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(Received for publication, September 15,
1995; and in revised form, February 5, 1996) From the
The hormone binding domain (HBD) of the glucocorticoid receptor
(GR) contains five cysteine residues, with three of them being spaced
close to one another in the steroid binding pocket. The HBD also
contains the contact region for the chaperone protein hsp90, which must
be bound to the GR for it to have a steroid binding conformation.
Binding of hsp90 to the receptor through its HBD inactivates the DNA
binding domain (DBD). The DBD contains a number of cysteines essential
to its DNA binding activity. Here, we assess the effects of hsp90
binding on the accessibility of cysteine residues in both the HBD and
DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding
activity of the immunopurified, untransformed GR
The steroid binding site of the glucocorticoid receptor (GR) ( Steroid binding activity of the
GR is inactivated by a variety of sulfhydryl-reactive agents and by
redox conditions that promote disulfide bond formation (see Simons and
Pratt(1995) for review of GR thiols and steroid binding activity). A
series of studies from the Simons laboratory has demonstrated that
steroid binding activity is inactivated by the formation of disulfide
bonds between cysteine SH groups that are vicinally spaced in the HBD
when it is bound to hsp90 (Miller and Simons, 1988; Simons et
al., 1990, Chakraborti et al., 1990, 1992). Simons et
al.(1989) have shown that the GR can be cleaved with trypsin to a
16-kDa fragment of the HBD that binds glucocorticoids with One of the cysteines in the vicinal thiol cluster
(Cys-656) is the site that is covalently labeled by the site-specific
affinity label dexamethasone 21-mesylate (Simons et al.,
1987), and the steroid binding activity of the GR is abrogated by
arsenite at low concentrations where it acts as a vicinal
thiol-specific reagent (Chakraborti et al., 1990; Lopez et
al., 1990; Simons et al., 1990), reacting specifically
with Cys-656 and Cys-661 in the thiol cluster (Chakraborti et
al., 1992). We have previously compared the effects of the
reversible thiol-reactive agents arsenite and methyl
methanethiosulfonate on both the steroid binding activity of the
unliganded GR and dissociation of steroids from preformed
steroid-receptor complexes (Stancato et al., 1993). Our
observations were consistent with the concept that the thiol cluster
lies in a portion of the binding pocket that is critical for binding of
the D-ring of the steroid, and we proposed that labeling of the GR with
a derivatizing agent that reacts preferentially with vicinally spaced
dithiols might allow detection of conformational changes likely to
occur in a critical region of the HBD on dissociation of hsp90. Many
reagents have been used to label protein thiols, including radioactive
iodoacetic acid, iodoacetamide, and N-ethylmaleimide. However, N-iodoacetyl-3-[
Figure 1:
Effect of IAT on the steroid binding
activity of the glucocorticoid receptor. A, IAT inhibition of
steroid binding activity. Aliquots (100 µl) of L cell cytosol
containing steroid-free or steroid-bound receptors were immunoadsorbed
with BuGR to protein A-Sepharose, the immune pellets were incubated for
1 h at 0 °C with the indicated concentrations of nonradioactive
IAT, and binding of [
The concentration of the immunoadsorbed GR in the labeling mixture
is
Figure 5:
Dissociation of hsp90 from the
immunoadsorbed GR renders the receptor less susceptible to cleavage by
trypsin. Replicate nonimmune (N) and BuGR (I)
immunopellets were incubated for 2 h at 4 °C in HE buffer with
molybdate to preserve the GR
Figure 6:
Dissociation of hsp90 results in increased
[
Figure 4:
[
For quantitative Western blotting experiments
of Fig. 4and Fig. 6, the intact GR or GR fragments on
the immunoblots were identified by developing them first with
horseradish peroxidase-conjugated counterantibody. Reflected UV
photographs were taken of the peroxidase-stained gel to produce the
panels labeled immunoblot in the figures. The GR bands identified by
color were excised and counted for [
These data are consistent with IAT inactivation of glucocorticoid
binding activity through covalent reaction with the receptor. Thus, we
prepared the radioiodinated derivative of IAT and incubated it with the
immunoadsorbed GR
Figure 2:
Tryptic fragments of the
[
Fig. 3demonstrates the
immunoreactivity of each of the fragments generated by trypsin
digestion to confirm their identity. When the immunoblot is probed with
BuGR (condition 2), 44-, 42-, and 15-kDa fragments are seen in
the immunopellet. When the immunoblot is probed with aP1 (condition
3), the 44- and 42-kDa species are seen in the immunopellet as
well as 30-kDa and 16-kDa fragments that have been released into the
supernatant. There is an
Figure 3:
Western blot of the tryptic fragments of
the GR. Replicate nonimmune (N) and BuGR (I)
immunopellets were cleaved by incubating for 1 h at 0 °C with 1
µg/ml trypsin. Both polypeptides remaining with the immunopellet (P) and those released into the supernatant cleavage buffer (S) were resolved by SDS-PAGE and Western blotting with either
BuGR, aP1, or both BuGR and aP1 as indicated under the number for each
condition. Condition 1, uncleaved immunopellet probed with
BuGR; condition 2, trypsin-cleaved receptor probed with BuGR; condition 3, trypsin-cleaved receptor probed with aP1; condition 4, trypsin-cleaved receptor probed with both BuGR
and aP1.
In the
experiments summarized in Fig. 6,
[ We had hoped to
examine specifically the [
It has been proposed that binding
of hsp90 to the HBD causes the polypeptide as a whole to assume a
partially ``unfolded'' conformation that is reversed on hsp90
dissociation (Picard et al., 1988). It seems unlikely to us
that hsp90 causes the DBD to have a different conformation via a
folding mechanism. Spanjaard and Chin(1993) have demonstrated
reconstitution of hormone-mediated activity by expressing as individual
proteins an amino-terminal fragment of the GR containing the trans-activation and DNA binding domains and a fragment
containing the COOH-terminal HBD, with each fragment being fused to
either a c-Jun or c-Fos leucine zipper. As each fragment was translated
and folded independently, this observation argues strongly against a
model in which hsp90 binding to the HBD causes the DBD to assume an
unfolded conformation. At this time, we do not know which of the
cysteines in the 15-kDa fragment are derivatized after hsp90
dissociates. As shown in Fig. 2, the 15-kDa fragment contains 8
cysteines in a vicinal thiol arrangement coordinating two atoms of
zinc, and there are 2 or 3 additional cysteines, depending on the
amino-terminal trypsin cleavage site. Given that half of the cysteines
of the GR are in the 15-kDa fragment, the 2.3-fold increase in labeling
of this fragment occurring with hsp90 dissociation (Fig. 6) may
account for the overall 1.9-fold increase in labeling that is observed
in the intact GR (Fig. 4) despite the 50% reduction in labeling
of the 30-kDa fragment of the HBD which contains 5 cysteines (Fig. 6). However, as with arsenite, the presence of zinc should
prevent cysteine labeling. In this event, increased labeling of the
15-kDa fragment may reflect increased availability of only Cys-438 and
-448 to [ We know that
hsp90 is a component of a multiprotein chaperone system that folds the
HBD of the GR into a high affinity steroid binding conformation (for
review, see Pratt(1993)). It is thought that the steroid receptors have
evolved a tight interaction with hsp90 and that the heterocomplex
probably represents a normal transition state in a general folding
process. The hsp90 can be conceived as trapping the HBD in a partially
unfolded state, with the binding of steroid favoring the naturally
folded conformation of the HBD and disruption of its complex with hsp90
(Hutchison et al., 1992b). In essence, procession from the
folding intermediate (i.e. the GR In this model, the hsp90 binding
region of the HBD assumes a folded conformation upon dissociation of
the hsp. Because [
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8831-8836
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
I
iodotyrosine to
Demonstrate Conformational Differences between the Unbound and
hsp90-bound Glucocorticoid Receptor Hormone Binding Domain (*)
hsp90 complex in
a manner that is prevented by the sulfhydryl reagents cysteine and
dithiothreitol but is not reversed by them. The 
I-labeled
IAT derivative N-iodoacetyl-3-[I]iodotyrosine
([I]IAIT) covalently labels the immunopurified,
hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90
leads to an 
2-fold increase in [I]IAIT
labeling of the full-length, 100-kDa GR. The increase in thiol labeling
is related to the presence of hsp90 because it is blocked by molybdate,
which prevents hsp90 dissociation. Cleavage of the
[I]IAIT-labeled receptor with trypsin yields a
15-kDa labeled fragment containing the DBD and a 30-kDa labeled
fragment containing all of the cysteines in the HBD and the contact
region for hsp90. Dissociation of hsp90 from the GR results in a
2.3-fold increase in [I]IAIT labeling of the
15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment.
These data are consistent with the proposal that dissociation of hsp90
from the GR produces a conformational change in the HBD such that some
of the thiols that are exposed in the GRhsp90 complex become
buried and are no longer accessible to the
[I]IAIT probe. In contrast, binding of the GR
to hsp90 restricts access of cysteines in the DBD to this small
thiol-derivatizing agent, a restriction that is relieved as a result of
unmasking or conformational change accompanying hsp90 dissociation.
)is located in its COOH-terminal one-third in a region
called the hormone binding domain (HBD). The HBD must be properly
folded for there to be a high-affinity steroid binding cavity, and the
GR must be associated with the 90-kDa heat shock protein (hsp90)
component of the protein folding system for there to be an appropriate
steroid binding site (see Pratt(1993) for review). It is known that the
HBD is both necessary and sufficient for binding of the GR to hsp90
(Pratt et al., 1988; Denis et al., 1988a; Cadepond et al., 1991; Scherrer et al., 1993), and
dissociation of hsp90 from the unliganded HBD yields a conformation
with either no steroid binding activity or with very low affinity
binding activity (Bresnick et al., 1989, Nemoto et
al., 1990). The HBD can be returned to the high-affinity steroid
binding conformation by incubating the GR with rabbit reticulocyte
lysate, which contains a protein folding system that restores the
receptor to its heterocomplex state with hsp90 (Scherrer et
al., 1990; Hutchison et al., 1994a, 1994b). Although the
loss of steroid binding activity suggests that a major conformational
change occurs within the HBD upon hsp90 dissociation, an analysis of
peptides produced by limited proteolysis of the untransformed
(hsp90-bound) and the transformed (hsp90-free) GR did not reveal any
differences suggestive of conformational change dependent upon hsp90
(Reichman et al., 1984). To date, no studies have been
published using chemical reagents to probe the conformation of the
hsp90-bound and hsp90-free GR HBD.23-fold
lower affinity than the intact 98-kDa receptor. This fragment of the
HBD (amino acids 537-673 of the rat GR) is bound to hsp90
(Chakraborti and Simons, 1991), and it contains three cysteines (640,
656, and 661 in the rat GR, or 628, 644, and 649 in the mouse GR), of
which any two can form an intramolecular disulfide (Chakraborti et
al., 1992), suggesting that there is a cysteine cluster in this
region of the steroid binding site. A variety of observations indicate
that a short region of the HBD containing this cysteine cluster
directly contacts hsp90 in the untransformed GR heterocomplex (Dalman et al., 1991; Chakraborti and Simons, 1991; Cadepond et
al., 1991).I]iodotyrosine
([I]IAIT) has a much higher reactivity with
protein thiols and has the advantage of the I label
(Gitler et al., 1994). [I]IAIT is a
thiol-specific reagent used to label protein thiols, and it has the
unusual property that its reaction with protein thiols is not affected
by up to 10 mM dithiothreitol (DTT) or 2-mercaptoethanol
(Gitler et al., 1994). Its reaction with vicinal thiols is
blocked by arsenicals, and labeling with
[I]IAIT in the presence and absence of a
compound such as arsenite or phenylarsine oxide is a sensitive method
of distinguishing between reaction with monothiols and vicinally spaced
dithiols. In this work, we have examined the accessibility of thiols to
[I]IAIT derivatization in both the DNA binding
domain (DBD) and the HBD of both the hsp90-bound and hsp90-free GR.
Materials
[6,7-
H]Triamcinolone acetonide (42.8
Ci/mmol) and I-conjugated goat anti-mouse and anti-rabbit
IgGs were obtained from DuPont NEN. Twice crystallized bovine
pancreatic trypsin (Type III), nonimmune mouse IgG, protein
A-Sepharose, and goat anti-mouse IgG-horseradish peroxidase conjugate
were from Sigma. Donkey anti-rabbit IgG-horseradish peroxidase
conjugate was from Pierce. Immobilon-P was from Millipore. The BuGR2
IgG monoclonal antibody against the GR was from Affinity Bioreagents
(Neshanic Station, NJ), and the AC88 monoclonal IgG against hsp90 was
from StressGen (Victoria, BC). The aP1 rabbit antiserum raised against
amino acids 440-795 of the rat GR has been described previously
(Hoeck et al., 1989).Methods
Cell Culture and Fractionation
L929 mouse
fibroblasts (L cells) were grown in monolayer culture in
Dulbecco's modified Eagle's medium supplemented with 10%
iron-supplemented calf serum. The WCL2 line of Chinese hamster ovary
cells overexpressing the mouse GR was established by Hirst et
al.(1990). WCL2 cells were grown in monolayer culture in
Dulbecco's modified Eagle's medium plus 3 µM methotrexate, 40 µg/ml proline, and 10% iron-supplemented calf
serum. All cells were harvested by scraping into Earle's balanced
saline followed by a second wash and centrifugation at 500 
g. The washed cells were suspended in 1.5 volumes of HE buffer
(10 mM HEPES, 1 mM EDTA, pH 7.4) and ruptured by
Dounce homogenization. Homogenates were centrifuged for 1 h at 100,000
g, with the supernatant from this step being the
``cytosol'' from which the GR was immunoadsorbed.Receptor Immunoadsorption
Prior to
immunoadsorption, the BuGR antibody was prebound to protein A-Sepharose
by incubating 40 µl of a 20% slurry of protein A-Sepharose for 1 h
at 4 °C with 1 µg (Fig. 1) or 3 µg (all other
figures) of BuGR and 150 µl of TEG buffer (10 mM TES, 50
mM NaCl, 4 mM EDTA, 10% glycerol, pH 7.6), followed
by centrifugation and washing with TEG. GR was immunoadsorbed from
100-µl aliquots of L cell cytosol (for experiments of Fig. 1) or 200 µl of WCL2 cell cytosol (all other
experiments) by rotation for 2 h at 4 °C with 8 µl of protein
A-Sepharose-prebound BuGR.
H]triamcinolone acetonide
was assayed as described under ``Methods.'' 
,
immunoadsorbed, steroid-free GR; , immunoadsorbed, steroid-bound
GR. B, effect of cysteine or DTT on IAT inhibition.
Immunoadsorbed, steroid-free receptors were suspended in 100 µl of
TEGM buffer for 1 h at 0 °C with 2 mM IAT (black
bar), with IAT plus 30 mML-cysteine or DTT (stippled bars), or with IAT followed after 1 h by 30 mM cysteine or DTT after 1 h (hatched bars). Samples were
then washed and assayed for binding of
[H]triamcinolone acetonide. Binding is expressed
as percent of untreated control, mean ± S.E. for three
experiments.
Treatment with Thiol-reactive Reagents and Steroid
Binding Assay
In the experiment of Fig. 1, the immune
pellets were incubated in 100 µl of TEGM buffer (TEG plus 20 mM sodium molybdate) and various concentrations of IAT for 1 h at 0
°C. Samples were then washed twice in TEGM buffer and incubated 2.5
h at 0 °C in 100 µl of TEGM buffer and 100 nM [H]triamcinolone acetonide, and, after
washing 3 times with 1 µl of TEG buffer, the immune pellets were
assayed for radioactivity. To normalize data between separate
experiments, the counts/min of [H]triamcinolone
acetonide bound in each treated sample was expressed as a percent of
the untreated control, and values from three experiments were averaged. Synthesis of IAT and
Radioiodination
N-Iodoacetyltyrosine (IAT) was
synthesized exactly as described by Gitler et al.(1994) and
radioiodinated to [I]IAIT according to the
procedure of Bolton and Hunter(1973), modified in that acid was added
to allow extraction of IAIT into the benzene-dimethyl formamide.
Because of a chemical decay, the compound must be used within 2 to 3
weeks after radioiodination. The parent IAT is stable for years. To
label the receptor, immunoadsorbed GR was washed once in 1 ml of TEGM
buffer with 1 mM DTT and then incubated with 3 µl (3
µCi) of [I]IAIT in 100 µl of TEGM
buffer containing 1 mM DTT and 3% dimethyl sulfoxide for 1 h
at 4 °C with constant rotation. The final concentration of
[I]IAIT was 1 µM. The
immunopellets were then washed 3 times with 1 ml of TEG buffer plus 0.5 M NaCl and 1% Triton X-100. For samples to be cleaved with
trypsin, the immunopellets were washed once with 1 ml of TEGM buffer. 20 nM, or 400 nM with respect to GR
sulfhydryls. The concentration of total sulfhydryls in the immune
pellet, which includes the sulfhydryls in 3 µg of BuGR antibody, is
in considerable excess of the 1 µM [I]IAIT used for labeling. It should also
be noted that washing the immune pellets with 0.5 M NaCl and
1% Triton X-100 eliminates hsp90 and other receptor-associated
proteins.Trypsin Cleavage of the Immunoadsorbed GR
Protein
A-Sepharose pellets containing immunoadsorbed
[I]IAIT-labeled or unlabeled GR were incubated
in 100 µl of TEGM buffer containing 1 µg/ml tosylphenylalanyl
chloromethyl ketone-treated trypsin. For the untransformed, hsp90-bound
GR the incubation in the trypsin was for 1 h at 0 °C and for the
transformed, hsp90-free GR the incubation was for 1 h at 20 °C. The
supernatants containing the GR fragments released from the immune
pellets were treated with SDS sample buffer, and proteins were resolved
by SDS-PAGE. The trypsin-treated immunopellets were washed 3 times with
1 ml of TEG buffer containing 0.5 M NaCl and 1% Triton X-100
prior to resolving the BuGR antibody-bound fragments by SDS-PAGE.Gel Electrophoresis and Western Blotting
Immune
pellets and supernatants from trypsin-digested immunopellets were
boiled in SDS sample buffer with 10% 
-mercaptoethanol, and
proteins were resolved in a 12% SDS-polyacrylamide gel as described in
Hutchison et al. (1992a). To optimize the transfer to an
Immobilon membrane of 15-kDa, 16-kDa, and 30-kDa tryptic fragments of
the GR in the experiments of Fig. 5and Fig. 6, proteins
were transferred for 16 h at 0.1 A. After transfer to an Immobilon-P
membrane, GR and receptor fragments were probed both with 2 µg/ml
BuGR2 monoclonal IgG and with 0.05% aP1 rabbit anti-GR serum. hsp90 was
detected by probing with 1 µg/ml AC88. The immunoblots were then
incubated a second time with the appropriate I-labeled or
horseradish peroxidase-conjugated counter antibody to visualize the
immunoreactive bands.
hsp90 complex or incubated for 2 h in
buffer with 0.5 M NaCl to dissociate hsp90. After washing, the
immunopellets were incubated for 1 h with 1 µg/ml trypsin at 0
°C or 20 °C. Both the fragments remaining with the immunopellet (P) and those released into the supernatant cleavage buffer (S) were resolved by SDS-PAGE and Western blotting with both
BuGR and aP1. The immunoblot was then incubated a second time with the
appropriate I-labeled counterantibodies and visualized by
autoradiography. Condition 1, GRhsp90 complex cleaved
with trypsin at 0 °C; condition 2, hsp90-free GR cleaved
with trypsin at 0 °C; condition 3, hsp90-free GR cleaved
with trypsin at 20 °C.
I]IAIT labeling of the 15-kDa fragment
containing the DBD and decreased labeling of the 30-kDa fragment of the
HBD. Replicate nonimmune (N) and BuGR (I)
immunopellets were incubated for 2 h at 4 °C in HE buffer with
molybdate to preserve the GRhsp90 complex (condition 1)
or in buffer with 0.5 M NaCl to dissociate hsp90 (condition 2). After washing, the immunopellets were incubated
for 1 h at 0 °C with [I]IAIT. The
immunopellets were then washed with TEGM and incubated for 1 h with 1
µg/ml trypsin at 0 °C or 20 °C. Both polypeptides remaining
with the immunopellet (P) and those released into the
supernatant cleavage buffer (S) were resolved by SDS-PAGE and
Western blotting. A shows the immunoblot and
[I]IAIT-labeled fragments from one experiment. B shows the [I]IAIT radioactivity from
several experiments corrected for the relative amount of fragment
protein and expressed as a percent of the condition 1 control
as described in the legend to Fig. 4. The data represent the
mean ± S.E. from six experiments for the 15-kDa fragment and
seven experiments for the 30-kDa fragment. For both panels, condition 1 is the GRhsp90 complex cleaved with trypsin
at 0 °C, and condition 2 is the hsp90-free GR cleaved at
20 °C. For the 15-kDa fragment, condition 1 is different
from condition 2 at p < 0.01 and for the 30-kDa
fragment p < 0.001.
I]IAIT labeling
of the intact GR in the presence and absence of hsp90. Replicate
nonimmune (N) and BuGR (I) immunopellets were
incubated for 2 h at 4 °C in 100 µl of HE buffer with 20 mM sodium molybdate (condition 1), in buffer with 0.5 M NaCl without molybdate (condition 2), or in buffer with
0.5 M NaCl and 20 mM molybdate (condition
3). Samples were assayed for GR-associated hsp90 by SDS-PAGE and
immunoblotting. Replicate samples were incubated for 1 h at 0 °C
with [I]IAIT, and proteins were resolved by
SDS-PAGE and immunoblotting. The row labeled Immunoblot is a
photograph of the Western blotted receptor developed with horseradish
peroxidase-conjugated counter antibody, and the row below is an
autoradiogram of the immunoblot reflecting the radioactivity from
[I]IAIT. To prepare the graph, the GR bands
identified by color, on the Western blot, were excised and counted for
[I]IAIT radioactivity. The cut pieces were then
counterblotted with I-conjugated goat anti-mouse IgG,
washed, and counted again to determine the relative amount of GR after
subtracting the radioactivity derived from
[I]IAIT (less the 10%) from the total. After
correction for the relative amount of GR protein, the values were
expressed as a percent of the condition 1 control. The data
represent the mean ± S.E. from six experiments. Condition 2 is different from conditions 1 and 3 at p = 0.011.
I]IAIT
radioactivity, as was an equivalent region from the nonimmune sample.
The cut pieces of immunoblot were then counterblotted with I-conjugated goat anti-mouse or anti-rabbit IgG, washed,
and counted again in order to determine the relative amount of GR or
fragment in each sample after subtracting the radioactivity derived
from [I]IAIT (less than 10%) from the total.
After correction for the relative amount of GR (or GR fragment)
protein, the values were expressed as a percent of condition 1 control.
Effect of IAT on Steroid Binding Activity of the
GR
In a previous report (Stancato et al., 1993), we
studied the effects of two thiol-reactive reagents, MMTS and arsenite,
on the steroid binding activity of the GR in L cell cytosol. These
reagents inactivate steroid binding activity of the unliganded GR at
concentrations much lower than those required for release of steroid
from the prebound receptor. Fig. 1A shows the IAT
inhibition of the steroid binding activity of the immunoadsorbed
unliganded GRhsp90 complex (open circles), which is
affected by concentrations 100-fold lower than the steroid-bound
receptor complex (closed circles). Inactivation of the
immunopurified receptor is blocked if 30 mM cysteine or DTT is
added prior to the IAT (Fig. 1B, stippled
bars); however, in contrast to the reversible inhibitors MMTS or
arsenite, inactivation by IAT is not reversed by subsequent addition of
cysteine or DTT (Fig. 1B, hatched bars). hsp90 complex. The GR was resolved by gel
electrophoresis, and autoradiography showed
[I]IAIT labeling of the receptor.
[I]IAIT labeling was not affected by
preincubation with 1 mM DTT, but was markedly reduced by 30
mM DTT or 1 mM arsenite (data not shown). The
inhibition of receptor labeling by 1 mM arsenite is consistent
with [I]IAIT reaction with cysteine SH groups
in the GR (Gitler et al., 1994).Tryptic Cleavage of the
[
In the experiment
shown in Fig. 2, the immunoadsorbed GRI]IAIT-labeled GRhsp90 complex was
labeled with [I]IAIT, washed, and cleaved with
trypsin. The [I]IAIT-labeled polypeptides were
resolved by SDS-PAGE and autoradiography. The major fragments produced
by trypsin cleavage are shown in the diagram at the top of the figure. Trypsin cleavage produces 44-kDa (30 kDa + 15
kDa) and 15-kDa fragments that contain the BuGR epitope and remain in
the immunopellet (lane 4), while the 30-kDa and 16-kDa
fragments are released into the supernatant cleavage buffer (lane
6). All four major fragments are labeled with
[I]IAIT. It can also be seen that this
incubation with trypsin does not cleave the immunoglobulin, which is
also labeled with [I]IAIT. This differential
trypsin sensitivity is very useful.
I]IAIT-labeled GR. Aliquots of WCL2 cell
cytosol were immunoadsorbed with nonimmune IgG or with BuGR.
Immunopellets were labeled with [I]IAIT and
washed. One set of immunopellets was incubated at 0 °C for 1 h with
1 µg/ml trypsin as described under ``Methods.'' Both the
polypeptides remaining with the immunopellet and those released into
the cleavage buffer were resolved by SDS-PAGE and autoradiography. Lane 1, BuGR immunopellet without trypsin; lane 2,
nonimmune pellet without trypsin; lanes 3 and 4,
nonimmune and BuGR immunopellets, respectively, digested with trypsin; lanes 5 and 6, supernatants from samples 3 and 4,
respectively. The 100-kDa intact GR and the tryptic fragments are
diagrammed above the autoradiogram. Cysteines are assigned according to
the primary sequence of the mouse GR by Danielsen et al. (1986) and tryptic fragments are according to Simons et al. (1989). The 30-kDa and 15-kDa fragments are derived from cleavage
of the 44-kDa fragment shown in the autoradiogram. The hormone binding
domain is stippled, the DNA binding domain is hatched, and the black band designates the BuGR
epitope. The conserved cysteines involved in the tetrahedral
coordination of zinc in the DBD are indicated. The thiol cluster in the
HBD is comprised of Cys-628, -644, and -649, hc =
antibody heavy chain.
30-kDa fragment in the immunopellet
migrating a little bit slower than the light chain of the BuGR antibody
that reacts with both the BuGR and aP1 antibodies. This fragment is
recovered only occasionally, and it likely represents an uncleaved
combination of the 15-kDa and 16-kDa segments. The BuGR monoclonal
antibody does not recognize the 30-kDa and 16-kDa fragments released
into the supernatant, but the aP1 antiserum does react very faintly
with a 15-16-kDa species in the immunopellet (condition
3), which likely represents a trace of 16-kDa fragment remaining
in the immunopellet.
[
In the experiments of Fig. 4, we examined
labeling by [I]IAIT Labeling of the Intact
hsp90-free GRI]IAIT of three states of the GR.
In condition 1, the immunoadsorbed GRhsp90 complex was
labeled. In condition 2, the GR was stripped of hsp90 (cf.
lanes 2 and 4) by incubating the immunopellet with 0.5 M NaCl. In condition 3, the immunoadsorbed GR was
incubated with salt but in the presence of molybdate to inhibit hsp90
dissociation. The relative amount of [I]IAIT
labeling of the GR under each condition is expressed in the bar
graph. It can be seen that the intact 100-kDa GR that is
dissociated from hsp90 (condition 2) is labeled by
[I]IAIT 1.9-fold more with respect to the GR
that is associated with hsp90 (condition 1). The increase is
related to dissociation of hsp90, because it is blocked by molybdate,
which prevents hsp90 dissociation (condition 3). The same
increase in labeling is seen if hsp90 is dissociated by heating the
immunopellet in buffer at 25 °C and after hsp90 dissociation from
the dexamethasone-bound receptor (data not shown).Tryptic Fragments Derived from hsp90-bound and hsp90-free
[
The hsp90-free GR is
less sensitive to trypsin digestion than the hsp90-bound GR. As shown
in Fig. 5(condition 2), when the salt-stripped,
hsp90-free GR is incubated for 1 h at 0 °C with 1 µg/ml
trypsin, less 15-kDa fragment and 30-kDa fragment is obtained than
after an identical digestion of the hsp90-bound GR (cf. with condition 1) and no 16-kDa fragment is recovered. The best way
we have found to get substantial amounts of the 15-kDa and 30-kDa
fragments is to incubate the immunoadsorbed salt-stripped GR with the
same concentration of trypsin for 1 h at 20 °C (condition
3). As reported in 1984 by Reichman et al. for the
transformed and untransformed GR, we find that the same tryptic
fragments are derived with the hsp90-free as the hsp90-bound GR.
However, the hsp90-free GR requires more intensive cleavage conditions
to generate the fragments. This is true regardless of whether the
immunoadsorbed GR was stripped of hsp90 by salt (as in Fig. 5)
or by incubation in buffer at 25 °C (data not shown).I]IAIT-labeled GRI]IAIT-labeled GRhsp90 complex (condition 1) or hsp90-free GR (condition 2) was
cleaved with trypsin and the radioactivity in the 15-kDa and 30-kDa
fragments was assayed. Fig. 6A shows an immunoblot and
an autoradiogram of [I]IAIT radioactivity from
the same immunoblot. Fig. 6B presents a bar graph of
the relative amount of [I]IAIT labeling of the
fragments under each condition after averaging data from several
experiments. We find that the 15-kDa fragment containing the DNA
binding domain from the hsp90-free GR is labeled 2.3-fold more by
[I]IAIT than the 15-kDa fragment from the
hsp90-bound GR. In contrast, the 30-kDa fragment containing the HBD of
the hsp90-free GR is labeled only half as much as the 30-kDa fragment
from the hsp90-bound GR. It should be emphasized that 1 mM DTT
was present during labeling with [I]IAIT; thus,
differential oxidation is not responsible for differences in
incorporation of label. These data are consistent with the proposal
that dissociation of the hsp90 from the GR produces a conformational
change that allows increased access of the IAIT probe to thiols of the
DBD and decreased access to thiols in the HBD.I]IAIT labeling of
the three cysteines in the thiol cluster of the 16-kDa fragment. This
proved not to be possible for two reasons. First, the recovery of
sufficient 16-kDa fragment from the hsp90-bound GR to visualize
[I]IAIT labeling as in Fig. 2is erratic (cf. Fig. 2and Fig. 6A). Secondly, as
illustrated in Fig. 5, even when we recover a significant amount
of 16-kDa fragment from the hsp90-bound GR, only trace amounts are
recovered from the hsp90-free GR. We were able, however, to
consistently recover ample amounts of the 30-kDa fragment from both the
hsp90-bound and hsp90-free GR to accurately determine thiol
accessibility to [I]IAIT. As shown in Fig. 2, the 30-kDa fragment of the mouse GR contains two
cysteine residues at positions 671 and 742, in addition to the three
cysteines in the thiol cluster. Thus, the 50% decrease in
[I]IAIT labeling of the 30-kDa fragment of the
GR that we see after hsp90 dissociation could involve any of these
cysteines, and we can conclude only that thiols lying in the HBD become
protected from IAIT attack as a result of conformational change in the
receptor upon hsp90 dissociation.Models of Folding Changes Occurring upon hsp90
Dissociation
It has been known for some time that dissociation
of hsp90 from the GR is accompanied by acquisition of DNA binding
activity (Sanchez et al., 1987; Denis et al., 1988b),
with the rates of the two processes being identical (Meshinchi et
al., 1990). It is not known whether hsp90 abrogates DNA binding
activity solely by physically masking the DBD or whether the DBD of the
hsp90-bound GR is in a different conformation from the hsp90-free GR.
The [I]IAIT probe used here is small, and it
can access and derivatize cysteine thiols in the DBD even when the GR
is bound to hsp90. Yet, dissociation of hsp90 allows a 2.3-fold
increase in labeling of the 15-kDa, DBD-containing GR fragment (Fig. 6). In the event hsp90 inhibits the DNA binding activity
of the GR solely by physically blocking access of DNA to the DBD, we
infer from the fact that hsp90 also impedes the access of the very
small IAIT reagent to DBD thiols that there must be rather close
association of hsp90 with the DBD.I]IAIT. Because
[I]IAIT labeling of the hsp90-bound receptor is
reduced about 70% by 1 mM arsenite (data not shown), it is
likely that cysteine SH groups are the predominant labeled moiety in
the intact 100-kDa receptor. However, it is at least theoretically
possible that [I]IAIT could react with
non-thiol groups in the DBD of the hsp90-bound GR.hsp90 complex) has been
brought under hormonal control.I]IAIT is small and
specifically derivatizes thiol moieties, we can show that dissociation
of hsp90 is accompanied by decreased accessibility of thiols in the HBD
to derivatization by the reagent (Fig. 6). This is the first
demonstration that specific sites that were previously accessible in
the HBD become inaccessible upon receptor transformation. We have
worked here with the steroid-free receptor because we wanted to
eliminate the possibility that the presence of steroid in the hormone
binding site could sterically block access of the reagent to thiols
lying in the binding pocket. The fact that the change in the HBD is
from accessibility to reagent in the hsp90-bound state to less
accessibility when hsp90 is not bound is consistent with an
internalization of thiol moieties occurring as the unliganded receptor
HBD assumes a more folded conformation. In this folded conformation,
there is no steroid binding activity until the receptor is reassociated
with the hsp by the hsp90/hsp70-based chaperone system.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
