suggesting that this part of the ligand conveys the activation
signal. The A, B, and C rings, which are identical to the full agonists
and similar to receptor-binding antagonists, may convey affinity
information.
The aim of this study was to model the LBD of the GR, by its homology with the progesterone receptor (PR), and further to identify amino acids within the ligand-binding pocket that may differentiate between agonists, partial agonists, and antagonists.
In this work we have identified tyrosine 735 as important for
ligand binding and ligand-dependent transactivation. The hydroxyl
side chain contributes to receptor activation but not to ligand binding
as evidenced by mutation to phenylalanine, which results in unchanged
ligand binding affinity and transrepressive capacity, but reduced
transactivation potential. Mutation to valine results in a minimal
reduction in ligand binding affinity, no change in transrepression, but
similar changes in transactivation as the phenylalanine mutant. In
contrast, mutation to the small polar side chain amino acid serine
results in reduced ligand binding affinity, unaltered ability to
transrepress NF-B, but a marked right shift in the transactivation
dose response and a further blunting of the dose response. Therefore,
the hydrophobic benzene ring of tyrosine contributes to ligand binding,
and the tyrosine hydroxyl side chain contributes to
transactivation.
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RESULTS |
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Evaluation of the 3D Structure
The quality verification of this structure was performed
using the PROCHECK program (25), which generates a Ramachandran plot.
About 99% of the -
angles in this model were placed within the
most favored and also within allowed regions of the conformational
space. The Protein Structure Analysis (Prosa) program (26) was used for
energy analysis of this model. This predicts that each residue
interaction energy in the structure was negative. The 3D structure
predicted for the LBD of the human GR could be superimposed upon that
for the PR with a root mean square deviation (rms) of 0.38 Å for 250
C
atoms.
The LBD of the hGR is outlined by helices 5, 7, 11, and 12, the
ß-turn, and loops L67 and L1112 (Fig. 1). The ligand-binding
pocket is predicted to be lined by 18 amino acids. Of these, 15 are
predicted to contribute to the hydrophobic environment of the pocket:
Met560, Leu563, Leu566, Gly567, Trp600, Met601, Met 604, Ala605,
Leu608, Phe623, Met 646, Leu732, Tyr735, Thr739, Phe749. There are
three polar residues, two at one end of the pocket, Gln570 and Arg611,
and the other, Cys736, at the opposite end (Fig. 2a
).
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Affinity of Mutant Human GR for Dexamethasone
As Tyr735 was predicted to have hydrophobic interaction with the D
ring of dexamethasone, it was important to identify changes in ligand
binding affinity caused by the mutations at position 735. Site-directed
mutagenesis to phenylalanine (Tyr735Phe) resulted in no alteration in
ligand binding affinity (4.3 nM compared with 4.6
nM for wild-type), in keeping with the hypothesis that the
benzene ring is sufficient to generate a hydrophobic surface for ligand
interaction. Change to valine (Tyr735Val) resulted in lower affinity
binding compared with wild-type, but only to a minor degree (6
nM). The change to serine (Tyr735Ser), however, resulted in
a 2-fold reduction in ligand binding (10.4 nM) (Table 1 and Fig. 3
).
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Transactivation by Mutant GR
All three mutant GR molecules were capable of transactivating the
mouse mammary tumor virus (MMTV) promoter (Fig. 4 and Table 1
). In response to
dexamethasone, Tyr735Phe had a similar EC50 to the
wild-type (8.6 nM compared with 6 nM) and
Tyr735Val a lower EC50 14.8 nM. However, the
maximal transactivation potential of both of these two mutant GR
molecules was less than the wild type (Fig. 4
and Table 1
).
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Further, the physiological ligand hydrocortisone (100 nM) induced 18-fold induction of MMTV via the wild-type GR, 11-fold with the Tyr735Phe, 10.6-fold with the Tyr735Val, and failed to induce the reporter via the Tyr735Ser. Thus, the mutated receptors have the same rank order of activity with both agonist ligands.
Transrepression by Mutant GR
Transrepression by activated GR usually has a lower
EC50 than transactivation and places less stringent
requirements on the receptor. Hence, ligands have been identified that
promote transrepression in the absence of transactivation, but not vice
versa. We examined the ability of the mutant GR molecules to inhibit
NF-B p65-mediated transactivation through a NF-
B response element
linked to luciferase (Fig. 5
). The
reporter was driven by cotransfection of a p65 expression vector. The
wild-type receptor achieved significant suppression at 0.1
nM dexamethasone and maximal suppression at 1
nM, as did Tyr735Phe and Tyr735Val. Tyr735Ser had a minor,
but consistent, increase in IC50 for this effect, with no
suppression at 0.1 nM dexamethasone (Fig. 5
). These data
are compatible with the observed Kd for binding to
dexamethasone and show the dose-response curve for transrepression to
be left-shifted in comparison with transactivation (Figs. 4
and 5
). In
contrast to the transactivation data, Tyr735Phe and Tyr735Val performed
similarly to the wild-type GR, showing that the substitution of Tyr735
results in selective impairment of transactivation in the absence of
significant changes in ligand binding affinity and transrepression.
Tyr735Ser has a slightly higher IC50 for transrepression
compared with the other three GR molecules examined, compatible with
the observation that its affinity for Dex is reduced (Fig. 5
and Table 1
). However, at 100 nM Dex Tyr735Ser has achieved maximal
suppression, which is close to that observed with the wild-type GR, in
striking contrast to the results seen on transactivation (Figs. 4
and 5
and Table 1
).
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DISCUSSION |
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The C3 ketone group of the steroid A ring formed two hydrogen bonds with Arg611 (helix 5), bond length 3.00 A, and Gln570 (helix 3), bond length 3.16 A. These two amino acids are found to be conserved in the mineralocorticoid receptor (MR) (Arg817 and Gln776), and the orientation of ligand within both the MR, determined by mutagenesis experiment (28), and the PR, determined by crystal structure (9), is the same as in our model. It is relevant that mutation of the Arg817 to Ala in the MR resulted in undetectable binding to cortisol, and Gln776 to Ala resulted in 40-fold lower affinity for cortisol, strongly suggesting their involvement in ligand binding (28).
The model predicts that Cys736, within helix 11, makes contact with the ligand by formation of a hydrogen bond with the steroid D ring C20 ketone group. The bond length is calculated to be 3.04 A. Cys736 is conserved in the related MR (Cys942), and the PR. Affinity labeling experiments suggest that Cys736 lies in close proximity to the ligand. Natural mutation of the homologous residue in mouse, mGR (Cys742Gly), which was found in dexamethasone-resistant lymphoma, results in a receptor molecule with reduced activity (30). In addition, extensive mutagenesis of Cys736 has been performed in yeast and mammalian cells (31). Change to alanine results in no change in receptor function. In contrast, change to Ser results in near-absent transactivation in response to cortisol, and a right shift in dose response to triamcinolone. Interestingly, Cys736Thr results in a molecule with enhanced transactivation response to triamcinolone and reduced response to cortisol (31). These data suggest that different steroids may interact with the pocket-lining amino acids in subtly different ways. All other amino acid substitutions resulted in absent receptor activity. These studies make it clear that the relatively hydrophobic residues, Ala and Cys, function better than Ser or Thr, which have polar hydroxyl side groups, except that Thr functions better with triamcinolone as the ligand.
Affinity labeling experiments with the GR have suggested that Met604 (helix 5), Cys638 (loop 67) and Cys736 (helix 11) are in close proximity to the ligand binding surface (32). In our model Met604 and Cys736 are within 4.5 A of dexamethasone, but Cys638 is not. Met604 and Cys736 were identified using labeled triamcinolone and the Cys638 by dexamethasone mesylate, and the different molecular structure of this steroid may explain the discrepancy.
The adjacent amino acid Tyr735 appears to contribute to the hydrophobic internal surface of the ligand-binding pocket, but the hydroxyl group is too far away to interact with ligand. It is relevant that Tyr735 is tightly conserved through vertebrate evolution, with substitution to Phe in Xenopus and Tilapia, and to isoleucine in guinea pig and rainbow trout. The change to Phe is conservative, in that the benzene ring is preserved. The human PR also has Tyr at the equivalent position. It appears that the steroid D ring carbons C15, C16, and C22 of dexamethasone would make close contact with Tyr735, but the hydroxyl group could not be accommodated in the ligand binding interaction. Dexamethasone is a more potent glucocorticoid agonist than cortisol and has a larger, hydrophobic region in close proximity to the Tyr735, with the C22 attached to C16. Cortisol has a single hydrogen in this position and so would be expected to make weaker contact with the Tyr735 benzene ring. Progesterone, a steroidal GR antagonist, also lacks a C16 substituent, but also has a smaller, less polar C17 attachment with no hydroxyl groups on C21, C17, and C11, which are all orientated on the opposite side of the molecule. Progesterone, therefore, is a less bulky molecule, with a calculated Van der Waals volume of 304.8 A3 compared with 318 A3 for cortisol (33), and this may reduce the ligand interaction with Tyr735.
To identify amino acid residues that could potentially distinguish agonists from antagonists, we looked at residues predicted to interact with the steroid D ring. The steroid recognizing amino acid should also potentially be capable of transmitting a signal to the rest of the molecule, perhaps by hydrogen bond formation, and Tyr735 fulfilled these criteria. Mutation of such a residue would be predicted to alter transactivation but not ligand binding affinity. Previously identified mutants with this characteristic include mutation of a highly conserved Gln755 within the AF-2 helix, which reduces binding to coactivators (34, 35, 36). Clearly Gln755 could not be involved in ligand recognition as it is directed away from the ligand-binding pocket.
Mutation of Tyr735 confirms the importance of this residue for ligand binding and transactivation. Substitution by phenylalanine results in no change to ligand binding affinity, as predicted by the model, but does reduce ligand-mediated transactivation. More severe disruption of Tyr735 to valine and to serine indicates the importance of the aromatic ring and hydrophobicity, respectively, for efficient binding to dexamethasone. The change in transactivation ability shown by the mutants is not predictable from their ligand binding affinities, which are, however, concordant with their ability to transrepress. This suggests that the Tyr735 does subserve two functions, not only in mediating ligand binding but also contributing to the conformational change of helix 12 that is predicted to be required for efficient recruitment of transcriptional coactivator molecules.
We propose that there are two separate and separable signals encoded within the ligand: one directs high-affinity binding to a specific receptor, and the second induces receptor conformational change. Therefore, steroidal GR antagonists, such as progesterone, contain signal 1, but not signal 2. It is proposed further that partial agonists, such as RU24858, encode high-affinity binding but only allow partial receptor conformational change such that they can interact with other transcription factors but not efficiently with the coactivators. Full agonists and steroidal antagonists share structural features at the steroid A, B, and C rings, but differ at the D ring. Therefore, it seems likely that the signal to activate the receptor lies within the steroid D ring, and that this promotes receptor conformational change by interaction with a recognition motif within the ligand-binding pocket.
In summary, we have identified Tyr735 as an important amino acid for ligand binding, and for mediating ligand-dependent transactivation an activity conferred by its hydroxyl group. Mutations at this residue have a disproportionate impact on transactivation compared with either ligand binding affinity or transrepression, suggesting that Tyr735 forms part of the mechanism for ligand-dependent conformational change.
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MATERIALS AND METHODS |
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Generation of Mutant GR
An EcoRI fragment of the human GR cDNA (16302377)
was subcloned into pGEM 11Zf(+), and codon 735 was mutated using a
GeneEditor kit from Promega Corp. (Madison, WI) and
synthetic mutagenic oligonucleotides synthesized by Perkin Elmer Corp. (Norwalk, CT). The identity of all mutants was verified by
sequencing. Mutated fragments were subcloned back into the hGR cDNA in
pcDNA3, and orientation was confirmed by enzyme digestion.
Ligand Binding
COS 7 cells, obtained from European collection of animal cell
cultures (ECACC), were transfected in 10-cm plates with 5 µg of the
GR expression vectors using Lipofectamine plus reagent, as suggested by
the manufacturer. Cells were split after 24 h into 24-well plates
and were serum starved for 16 h before study. Cells were incubated
with tritiated dexamethasone (Amersham International,
Bucks, UK) at increasing concentrations between 0.1 and 20
nM. Incubations were performed in multiples of 3, and at
each concentration three wells were incubated with 100-fold excess of
cold dexamethasone to measure nonspecific binding. All experiments were
performed on four separate occasions. After 1 h incubation at 37
C, cells were washed three times in serum-free medium and were lysed in
Tris-Cl (pH 7.8), 150 mM NaCl, 1% Triton-X 100. The cell
lysate was counted in a Packard ß-counter (Packard Instruments,
Meriden, CT). Specifically bound counts were calculated and bound
ligand was determined from the measured specific activity of the
tritiated dexamethasone. Ligand binding affinity was calculated by
nonlinear regression analysis using the Simfit package (WF Bardsley,
University of Manchester, UK), as previously described (38).
Transactivation Analysis
COS 7 cells were transfected with 2 µg MMTV-luc (39), 1 µg
CMV-ßGAL, and 1 µg CMV-hGRwt, phe, ser, or val using Lipofectamine
plus in 10-cm tissue culture dishes. Cells were divided and treated in
triplicate. Cells were harvested after 24 h, and the luciferase
activity was measured as previously described (39). Results were
normalized to ß-galactosidase as measured using the
O-nitrophenyl ß-D-galactopyranoside
assay, by dividing light units by optical density units, as described
previously (28). Experiments were repeated on four occasions with
similar results.
Transrepression Analysis
COS 7 cells were transfected with an NRE-luc reporter (17), an
NF-B p65 expression vector (17), CMV-ßgal, and GR expression
vectors. Cells were then split and treated with dexamethasone for
48 h before harvest, and luciferase/lacZ assays. Results are
expressed as light units per unit lacZ, to control for transfection
efficiency (28).
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FOOTNOTES |
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David Ray was supported by a Glaxo-Wellcome Research Fellowship.
Received for publication May 21, 1999. Revision received July 27, 1999. Accepted for publication August 4, 1999.
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REFERENCES |
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