Identification of Amino Acids in the
2-Region of the Mouse Glucocorticoid Receptor That Contribute to Hormone Binding and Transcriptional Activation
Jon Milhon,
Sunyoung Lee,
Kulwant Kohli,
Dagang Chen,
Heng Hong and
Michael R. Stallcup
Departments of Pathology and of Biochemistry and Molecular
Biology University of Southern California Los Angeles,
California 90033
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ABSTRACT
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The
2-region of steroid hormone receptors
is a highly conserved region located at the extreme N-terminal end of
the hormone-binding domain. A protein fragment encoding
2 has been
shown to function as an independent transcriptional activation domain;
however, because this region is essential for hormone binding, it has
been difficult to determine whether the
2-region also contributes to
the transactivation function of intact steroid receptors. In this study
a series of amino acid substitutions were engineered at conserved
positions in the
2-region of the mouse glucocorticoid receptor (mGR,
amino acids 533562) to map specific amino acid residues that
contribute to the hormone-binding function, transcriptional activation,
or both. Substitution of alanine or glycine for some amino acids
(mutations E546G, P547A, and D555A) reduced or eliminated hormone
binding, but the transactivation function of the intact GR and/or the
minimum
2-fragment was unaffected for each of these mutants.
Substitution of alanine for amino acid S561 reduced transactivation
activity in the intact GR and the minimum
2-fragment but had no
effect on hormone binding. The single mutation L550A and the double
amino acid substitution L541G+L542G affected both hormone binding and
transactivation. The fact that the S561A and L550A substitutions each
caused a loss of transactivation activity in the minimum
2-fragment
and the full-length GR indicated that the
2-region does contribute
to the overall transactivation function of the full-length GR. Overall,
the N-terminal portion of the
2-region (mGR 541547) was primarily
involved in hormone binding, whereas the C-terminal portion of the
2-region (mGR 548561) was primarily involved in transactivation.
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INTRODUCTION
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Steroid hormone receptors are hormone-regulated transcriptional
activator proteins that consist of three major functional domains: a
C-terminal hormone binding domain (HBD), a DNA binding domain (DBD)
located in the central region of the polypeptide chain, and an
N-terminal transcriptional activation domain (1, 2, 3). The HBD of steroid
hormone receptors has many functions, in addition to hormone binding.
In the absence of hormone it is responsible for binding hsp90. The HBD
contains nuclear localization and dimerization functions that are
activated after hormone binding causes dissociation of hsp90. After the
receptor dimer binds to its cognate enhancer element on DNA, the HBD
also contributes to the transcriptional activation function of the
intact receptor, apparently by protein-protein contacts with basal
transcription factors and/or transcriptional coactivators (4, 5, 6, 7, 8, 9).
The transcriptional activation function (AF) of intact steroid
receptors is thought to reside primarily in two regions: AF-1 located
in the N-terminal activation domain, and AF-2 located near the C
terminus of the HBD (10, 11). In addition, several studies suggest that
the
2-region of steroid receptors, found at the N-terminal end
of the HBD [amino acids 533562 of mouse glucocorticoid receptor
(mGR)], may contribute to transactivation (10, 12). This
2-fragment
includes the N-terminal boundary of the HBD and contains a sequence
that is highly conserved among steroid receptors, but only a few
residues in this region are conserved between steroid receptors and
other nuclear receptors (13, 14). A translocated protein fragment
containing the
2-region exhibits an autonomous transactivation
activity when attached to a DBD (10). However, in the context of the
intact HBD, deletions and insertions in this region eliminate hormone
binding; this has prevented determination of whether the
2-region
actually contributes to the transactivation function of intact steroid
receptors (3, 15, 16). In the study reported here, point mutations were
used to circumvent the problem of interdigitated hormone binding and
transactivation functions. Numerous single and one double amino acid
substitutions in the mGR were tested for their effects on hormone
binding and transcriptional activation in the context of the intact
glucocorticoid receptor (GR) HBD. However, in this context the ability
to observe transactivation activity depends on the ability of the
mutant HBD to bind hormone; i.e. if a mutation eliminates
hormone binding, it is not possible to assess the effect of that
mutation on the transactivation function. Therefore, each mutation was
also tested for its effect on transcriptional activation in the context
of the minimal
2-domain, where its activity was independent of
hormone-binding activity. The resultant data provided a detailed
functional map of the
2 domain and indicated that the
2 region
contributes to the transactivation function of the intact GR HBD.
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RESULTS
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Hormone Responsiveness and Hormone Binding Affinity of mGR
2
Mutants
Several point mutations in the
2 region of mGR (residues
533562) have previously been shown to reduce or eliminate hormone
binding (13); these include a double substitution at amino acids 541
and 542, and single substitutions at positions 543, 544, 546, 547, and
549 (Fig. 1
). The present study extended
the analysis of some of these mutants and also examined the effects of
alanine or glycine substitutions at six additional highly or partially
conserved positions in the
2 region, i.e. amino acids
548, 550, 555, 556, 560, and 561 (Fig. 1
).

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Figure 1. The 2-Region of mGR
The amino acid sequence of the 2-region of mGR (18) is shown with
the specific amino acid substitutions (arrows) used in
this study. Homology with other steroid receptors is indicated: hMR,
human mineralocorticoid receptor (30); hPR, human progesterone receptor
(31); hAR, human androgen receptor (32); hER, human estrogen receptor
(33); dashes () indicate the same amino acid as mGR;
dots indicate a gap introduced to optimize the alignment
of sequences. The N-terminal end of the HBD is at approximately mGR
L538 (3, 16, 28). Numbers above the substituted amino
acids indicate previously published mutations: 1, Ref. 13; 2, Ref. 18;
3, Ref. 19.
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The newly engineered mutant GR species were expressed in CV-1 cells by
transient transfection to test their ability to activate a mouse
mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT)
reporter gene in response to various concentrations of dexamethasone
(Dex); the EC50 value for Dex was determined as the
concentration of Dex required to produce 50% of the maximum CAT
activity achieved with saturating Dex concentrations. The
EC50 values for four of the new mutants (E548A, L550A,
D555A, and S561A) were 5- to 20-fold higher than that for wild type
mGR. In contrast, mGR D560G was unresponsive to Dex; and the
EC50 of S556A was only slightly, if at all, higher than
that of wild type mGR (Table 1
).
The new mutant GR species and some of the previously reported mutant
GRs were translated in vitro to produce protein fragments
that included the intact DBD and HBD, and Dex-binding studies were
conducted under stringent conditions (17). Traditionally,
hormone-binding studies in vitro have been performed at 0 C
overnight in the presence of 20 mM sodium molybdate. While
these conditions produce optimal binding, we have shown that they mask
some mutant phenotypes, since the low temperature and molybdate
maintain GR interaction with hsp90 and help prevent denaturation of
temperature-sensitive and activation-labile mutants. In contrast, when
binding is conducted at 26 C for 30 min in the absence of molybdate, GR
dissociates from hsp90; these more stringent conditions allow
phenotypes of temperature-sensitive and activation-labile mutants to be
observed (17). The dissociation constant (Kd value) for Dex
binding to wild type GR was 24 nM when measured at 0 C
with molybdate, but was 1530 nM when measured at 26 C
without molybdate. Under the stringent conditions, two GR mutants
(E548A and S561A) with moderately (5- to 10-fold) increased
EC50 values for Dex exhibited Kd values for Dex
near 20 nM, which was indistinguishable from the wild type
value (Table 1
). Thus, the elevated EC50 values observed
for these mutants (Table 1
) did not appear to be caused by reduced
hormone binding. Two other GR mutants (L550A and D555A) with 10- to
20-fold elevated EC50 values exhibited Kd
values that were 2 and 5 times higher, respectively, than those for
wild type GR; these modest reductions in the hormone-binding affinity
account at least partially for the increased EC50
values.
Three previously reported mutants with severe functional impairment
were tested for their ability to bind hormone in vitro at 0
C with molybdate and, in one case, at 26 C without molybdate. E546G
(18) and the double mutant L541G+L542G (13) did not exhibit any Dex
binding at a concentration of 20 nM Dex even at 0 C (Fig. 2
). Both of these mutants were previously
shown to be unresponsive to Dex and did not bind hormone in transfected
cell extracts or whole cell binding assays. Mutant P547A was previously
reported to have an EC50 value 300 times higher than that
for wild type GR (19). Hormone binding studies in vitro
(Fig. 2
) indicated that P547A exhibited substantial Dex binding at 0 C
in the presence of molybdate, but failed to bind hormone at 26 C in the
absence of molybdate; thus, P547A appears to be a temperature-sensitive
or activation-labile type of mutant, similar to other previously
described GR mutants (17, 20). When the mutant and wild type GR
fragments were translated in vitro for the hormone-binding
studies, parallel translation reactions were conducted with
[35S]methionine; SDS-PAGE analysis of these products
demonstrated that approximately equivalent amounts of mutant and wild
type GR fragments were produced in the cell-free translation reactions
(data not shown).

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Figure 2. GR Mutants That Fail to Bind Dex in
Vitro
Unlabeled and 35S-labeled GR DBD-HBD fragments containing
the indicated amino acid substitutions were synthesized in parallel
cell-free reactions. The unlabeled products were incubated in
triplicate reactions with 20 nM [3H]Dex under
two conditions: overnight at 0 C with molybdate or for 30 min at 26 C
without molybdate; unbound Dex was removed by charcoal adsorption, and
the supernatant was counted to determine total Dex binding. Samples
from control translation reactions incubated without GR mRNA were used
to determine background Dex binding, and this background value was
subtracted from total Dex binding to determine specific Dex binding by
each GR species. Samples of the 35S-labeled GR products
were analyzed by SDS-PAGE and autoradiography; the results indicated
that approximately equal amounts of mutant and wild type GR species
were synthesized in the cell-free reactions (not shown).
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Effects of Mutations on Transactivation Activities of Intact GR and
Minimum
2-Fragments
CV-1 cells transiently expressing the newly engineered GR mutants
were treated with saturating concentrations of Dex to determine the
maximum level of MMTV-CAT reporter gene activity that each mutant GR
could produce. At saturating Dex concentrations, the transactivation
potential of mutants E548A, D555A, and S556A was essentially equivalent
to that of wild type GR, whereas reporter gene activation by L550A and
S561A was only about 30% and 20%, respectively, of that by wild type
GR; D560G produced no reporter gene activity (Fig. 3a
). When the same full-length GR
expression vectors were transfected into COS7 cells, immunoblots
demonstrated that D560G was not expressed, i.e. the protein
was presumably unstable; the other five mutant GR species were
expressed at levels approximately equivalent to that of wild type GR
(Fig. 3b
).

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Figure 3. Activation of MMTV-CAT Reporter Gene by Full-Length
Mutant and Wild Type GR at Saturating Dex Concentrations
a, CV-1 cells in 60-mm dishes were transiently transfected with
0.5 µg of the indicated mGR expression vector, 2.0 µg pMMTV-CAT,
and 0.5 µg pCMV-ßgal. Cells were harvested after 4872 h and
treated with 1 µM Dex during the final 24 h before
harvest. The ßgal activities in the cell extract were determined and
used to balance the quantities of extracts assayed for CAT activity,
thus normalizing for transfection efficiencies. In each experiment
triplicate transfections were conducted for each GR species, and the
results shown are the mean and SEM for two or more
independent experiments. b, COS7 cells in 60-mm dishes were transiently
transfected with 8 µg of the indicated wild type or mutant mGR
expression vector, and after 4872 h, cell extracts were made and
analyzed by immunoblot analysis with the BUGR2 antibody against GR. Two
independent transfection experiments are shown. WT, Wild type GR; 0,
mock transfected control, showing nonspecific bands; mGR, the position
of the intact mGR protein (the lower molecular weight GR species also
seen here are presumably degradation products or incomplete translation
products).
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Some amino acid residues presumably contribute either to hormone
binding function or to transactivation, while other residues
(e.g. those involved in forming three-dimensional structure)
may contribute to both activities. The failure of some of the mutant
GRs to bind hormone made it impossible to test whether those mutations
also have a direct effect on the function of the
2-transactivation
domain in the context of the intact GR. Thus, we established a system
for testing the transactivation potential of the minimum
2-fragment
containing various mutations. Three different wild type mGR fragments
were expressed transiently in CV-1 cells to test their relative
abilities to activate the MMTV-CAT reporter gene (Fig. 4
): one fragment
contained the intact DBD, hinge region, and HBD (mGR 395783); the
second fragment included the intact DBD, hinge region, and the
2-portion of the HBD (mGR 395562); and the third fragment
consisted of the intact DBD and hinge region (mGR 395533), but none
of the HBD or the
2 region. The longest fragment was tested in the
presence of hormone, while the two shorter fragments were tested in the
absence of hormone, since they do not bind hormone. Nonsaturating
amounts of the GR expression vectors were used (data not shown) to
ensure that the reporter gene activity observed was a true measure of
the specific transactivation activity of each GR species. The activity
of the DBD fragment lacking
2 was less than 1% that of the fragment
with an intact DBD and HBD, whereas the fragment containing the DBD and
2 produced 11% of the activity of the fragment containing an intact
HBD (Fig. 4a
). This experiment demonstrated the transactivation
activity of the minimum
2-fragment.

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Figure 4. Activation of MMTV-CAT
Reporter Gene by Minimum mGR 2-Fragments Attached to the GR DBD
a and b, Expression vectors (0.07 µg) for the indicated GR
species were transiently transfected into CV-1 cells in six-well
plates, along with 0.5 µg pMMTV-CAT and 0.25 µg pCMV-ßgal. Where
a full-length HBD was present, 1 µM Dex was added 24
h before harvest, and CAT activities were determined and normalized for
ßgal activities as described in Fig. 3 . In each experiment,
triplicate transfections were conducted for each GR species, and the
results shown are the mean and SEM for two or more
independent experiments. a, Relative CAT activities stimulated by three
different fragments of wild type GR were compared: full-length DBD and
HBD (mGR 395783); DBD with 2 (shaded) (mGR
395562); and DBD without 2 (mGR 395533). b, CAT activities
stimulated by GR DBD- 2-fragments (mGR 395562) containing the
indicated amino acid substitutions were compared with those of wild
type fragments containing and lacking 2. c, COS7 cells were mock
transfected (no GR) or transfected with vectors coding for mGR 395533
(no tau2) or mGR 395562 containing wild type (wt) 2 or 2 with
the indicated amino acid substitution(s); GR fragment expression was
observed by immunoblot analysis of the cell extracts as in Fig. 3 .
Positions of GR fragments are indicated on the right.
Results of two independent transfection experiments are shown, and
within each experiment extracts from duplicate transfected cultures are
shown for each GR expression vector.
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To test the effect of various mutations on the
2 transactivation
activity, DBD-
2-fragments containing the mutations were compared
with wild type fragments containing and lacking
2 by transient
transfection in CV-1 cells (Fig. 4b
). Four of the mutations (E546G,
E548A, L550A, and D555A) caused a modest 2550% reduction in
transactivation activity of the mGR 395562 fragment; the single
mutation S561A and the double mutation L541G+L542G caused dramatic
losses of transactivation activity, 85% and 95%, respectively; and
changing proline 547 to alanine caused an unexpected moderate 2- to
3-fold increase in transactivation activity. When the same expression
vectors were transfected transiently into COS7 cells, immunoblots of
the transfected cell extracts indicated that all mutants were expressed
at levels similar to those of the wild type mGR 395562 fragment (Fig. 4c
). Interestingly, the mGR 395562 fragment containing the double
substitution L541G+L542G migrated slightly slower than the wild type
fragments and the other mutants; the loss of the two hydrophobic
leucine side chains may have reduced the amount of SDS that bound to
the protein, and in such a small (167-amino acid) polypeptide chain the
reduction in net charge may have been enough to affect the rate of
migration during electrophoresis.
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DISCUSSION
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In addition to
2, several other regions of steroid receptor
HBDs have been implicated in transactivation. The conserved AF-2 region
near the C terminus of steroid receptors is the most well documented
transactivation region in the HBD; amino acid substitutions in this
region and nearby reduce or eliminate transactivation without affecting
hormone binding (21, 22). In addition, some point mutations in the
estrogen receptor just downstream from
2 have also been shown to
reduce transactivation but not hormone binding (23). The mechanism of
2-transactivation is unknown but presumably involves either
intermolecular interactions with coactivators or component(s) of the
transcription initiation complex or intramolecular interactions that
stabilize the appropriate structure of another transactivation domain
of the steroid receptor. For example,
2 could interact with a
transcriptional coactivator, such as the recently identified steroid
receptor coactivator-1 (SRC-1) (7) and glucocorticoid receptor
interacting protein 1 (GRIP1)/transcriptional intermediary factor 2
(TIF2) (8, 9), which mediate transactivation by nuclear receptor AF-2
domains. However, in yeast two-hybrid system assays, we failed to
detect any interaction between the minimum
2-domain and GRIP1;
furthermore, an mGR DBD-HBD fragment containing either the L550A or the
S561A mutation retained the ability to interact strongly with GRIP1
in vitro (our unpublished data). Thus, we find no evidence
that
2 interacts with this family of nuclear receptor
coactivators.
The
2-region of steroid receptors was originally defined as amino
acids 533562 of mGR (10). A portion of this region, corresponding to
mGR 541561, is highly conserved among all five steroid receptors
(Fig. 1
), and this homology is also partially shared with a few orphan
receptors, e.g. the estrogen receptor-related (ERR) and
chicken ovalbumin upstream promoter/seven-up proteins (14). However,
in spite of the overall homology in the HBD domain between steroid
receptors and the class II (thyroid hormone and retinoid) nuclear
receptors, the amino acid sequence conservation in the
2-region
between the steroid and the class II nuclear receptors is very low
(24). Although the three-dimensional structures of steroid receptor
HBDs have not yet been experimentally determined, recent x-ray
crystallographic studies of thyroid hormone receptor (TR) (25),
retinoic acid receptor (26), and retinoid X receptor (27) HBDs have
allowed structural predictions to be made for the HBDs of steroid
receptors (24) (Richard L. Wagner, University of California at San
Francisco, personal communication). The canonical nuclear receptor HBD
is a layered structure composed of 12
-helices and four short
ß-strands. In terms of the nomenclature designated for
TR
1 (25), helices H1, H3, H5-H6, H9, H10, and H11
cooperatively form the hydrophobic core of the HBD (Fig. 5A
). The bound hormone is in direct
contact with, and apparently influences the structure of, the
hydrophobic core. In the predicted structure for mGR based upon these
models (R. L. Wagner, personal communication) the N-terminal half
of the
2-region, including mGR amino acids 532547, is predicted to
be part of
-helix H1. One face of H1 makes contact with
-helices H4, H5-H6, H9, and H10 that are part of the highly
conserved hydrophobic core of nuclear receptor HBDs; another face of H1
contacts helix H3 (Fig. 5
). H1, through its interaction with these
other helices, is predicted to help stabilize the hydrophobic core and
thus the hormone-binding pocket of the HBD. Another face of H1 is
exposed on the surface of the HBD and accessible for interactions with
other proteins and thus could potentially play a role in
transactivation. The C-terminal portion of
2 in steroid receptors is
predicted to form a loop, whose structure cannot presently be predicted
because of the low homology and variable length of this region between
steroid and class II receptors. However, much of this loop is predicted
to be on the surface of the HBD and thus potentially accessible for
intermolecular protein-protein interactions. The extreme C terminus of
the
2-region (mGR amino acids 561562) is predicted to be at or
near the beginning of a long, conserved
-helical region called H3,
which is part of the hydrophobic core and also contributes to the
formation of the hormone-binding pocket (Fig. 5A
shows the
TR
1 HBD). The N-terminal end of H3 is predicted to be on
the surface of the HBD near the hormone-binding pocket and potentially
available for intermolecular interactions (24, 25) (R. L. Wagner,
personal communication). While direct structural studies of the steroid
receptor HBDs will be required to test these predictions, they provide
a useful model for comparison with the conclusions of the present
study.
The phenotypes of some of the GR mutants in this study correlate in an
interesting way with the predicted three-dimensional structure
described above. The N-terminal end of the mGR HBD has been defined
functionally as residue 538, although sequences upstream from this
residue help to stabilize the structure of the GR HBD (28). The two
leucines at mGR residues 541 and 542 and glutamic acid 546 are among
the most highly conserved residues in this region of steroid receptors,
estrogen receptor-related (ERR), and chicken ovalbumin upstream
promoter/seven-up proteins (14, 24). Substitution of glycines for the
two leucines (mutant L541G+L542G) or for glutamic acid 546 (mutant
E546G) eliminated hormone binding in vivo and thus the
ability of the intact GR to activate the MMTV-CAT reporter gene (13, 18). In this study, we demonstrated that the same GR mutants translated
in vitro also lacked hormone-binding function. Due to the
complete loss of hormone-binding activity, it was not possible to
determine whether mGR residues L541, L542, and E546 were important for
the transactivation function of the intact GR. To circumvent this
problem, the mutations were tested in the context of the minimum
2-fragment; in this context, the E546G substitution caused no
reduction in the ability of the mGR DBD-
2-protein fragment to
activate the MMTV-CAT reporter gene. However, the L541G+L542G mutation
essentially eliminated
2-transactivation activity. In the predicted
structure for mGR, these three residues are on the face of helix H1
that interacts with other helices of the hydrophobic core, namely H3
and H9 (Fig. 5B
shows helical wheel models of helix H1 for rat
TR
1 and mGR). The double mutation L541G+L542G eliminates
two hydrophobic contacts between H1 and the other helices. The mutation
E546G disrupts a predicted salt bridge to the buried, conserved residue
K673 in H9. None of these residues is predicted to interact directly
with hormone. Instead, the effect on hormone binding is indirect: the
mutations disrupt structural contacts between H1 and other helices in
the hydrophobic core and thus dislodge H1; this may affect the
integrity of the hydrophobic core and thus the hormone-binding pocket
(R. L. Wagner, personal communication).
Proline 547 is predicted to lie at the C-terminal end of
-helix H1
of mGR (24) (R. L. Wagner, personal communication). We previously
reported that a pro-to-ala substitution at this position caused a
300-fold increase in the EC50 for Dex, but when saturating
Dex concentrations were used, the mGR P547A mutant activated the
reporter gene to approximately the same extent as wild type GR (19). In
this study, hormone-binding assays in vitro confirmed that
the large change in EC50 was due to a severe reduction in
hormone-binding function. However, the lack of hormone binding was
observed primarily at elevated temperatures in the absence of
molybdate, conditions that favor dissociation of hsp90 from GR; at 0 C
in the presence of molybdate this mutant GR exhibited substantial
hormone binding. Thus the hormone-binding function of the P547A mutant
is either temperature-sensitive or activation-labile, i.e.
unable to retain its bound hormone after hsp90 dissociates or because
of reduced association with hsp90 at the elevated temperature (17, 20).
The P547A mutation in the minimum
2-fragment caused no loss and in
fact appeared to increase transactivation function (Fig. 4b
);
similarly, no loss of transactivation function was observed for this
mutation in the intact GR at saturating hormone concentrations
(19).
By analogy with the three-dimensional structures of class II nuclear
receptor HBDs, mGR amino acids 548560 are predicted to form an
exposed loop between
-helices H1 and H3 (Fig. 5A
shows
TR
1 HBD); because of the low homology in this region
between steroid and class II receptors, a more detailed structure
cannot yet be predicted for this region in steroid receptors (24)
(R. L. Wagner, personal communication). Substitution of alanine
for two of the less highly conserved residues in this region, E548 and
S556, caused little or no change in EC50 value for Dex,
Kd for Dex in vitro, or transactivation
function. In contrast, substitution for residues that are highly
conserved among the steroid receptors resulted in some loss of
function. The 13-fold increase in EC50 for Dex caused by
mGR mutation L550A can be attributed to a 2-fold increase in the
Kd for Dex observed in vitro and a 5070%
decrease in transactivation function, which was observed when the
mutation was included in the full-length GR and in the DBD-
2
fragment. A mutant mGR with a D555A substitution had an
EC50 for Dex 18-fold higher than that of wild type GR. A
5-fold increase in the Kd for Dex was the major factor
found to account for the increased EC50. The mutation had
no apparent effect on the transactivation function of the full-length
GR, although it caused a reduction of approximately 50% in the
transactivation function of the DBD-
2 fragment. The mGR mutant D560G
was unstable in cells, i.e. no protein was detected by
immunoblot; this explained why no reporter gene activation was observed
with this mutant GR. The instability of this mutant suggests that D560
may be involved in a structurally crucial salt bridge or hydrogen bond
with another part of the HBD.
The position analogous to mGR S561 is conserved as ser or thr in all
five steroid receptors. The mGR S561A mutant had an EC50
for Dex 9 times higher than that of wild type GR; this was accompanied
by a 5- to 7-fold decrease in transactivation activity, observed in the
context of the full-length GR and the DBD-
2-fragment. The
hormone-binding affinity of this mutant GR in vitro was
normal. This residue is predicted to be at or near the N-terminal end
of
-helix H3 (see Fig. 5A
), an exposed region near the
hormone-binding pocket with the potential to engage in intermolecular
interactions (R. L. Wagner, personal communication).
Whereas the transactivation function of the
2-region has been
defined in small fragments of steroid receptors and other types of
artificial constructions (10), it has been difficult to determine
whether this region actually contributes to the overall transactivation
function of full-length steroid receptors, because deletion of this
region causes loss of hormone-binding function and thus inactivates the
receptor (3, 16). Our results with mGR mutations L550A and S561A now
suggest that the
2-region does indeed contribute to the
transactivation function of the full-length GR; i.e. while
these mutations had little or no effect on hormone binding affinity,
each mutation caused similar reductions in the transactivation function
of the full-length GR and the DBD-
2-fragment. These results provide
direct evidence that
2, as well as the more well characterized AF-2
region (11, 21), contributes to the transactivation function of the
intact HBD of steroid receptors.
Figure 6
provides a summary of the
phenotypic changes caused by mutations in the mGR
2-region, based on
results from this and previous studies. The
2-region is essential
for hormone-binding function and also contributes to transactivation
function; the analyses reported here now allow the
2-region to be
divided into two functional subdomains. Amino acid substitutions that
affect hormone binding are found primarily in the mGR region 541547
of
2, while substitutions that affect
2-transactivation function
are located primarily in the region 548561. This conclusion is
consistent with structural predictions for GR based upon the known
three-dimensional structures of class II receptors (24) (R. L.
Wagner, personal communication). Amino acids 541547 of mGR are
predicted to form the C-terminal end of
-helix H1, which interacts
with other
-helices of the hydrophobic core that directly form the
hormone-binding pocket. Thus, although H1 does not directly make
contact with the bound hormone, our data suggest that H1 indirectly
plays a role in hormone binding through its contacts with the other
helices in the hydrophobic core that form the pocket. Amino acids
548560 are predicted to form an exposed loop, and residue S561 is
predicted to be near the N-terminal end of
-helix H3. In this
exposed position near the hormone-binding pocket, S561 could
potentially participate in intermolecular interactions that contribute
to transcriptional activation.

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Figure 6. The Effect of Amino Acid Substitutions on
EC50 for Dex, Dex Binding, and Transactivation by mGR
The sequence of the conserved part of the 2 domain is shown, along
with the amino acid substitutions used in this study. GG
indicates the double substitution mutant. The effect of each amino acid
substitution on various mGR activities is represented schematically and
thus indicates which amino acids of mGR contribute to each specific GR
function. Open circles indicate near-wild type activity;
gray symbols indicate partial loss of function; and
black symbols indicate severe or complete loss of
function. X indicates that, although no activity was detected, the
result was not informative, because the mutant protein was unstable
(D560G) or completely lacked hormone-binding function (L541G+L542G and
E546G) thus making it impossible to assess transactivation function in
the context of the intact GR. Numbers below the activity
symbols indicate data reported previously: 1, Ref. 13; 2, Ref.
18; 3, Ref. 19. All other symbols represent data from this study.
EC50 and Kd values are from Table 1 ;
transactivation by minimum 2-fragment, from Fig. 4 ; and maximum
transactivation by intact GR, from Fig. 3 .
|
|
 |
MATERIALS AND METHODS
|
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Construction of Mutant GR Expression Vectors
Mutations were introduced into the full-length, wild type mGR
expression vector pKSX (13) as described previously (29), except that
the proofreading DNA polymerase Pfu (Stratagene, La Jolla, CA) was used
for all PCR reactions. Expression vectors coding for a GR fragment
equivalent to mGR 395533 (DBD and hinge region, but lacking
2) and
395562 (including
2) were constructed by modification of vector
pC7/g407C (kindly provided by Dr. K. Yamamoto, University of
California, San Francisco, CA), which includes codons 407795 of the
wild type rat GR (equivalent to mGR 395783) attached to a thymidine
kinase gene translation start signal and leader sequence, driven by a
cytomegalovirus promoter. A fragment of pC7/g407C extending from a
unique SphI site in the rGR DBD to a unique NotI
site in vector sequences after the GR termination codon was deleted and
replaced by a compatible PCR fragment encoding a truncated mGR fragment
designed with a termination codon after mGR codon 533 or 562. Vectors
encoding the equivalent fragment of mGR 395562 with various point
mutations in the
2-region were constructed in a similar manner. The
wild type and mutant SphI-NotI fragments used for
these constructions were generated by using pKSX or the appropriate
mutant form of pKSX as template in PCR reactions. The downstream primer
created a stop codon at the appropriate site and provided a
NotI site for the subsequent cloning step; the upstream
primer included the SphI site in the GR DBD that is
conserved between mouse and rat cDNAs.
Functional Analysis of Mutant and Wild Type GR
Transient transfection of CV-1 and COS7 cells by the calcium
phosphate method, chloramphenicol acetyltransferase (CAT) assays, and
ß-galactosidase (ßgal) assays were performed as previously
described (17). Immunoblots of extracts from transfected COS7 cells
were performed as described previously (13). Cell-free synthesis of GR
DBD-HBD fragments and analysis of hormone binding by these GR fragments
were conducted as described previously (17).
 |
ACKNOWLEDGMENTS
|
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We thank Dr. Richard L. Wagner (University of California, San
Francisco) for communicating his structural prediction of the mGR HBD
and providing Fig. 5
; Dr. Beatrice Darimont (University of California,
San Francisco) for help in analyzing the implications of the structural
predictions for our mutational analysis; and Zahid Iqbal for technical
assistance.
 |
FOOTNOTES
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Address requests for reprints to: Michael R. Stallcup, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90033.
This work was supported by USPHS Grant DK-43093 (to M.R.S.) from the
National Institute of Diabetes and Digestive and Kidney Disease.
Received for publication November 27, 1996.
Revision received July 11, 1997.
Accepted for publication August 5, 1997.
 |
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