Download The Glucocorticoid Antagonist 17a-Methyl testosterone Binds to the

The Glucocorticoid Antagonist
17a-Methyl testosterone Binds to the
10 S Glucocorticoid Receptor and
Blocks Agonist-Mediated
Dissociation of the 10 S Oligomer
to the 4 S Deoxyribonucleic
Acid-Binding Subunit
Bruce M. Raaka, Molly Finnerty, and Herbert H. Samuels
Division of Molecular Endocrinology
Departments of Medicine and Pharmacology
New York University Medical Center
New York, New York 10016
agonist for binding to the 10 S cytosolic receptor but
does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor. (Molecular Endocrinology 3: 332-341, 1989)
The glucocorticoid antagonist 17a-methyltestosterone inhibits binding of the agonist [3H]triamcinolone
acetonide to the glucocorticoid receptor in cytosol
prepared from rat pituitary tumor GH, cells. Competitive binding studies indicate that the dissociation
constant for 17a-methyltestosterone is about 1 MM.
After incubation of intact GHt cells with 10 nM [3H]
triamcinolone acetonide at 37 C and subsequent cell
fractionation at 4 C , three glucocorticoid receptor
forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent
incubation with 17«-methyltestosterone reduces the
amount of [3H]triamcinolone acetonide bound to
each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]
triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with
cytosolic 10 S receptor. Immunochemical analysis
using monoclonal antibody BuGR2 indicates that
17a-methy I testosterone does not cause substantial
accumulation of glucocorticoid receptors in GHn cell
nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with
agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17a-methyltestosterone does not reduce the
total amount of cellular glucocorticoid receptor and
does not reduce receptor half-life. These results are
consistent with a model for glucocorticoid receptor
transformation in which binding of agonist promotes
the dissociation of an oligomeric 10 S cytosolic
receptor protein to its DNA-binding 4 S subunit. The
antagonist 17a-methyltestosterone competes with
INTRODUCTION
When cells or tissues are deprived of steroid hormones,
steroid receptor proteins are generally found in the
soluble "cytosolic" fraction of broken cell preparations.
After exposure to steroid hormones, 60-90% of the
occupied receptor is found in the particulate "nuclear"
fraction. This apparent redistribution of receptor within
the cell suggests that binding of hormone transforms
receptor to a form capable of high affinity interactions
with chromatin components in the cell nucleus, where
the hormone-receptor complex acts to regulate the
expression of specific genes (1, 2). Results of studies
from this laboratory using rat pituitary tumor GH, cells
led to the proposal of a model for glucocorticoid receptor transformation (3, 4). According to this model, hormone binds initially to an oligomeric 10 S receptor
protein and shifts the apparent equilibrium between the
oligomer and its hormone- and DNA-binding 4 S subunit
in the direction of the subunit. The hormone-occupied
subunit then appears to interact reversibly with chromatin components in the cell nucleus. Evidence from a
variety of experimental systems supports the conclusion that steriod receptor transformation involves hormone-mediated dissociation of a receptor oligomer (59).
An early study by Samuels and Tomkins (10) described the effect of the antagonist 17a-
0888-8809/89/0332-0341 $02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
332
333
The Glucocorticoid Antagonist 17«-Methyltestosterone
methyltestosterone1 on the induction of tyrosine aminotransferase by glucocorticoid agonists in HTC cells.
These authors interpreted their results in the context
of a theoretically based allosteric model in which two
interconvertible forms of glucocorticoid receptor exist
with different abilities to induce expression of the enzyme. In the context of a model in which receptor
transformation requires agonist-mediated oligomer dissociation, antagonist might act by competing with agonists for binding to the oligomeric receptor without
promoting subunit dissociation. Recent direct ligandbinding studies with the high affinity glucocorticoid antagonist [3H]RU38486 document that this compound
binds to the oligomeric receptor but does not promote
dissociation (11, 12). In this report, we present experimental evidence from ligand-exchange assays showing
that 17a-methyltestosterone, an antagonist with low
affinity for the glucocorticoid receptor, also binds to the
10 S cytosolic receptor but, unlike agonists, does not
appear to convert receptor to the DNA-binding form.
Furthermore, we show for the first time that a glucocorticoid antagonist is able to competitively block oligomer dissociation mediated by an agonist. These results provide additional support for the conclusion that
agonist-mediated oligomer dissociation plays a central
role in glucocorticoid receptor transformation (3-9).
RESULTS
Binding of 17«-Methyltestosterone to the
Glucocorticoid Receptor
17a-Methyltestosterone is an antagonist of the glucocorticoid-mediated induction of tyrosine aminotransferase in HTC cells (10) and of the stimulation of GH
synthesis and mRNA accumulation in rat pituitary tumor
GHT cells (13). In both cases, the antagonist has no
detectable agonist activity and at sufficiently high concentrations is capable of fully blocking the action of
agonists. The concentration dependence of antagonist
activity along with other evidence suggested that both
agonists and antagonists interact with a specific glucocorticoid receptor system (10). A competitive binding
study confirms that 17a-methyltestosterone inhibits the
binding of the synthetic glucocorticoid agonist [3H]
triamcinolone acetonide to the glucocorticoid receptor
in cytosol prepared from GHT cells (Fig. 1). Under the
conditions used for this experiment, virtually all of the
cytosolic receptor is known to sediment at 10 S (3, 4)
and the protamine sulfate assay identifies only the 10
S receptor form (14). Thus, both steroids appear to
compete for the same binding site on the 10 S receptor.
The inhibition curve for 17a-methyltestosterone is
shifted more than three orders of magnitude toward
higher concentrations than the corresponding curve for
1
The trivial names used are: 17a-methyltestosterone, 17/3hydroxy-17«-methyl-4-androsten-3-one; triamcinolone acetonide, 9a-fluoro-11 j8,16a,17a,21 -tetrahydroxypregna-1,4diene-3,20-dione-16,17-acetonide.
17Q - M E T H Y L TESTOSTERONE
O"1O"1O 10-9 10-8 10-7 10"6 10~5 10"4
STEROID CONCENTRATION (M)
Fig. 1. Inhibition of [3H]Triamcinolone Acetonide Binding to the
Glucocorticoid Receptor by 17«-Methyltestosterone
Aliquots of cytosol prepared from GHi cells were adjusted
to contain 3 nM [3H]triamcinolone acetonide and the indicated
concentration of either unlabeled triamcinolone acetonide or
17«-methyltestosterone and incubated at 4 C for 18 h. Binding
of [3H]triamcinolone acetonide to the cytosolic glucocorticoid
receptor was measured by protamine sulfate precipitation (3).
The data are not corrected for nonspecific binding, which was
less than 3% of total specific binding.
triamcinolone acetonide. Since the dissociation constant (Kd) for triamcinolone acetonide binding to the
glucocorticoid receptor from GHi cells is about 1 nM
(3), this result suggests a Kd of about 1 HM for 17amethyltestosterone, in reasonable agreement with an
earlier estimate of 0.4 HM for binding to the glucocorticoid receptor from HTC cells (15).
Effects of 17a-Methyltestosterone on the
Intracellular Distribution of Glucocorticoid Receptor
Forms
Direct assessment of [3H]17a-methyltestosterone binding to the glucocorticoid receptor is not feasible because of the low affinity of receptor for this steroid.
Consequently a ligand-exchange assay in intact cells
was used to study effects of this antagonist on the
intracellular distribution of receptor forms. GHT cells
were exposed to unlabeled 17a-methyltestosterone at
37 C either alone or in combination with [3H]triamcinolone acetonide. The cells were then chilled to 0 C and
the culture medium was replaced several times over a
1- to 2-h period with medium without steroids. Finally,
the cells were incubated at 0 C for 4-5 h with 10-15
nM [3H]triamcinolone acetonide to exchange and reoccupy receptor binding sites with the radioactive ligand.
This 4- to 5-h incubation at 0 C is sufficient to achieve
virtually complete occupany of glucocorticoid receptor
in GHT cells not previously exposed to steroids (3). The
entire exchange assay was performed at 0 C which has
been shown to minimize interconversion of 10 S and 4
S cytosolic receptor forms and redistribution of receptor
forms between cytosolic and nuclear compartments (3).
GHT cells were incubated at 37 C for 1 h with 10 nM
[3H]triamcinolone acetonide or 20 HM 17a-methyltes-
MOL ENDO-1989
334
Vol 3 No. 2
amount of 4 S receptor was decreased in comparison
with cells receiving agonist alone. In cells that received
either no steroid or 17a-methyltestosterone alone at
37 C, the 10 S receptor predominated.
The amounts of 10 S and 4 S cytosolic receptors
were quantified from the gradient results shown in Fig.
2 and the amount of nuclear receptor was determined
in washed nuclei from each cell culture (Table 1). In
addition, Table 1 documents the efficiency of the exchange assay. Before the ligand exchange procedure
in cells treated with both 10 nM [3H]triamcinolone acetonide and 20 HM 17a-methyltestosterone, occupancy
of each receptor form by the labeled steroid was greatly
reduced and total occupancy was only 29% of that
found in control cells treated with the agonist alone.
This confirms that 17a-methyltestosterone inhibits
binding of the glucocorticoid agonist in intact cells. After
ligand exchange, total occupancy by [3H]triamcinolone
acetonide rose to 66% of control, indicating that the
exchange procedure is only partially effective. If occupied with the antagonist, it is not known whether ligand
exchange of 4 S or nuclear receptors is possible under
the conditions used for these experiments. However,
the amount of cytosolic 10 S receptor identified after
exchange increased more than 6-fold from 11 to 68
fmol/100 ng DNA, while cytosolic 4 S and nuclear
receptor forms increased only 1.6- and 1.3-fold, respectively. This suggests that most of the exchangeable bound 17a-methyltestosterone occupies 10 S cytosolic receptor.
This conclusion is supported by the results shown in
Table 1 from cells incubated with 20 ^M 17«-methyltestosterone alone at 37 C, where 113 fmol/100 ng DNA
or 87% of the receptor occupied after exchange was in
the cytosolic 10 S form. Similarly, 86% of total occupied
receptor was in the cytosolic 10 S form in control cells
that received no steroid at 37 C. Furthermore, the 113
fmol/100 HQ DNA of 10 S receptor in the cells treated
with 17a-methyltestosterone at 37 C is (113/185)100
= 6 1 % of the total glucocorticoid receptor identified in
tosterone or with both steroids. Based on dissociation
constants of 1 nM and 1 /XM respectively, these concentrations of steroid will each result in greater than 90%
occupancy of receptor binding sites at equilibrium. After
ligand exchange in intact cells, cytosol was prepared
and analyzed in sucrose gradients (Fig. 2). In cells
treated with [3H]triamcinolone acetonide alone, 10 S
and 4 S cytosolic receptors were found in roughly equal
abundance. In cells treated with both steroids, the
amount of 10 S receptor was increased while the
9
16 -
TREATMENT AT }?'
-
O--O 10nM[ 3 H]TA
:
0 - - 0 NO STEROID
• — • 10 nV [ 3 H ] TA + 20/iM MT
-O--
» - • 2 0 / i M MT
-
4 -
n
A
/
\
10
' " • •
15
20
25
10
15
20
25
FRACTION NUMBER
Fig. 2. Effect of 17a-Methylestosterone (MT) on the Sedimentation of Cytosolic Glucocorticoid Receptors
Monolayer cultures of GHT cells were incubated at 37 C for
1 h in steroid-depleted medium supplemented as indicated in
the figure. The cells were then chilled to 0 C by floating the
culture flasks in an ice-water bath and the medium was replaced two times with ice-cold serum-free medium without
steroids. After each replacement of medium, the cells were
incubated for 5 min at 0 C. Finally, the medium was replaced
with ice-cold steroid-depleted medium containing 10 nM [3H]
triamcinolone acetonide (TA) and incubation at 0 C was continued for 5 h. Cytosolic receptor forms were then analyzed in
low salt sucrose gradients as described in Material and Methods. The direction of sedimentation is from right to left in the
figure and only the bottom 25 of a total 39-40 fractions are
shown.
Table 1. Effects of 20 MM 17a-Methyltestosterone (MeTest) on the Intracellular Distribution of Glucocorticoid Receptors and
Documentation of the Ligand Exchange Assay in Intact Cells
Bound [3H]Triamcinolone Acetonide
(fmol/100 M g DNA)
Treatment
No steroid
20 HM MeTest
10nM[ 3 H]TA
10 nM [3H]TA + 20 M M MeTest
10 nM [3H]TA + 20 /XM MeTest
Total
(% of control)
Exchange
10S
Cytosolic
4S
Cytosolic
Nuclear
Total
160(86)
113(87)
24(12)
68 (53)
11 (20)
11 (6)
7 (5)
27(14)
11 (8)
7(12)
14 (8)
10 (8)
145(74)
50 (39)
38 (68)
185
130
196
129
56
100
70a
100
66'
29'
The experimental details and the ligand exchange procedures are described in the legend to Fig. 2. Cells that did not undergo the
exchange procedure were incubated at 0 C for the duration of that procedure in the same medium used for the 1 -h incubation at
37 C. Cytosolic 10 S and 4 S receptor forms were quantified by summing radioactivity in appropriate fractions of sucrose gradients.
Specific binding of [3H]triamcinolone acetonide (TA) to washed nuclei was determined by subtracting binding observed in cells
incubated with a 500-fold excess of the unlabeled steroid. Values in parentheses indicate 10 S or 4 S cytosolic receptor or nuclear
receptor as a percentage of total receptor identified with [3H]triamcinolone acetonide for each experimental condition.
a
Relative to "No steroid" control.
6
Relative to "10 nM [3H]TA" control.
335
The Glucocorticoid Antagonist 17a-Methyltestosterone
the control cells. In contrast, only (24/196)100 = 12%
of total glucocorticoid receptor found in cells treated
with 10 nM [3H]triamcinolone acetonide at 37 C was in
the 10 S form. This suggests that 20 HM 17a-methyltestosterone binds to the 10 S receptor but does not
effectively convert receptor to the cytosolic 4 S and
10
Me Test
10
15
20
nuclear forms. In addition to having more 10 S and less
4 S receptor, cells treated with both 17a-methyltestosterone and [3H]triamcinolone acetonide at 37 C have
more total cytosolic receptor after ligand exchange than
cells treated with the agonist alone. This suggests that
17a-methyltestosterone competes with [3H]triamcinolone acetonide for binding to the 10 S receptor form
and as a consequence inhibits the dissociation of 10 S
receptor and subsequent nuclear accumulation of the
4 S DNA-binding subunit which normally occurs in
response to agonist binding.
The competitive nature of this inhibition of agonistmediated 10 S cytosolic receptor dissociation by 17amethyltestosterone was confirmed by a dose-response
experiment. GHT cells were incubated at 37 C for 1 h
with 3 nM [3H]triamcinolone acetonide and various concentrations of the antagonist. After ligand exchange at
0 C in intact cells, cytosolic receptor forms were analyzed in sucrose gradients (Fig. 3). Cytosolic 10 S and
4 S receptor forms were quantified from the sucrose
gradients and nuclear receptor was measured in
washed nuclei (Table 2). With increasing concentrations
of 17a-methyltestosterone the amount of 10 S cytosolic
receptor steadily increases while cytosolic 4 S and
nuclear receptor forms decrease.
Effect of 17a-Methyltestosterone on Cytosolic and
Nuclear Glucocorticoid Receptors Determined by
Immunoblotting
25
Fraction Number
Fig. 3. Sedimentation of Cytosolic Glucocorticoid Receptors
from Cells Incubated with 3 PM [3H]Triamcinolone Acetonide
and Various Concentrations of 17a-Methyltestosterone
Monolayer cultures of GH: cells were incubated for 1 h at
37 C in steroid-depleted medium containing 3 nM [3H]triamcinolone acetonide and the indicated concentrations of 17«methyltestosterone (MeTest). After this incubation the flasks
were placed in an ice-water bath and the medium was replaced
two times with ice-cold serum-free medium without steroids.
After each replacement of medium, the cells were incubated
for 45 min at OC. Finally, the medium was replaced with
steroid-depleted medium containing 15 nM [3H]triamcinolone
acetonide and incubation at 0 C was continued for 4 h. Cytosolic receptor forms were then analyzed in sucrose gradients
as described in Fig. 2.
A reasonable explanation for the action of 17«-methyltestosterone as a glucocorticoid antagonist is that this
compound binds to the glucocorticoid receptor but
does not cause receptor to accumulate in the nucleus.
However, two technical limitations of the ligand exchange assay in intact cells make it difficult to conclude
with certainty that the antagonist does not convert a
fraction of receptor to the 4 S cytosolic form or to the
nuclear form. First, only about 70% of total receptor is
generally identified after ligand exchange (Table 1). This
failure to identify as much as 30% of the receptor
population probably results wholly or partially from residual 17a-methyltestosterone competing with [3H]
triamcinolone acetonide for receptor binding. In addi-
Table 2. Dose dependence of the Effects of 17«-Methyltestosterone (MeTest) on the Intracellular Distribution of Glucocorticoid
Receptors
MeTest
(MM)
0
0.33
1.0
3.3
10
20
Bound [3H]Triamcinolone Acetonide
(fmol/00 M g DNA)
10S
Cytosolic
4S
Cytosolic
Nuclear
Total
24(16)
28 (20)
39 (26)
58(41)
68(51)
70 (54)
32 (22)
28 (20)
30 (20)
24(17)
21 (16)
20(15)
92 (62)
86 (60)
79 (54)
59 (42)
43 (33)
41 (31)
148
142
148
141
132
131
The experiment is described in the legend to Fig. 3. Cytosolic and nuclear receptor forms were quantified as described in the
legend to Table 1. Values in parentheses indicate 10 S or 4 S cytosolic receptor or nuclear receptor as a percentage of total
receptor identified with [3H]triamcinolone acetonide for each concentration of 17a-methyltestosterone.
MOL ENDO-1989
336
tion, it is possible that cytosolic 4 S and nuclear receptor
forms, if occupied with antagonist, either do not
undergo ligand exchange or do so with considerably
lower efficiency than cytosolic 10 S receptor. Second,
the 4- to 6-h incubation required for intact cell ligand
exchange might allow redistribution of receptor forms
between nuclear and cytosolic compartments. To circumvent these limitations, an immunoblotting method
was used t o determine if 17«-methyltestosterone converts a detectable amount of receptor to the nuclear
form.
GHi cells were incubated at 37 C for 1 h with 10 nM
triamcinolone acetonide or 20 MM 17«-methyltestosterone or with both steroids. Control cells received no
steroid. Cells were then lysed at 0 C and cytosolic and
nuclear fractions were prepared immediately, thus minimizing the time available for possible redistribution of
receptor forms between cellular compartments after
cell lysis. The nuclear fraction was then extracted with
salt using a procedure which solubilizes about 5 5 % of
nuclear glucocorticoid receptors (14). The cytosol and
nuclear extract were then incubated with monoclonal
antibody BuGR2 followed by protein A-Sepharose to
immunoadsorb glucocorticoid receptors. The adsorbed
proteins were separated by electrophoresis and transferred to nitrocellulose.
Glucocorticoid receptor bound to the nitrocellulose
was detected by sequential incubations with monoclonal antibody BuGR2, rabbit anti-mouse immunoglobulin
G (IgG), and 125 l-protein A followed by autoradiography
(Fig. 4). The major glucocorticoid receptor band is positioned just below the 97 kilodalton (kDa) standard.
The t w o minor bands between the major receptor band
and the 66 kDa standard may correspond to receptor
forms produced from alternate translation initiation sites
within the receptor mRNA (16). The diffuse band seen
at the bottom of each gel lane is monoclonal antibody
BuGR2 from the immunoadsorption step. For control
and 17a-methyltestosterone-treated cells, receptor
was present in approximately equal abundance in the
cytosolic fractions but was not detected in the nuclear
fractions. By comparison, receptor in cells treated with
triamcinolone acetonide was less abundant in the cytosolic fraction and was easily detected in the nuclear
fraction. Cells treated with both steroids had increased
cytosolic and decreased nuclear receptors compared
with cells treated with agonist alone. These results
indicate that a concentration of 17«-methyltestosterone
that occupies more than 9 0 % of receptor binding sites
causes no detectable accumulation of glucocorticoid
receptors in the cell nucleus. Furthermore, when present together with agonist, the antagonist reduces the
nuclear accumulation of receptor observed with agonist
alone.
Dense Amino Acid Labeling Studies
A recent study from this laboratory used density labeling to document that [ 3 H]triamcinolone acetonide reduces the amount of glucocorticoid receptor in G H T
Vol 3 No. 2
CYTOSOLIC
NUCLEAR
MT
MT
MT CON + TA MT CON + TA
TA
TA
97K —
66K —
Fig. 4. Immunoblotting of Cytosolic and Nuclear Glucocorticoid
Receptors
GHi cells were incubated at 37 C for 1 h in steroid-depleted
medium supplemented with 20 nm 17a-methylstestosterone
(MT), 10 nM triamcinolone acetonide (TA), or with both steroids
(MT + TA). Control cells (CON) received no steroid. Glucocorticoid receptors in cytosolic and nuclear fractions were immunoadsorbed using mouse monoclonal antibody BuGR2. After
gel electrophoresis of the adsorbed protein and transfer to
nitrocellulose, glucocorticoid receptor was detected by an
immunoblotting procedure using sequential incubations with
BuGR2, rabbit anti-mouse IgG, and 125l-protein A as described
in Materials and Methods. The total immunoadsorbed receptor
obtained from either 10 mg cytosolic protein or 2 mg nuclear
protein was applied to each lane of the gel. Kodak XAR film
was exposed at - 7 0 C for 4 h using a Cronex lightning-plus
intensifying screen. Molecular weight standards were phosphorylase b (97K) and BSA (66K).
cells by decreasing receptor half-life without affecting
its rate of synthesis (14). The results from this study
were consistent with the conclusion that binding of [3H]
triamcinolone acetonide converted the 10 S receptor
form to the cytosolic 4 S and nuclear receptor forms
and that one or both of these latter forms was degraded
at a faster rate in the cell than the unoccupied 10 S
receptor (14). In view of these results, dense amino
acid labeling was used to assess possible effects of
17«-methyltestosterone on the half-life of the glucocorticoid receptor. GHT cells were exposed to 20 nM 17amethyltestosterone or 10 nM [3H]triamcinolone acetonide for a total of 24 h. This period is sufficient to
establish a new, lower steady state amount of receptor
in the agonist-treated cells when compared to control
cells cultured without steroids (14). Cells received
dense amino acids during the final 10 h of this 24-h
incubation. After harvesting the cells and preparing a
nuclear extract, the density composition of the pooled
cytosolic and nuclear receptor forms was analyzed in
sucrose gradients containing 0.4 M KCI (Fig. 5). The
sedimentation pattern of receptor from density labeled
cells treated with 17a-methyltestosterone closely resembles that of control cells that did not receive steroid
and is clearly different than the pattern in cells treated
for 24 h with [3H]triamcinolone acetonide. A second
density labeling experiment provided similar results.
The results from the gradients shown in Fig. 5 were
quantified and used to estimate values for the half-life
337
The Glucocorticoid Antagonist 17«-Methyltestosterone
10
15
20
25
15
20
25
10
15
20
25
Fraction Number
Fig. 5. Sedimentation of Glucocorticoid Receptors after Dense Amino Acid Labeling in the Presence of 20 HM 17a-Methyltestosterone
(MeTest) or 10 nM [3H]Triamcinolone Acetonide (TA)
After incubation for 24 h in steroid-depleted medium, monolayer cultures of GH, cells were supplemented with the indicated
steroid and incubation was continued for a total of 24 h. For the final 10 h of this incubation, the culture medium was replaced with
dense amino acid medium also supplemented with the indicated steroid (•). Control cells were maintained in normal amino acid
medium with the indicated steroid during this final 10-h period (O). Cells were then chilled to 4 C and the medium was replaced
twice with 6 ml serum-free medium at 4 C. After each replacement of medium, cells were incubated for 45 min at 4 C. Finally, the
cells were incubated for 30 min at 37 C in 5 ml steroid-depleted medium supplemented with 10 nM [3H]triamcinolone acetonide.
Cells were harvested in lysis buffer and the lysate was adjusted to contain 0.4 M KCI and incubated at 4 C for 15 min to extract
nuclear glucocorticoid receptors (14). After centrifugation for 10 min at 12,800 x g, the supernatant containing both cytosolic and
extracted nuclear receptors was analyzed in sucrose gradients containing 0.4 M KCI as described in Materials and Methods. The
direction of sedimentation is from right to left in the figure and only the bottom 26 of a total of 44 fractions are shown. The peaks
of radioactivity centered around fractions 17-19 and 12-14 correspond to normal and dense receptors, respectively.
and synthetic rate of the glucocorticoid receptor in the
presence of the indicated steroids (Table 3). Consistent
with previous results (14), cells incubated with [3H]
triamcinolone acetonide for 24 h had only (49/92)100 =
53% of the receptor found in control cells. In contrast,
cells treated with 17a-methyltestosterone had (81/
92)100 = 88% of the control value, indicating that the
antagonist caused little or no reduction in the amount
of receptor. The value of 88% is a minimum estimate
since ligand exchange is probably not complete, although the efficiency of exchange is higher in this
experiment than in those described earlier due to the
use of a 37 C incubation during exchange. The half-life
estimates from the 10-h density labeling period indicate
that 17a-methyltestosterone does not shorten the halflife of the glucocorticoid receptor compared with control
cells incubated without steroid. In contrast, [3H]triamcinolone acetonide significantly reduces receptor halflife. The values for synthetic rates shown in Table 3
indicate only small differences for the three experimental conditions. The calculated value for 17a-methyltestosterone-treated cells underestimates the synthetic
rate since it has not been corrected for the efficiency of
ligand exchange. The observation that the antagonist
17a-methyltestosterone does not reduce receptor halflife is consistent with the conclusion that only agonists
promote dissociation of the 10 S receptor and that the
4 S receptor form is degraded in the intact cell at a
faster rate than the oligomer (14).
DISCUSSION
The interactions of the antagonist 17a-methyltestosterone with the glucocorticoid receptor differ from those
of the agonist triamcinolone acetonide. First, the two
steroids appear to compete for binding to the same site
(or sites) in the 10 S cytosolic receptor, but the affinity
for the antagonist is at least 1000-fold lower than that
for agonist (Fig. 1). Second, based on exchange assay
results in intact GH, cells, the antagonist does not
appear to promote dissociation of the 10 S oligomer to
the 4 S subunit and, by competing with agonist for the
ligand binding site, inhibits oligomer dissociation mediated by the binding of agonist (Figs. 2 and 3). Third,
based on both exchange assay results and immunochemical detection, the antagonist does not cause substantial accumulation of glucocorticoid receptors in the
nuclear fraction and inhibits nuclear accumulation of
receptor promoted by the agonist (Tables 1 and 2, Fig.
4). Finally, while the agonist significantly shortens glucocorticoid receptor half-life, the antagonist does not
(Fig. 5, Table 3).
These observations are consistent with a model (3,
4) for receptor transformation in which the unoccupied
receptor exists in intact cells primarily as a 10 S oligomeric protein that is found in the soluble (cytosolic)
fraction of broken cell preparations. When the steroid
binding site is occupied with an agonist, the distribution
between the oligomer and its 4 S hormone- and DNAbinding subunit shifts toward the subunit. Experimental
evidence supporting this model came originally from
dense amino acid labeling studies and from studies of
the dose-dependent effects of a glucocorticoid agonist
on the intracellular amounts of 10 S and 4 S cytosolic
receptors and nuclear receptors in GH, cells (3). Furthermore, sodium molybdate stabilizes oligomeric steroid receptor forms in vitro (17) and was found to inhibit
the agonist-mediated nuclear accumulation of gluco-
MOL ENDO-1989
338
Vol 3 No. 2
Table 3. Effects of 17a-Methyltestosterone (MeTest) or [3 H]Triamcinolone Acetonide (TA) on the Half-Life and Synthetic Rate of
the Glucocorticoid Receptor
Condition
No steroid
20 MM MeTest
10nM[ 3 H]TA
Total Receptor
(fmol/100MgDNA)
Dense Receptor
(% of total)
t*
(h)
(fmol/h-IOOMgDNA)
92
81
34
31
16.9
18.8
3.8
3.0
49
48
10.6
3.2
Amounts of total receptor and dense receptor were calculated by summing the radioactivity in appropriate fractions of the sucrose
have been found to be in good agreement with more thorough time course studies (14). The receptor synthetic rate (ks) was
density receptor remaining in cells after a time (t) of incubation with dense amino acids, R,=o is the total amount of dense and
normal receptor found in the gradients, and K, = 0.693/tV2 (38). Since the estimation of t,/2 is dependent only on the relative amounts
of dense and normal receptor forms found in the gradients, measurements based on single times of dense amino acid labeling
have been found to be in good agreement with more thorough time course studies (14). The receptor synthetic rate (ks) was
estimated from the formula ks = kdRDt/1 -e~kdt, where RD,, is the amount of dense receptor found in the gradients after a time (t) of
incubation of cells with dense amino acids (38).
corticoid receptors in intact GH, cells by a mechanism
which appears to involve stabilization of intracellular
receptor oligomers (4).
The results presented here suggest that the glucocorticoid antagonist 17a-methyltestosterone binds to
the oligomeric 10 S receptor but does not appear to
promote dissociation of the 4 S DNA-binding subunit
from the oligomer. Consequently the antagonist does
not cause substantial accumulation of receptor in the
cell nucleus. By competitively inhibiting the binding of
agonist to the 10 S oligomeric receptor, the antagonist
effectively inhibits the subunit dissociation mediated by
agonist binding that is essential for the nuclear accumulation and biological actions of the glucocorticoid
receptor. The ability of the antagonist to block the
actions of an agonist therefore depends on both the
concentrations and the relative binding affinities of the
two ligands.
While making direct binding studies using [3H]17amethyltestosterone impractical, the low affinity of the
antagonist for the glucocorticoid receptor was essential
for the ligand exchange assay at 0 C used in this study.
Even at this low temperature, the antagonist dissociates from the receptor at a rate sufficiently rapid to
allow significant exchange with the high affinity agonist
[3H]triamcinolone acetonide. However, at 0 C the agonist does not promote the subunit dissociation and
nuclear accumulation that normally occurs rapidly at
37 C (3, 4). For this reason, it is assumed throughout
this study that the relative abundance and intracellular
location of receptor forms does not change during the
ligand exchange procedure at OC. The absence of
immunochemically detectable receptor in the nucleus
after rapid lysis and fractionation of cells treated at 37 C
with 17a-methyltestosterone (Fig. 4) appears to confirm
the conclusion from ligand exchange studies that the
antagonist does not cause significant nuclear accumulation of receptor in intact cells.
The conclusion that 17a-methyltestosterone and
other glucocorticoid antagonists which bind reversibly
to the receptor do not cause nuclear accumulation of
receptor is consistent with results from several direct
binding studies using 3H-labeled steroids. First, proges-
terone was found to be an effective glucocorticoid
antagonist in HTC cells (10). [3H]Progesterone did not
lower the amount of cytosolic receptor in HTC cells
(18), and did not cause nuclear accumulation of receptor
in mouse mammary tumor cells (19). Since [^progesterone elicits high levels of nonspecific binding in both
cytosolic and nuclear fractions from GHT cells (Raaka,
B. M. and H. H. Samuels, unpublished results), it was
not possible to use this steroid in the present study to
determine effects of an antagonist on 10 S and 4 S
receptor forms by direct binding assays. Second, two
groups concluded independently that [3H]cortexolone
(11-deoxycortisol) did cause nuclear accumulation of
glucocorticoid receptors in rat thymus cells (20, 21).
Because of the low affinity of receptor for this compound (15) and high nonspecific binding (20), these
studies did not provide quantitative data concerning the
fraction of total cellular glucocorticoid receptor that
accumulated in nuclei, and the available evidence suggests that the fraction was small. Since cortexolone
was shown to be a partial agonist in HTC cells (10), it
is reasonable to expect a fractional nuclear accumulation of receptor in proportion to the agonist activity of
this steroid. Finally, the synthetic glucocorticoid antagonist [3H]RU38486 has high affinity for the glucocorticoid receptor and causes little or no nuclear accumulation of receptor in rat thymus cells (22) or hepatoma
cell lines (23). Although several reports (24, 25) have
indicated that [3H]RU38486 accumulates in nuclei, it
has not clearly been shown that the labeled steroid
occupied glucocorticoid receptors. Recent studies have
shown that [3H]RU38486 does not promote dissociation of glucocorticoid receptor oligomers under conditions in which oligomer dissociation is promoted by
agonist [3H]triamcinolone acetonide (11,12).
The subunit structure and intracellular location of the
10 S oligomeric glucocorticoid receptor are not known
with certainty. Recent studies have indicated that oligomeric forms of steroid receptors contain a common
90 kDa heat shock protein that does not bind steroid
(26-28) and which dissociates from the hormone-binding subunit upon ligand-mediated transformation (9,
29). Evidence supporting a trimeric structure consisting
The Glucocorticoid Antagonist 17a-Methyltestosterone
of a single hormone-binding subunit and two 90 kDa
subunits has been presented for both glucocorticoid
(30) and progesterone (26) receptors. Controversy has
arisen concerning the intracellular location of untransformed steroid receptors. While immunochemical (31)
and rapid cell enucleation (32) studies have suggested
that the unliganded estrogen receptor may be located
in the cell nucleus, other immunochemical studies have
supported a cytoplasmic location for the unliganded
glucocorticoid receptor (33, 34). The possible nuclear
localization of certain unliganded steroid receptors does
not negate the concept of ligand-mediated receptor
transformation but indicates that this process may occur in the nucleus rather than in the cytoplasm.
While direct evidence of the intracellular existence of
steroid receptor oligomers has not been obtained to
date, evidence from a variety of experimental systems
(3-9) supports the conclusion that transformation of
steroid receptors in situ involves the hormone-mediated
dissociation of a receptor oligomer to its hormone- and
DNA-binding subunit. In particular, a recent study has
shown that certain glucocorticoid receptor deletion mutants which act as constitutive activators of transcription (35, 36) are unable to form oligomeric receptor
forms (37). This suggests that intracellular steroid receptor oligomers may serve to mask the DNA binding
and transcription-enhancing domains of the glucocorticoid receptor. By competing with agonists for binding
to an intracellular receptor oligomer, glucocorticoid antagonists would effectively block the agonist-mediated
oligomer dissociation required to unmask functional
domains of the receptor.
339
resulting particulate fraction, referred to as washed nuclei, was
incubated with 0.25 ml of ethanol for 30 min at 37 C to
extract [3H]triamcinolone acetonide. Specific binding to receptor proteins was determined by comparing radioactivity in
nuclear fractions derived from cells treated with the labeled
steroid and with or without a 500-fold excess of the unlabeled
steroid. Radioactivity in ethanol extracts was determined by
scintillation counting at 43% efficiency. The DNA content of
the nuclear fraction was determined by the method of Burton
(39).
Sucrose Gradient Centrifugation
For resolution of 10 S and 4 S cytosolic receptor forms,
discontinuous gradients were made of 1.3 ml each of 30%,
20%, and 10% sucrose solutions (wt/vol) prepared in buffer,
pH 7.4 at 25 C, containing 20 M Tris-HCI, 20 M sodium molybdate, and 2 mM dithiothreitol. After centrifugation for 16 h at
58,000 rpm and 4 C in an SW 60 Ti rotor, 39 or 40 fractions
were collected from each gradient. Using these conditions, the
molybdate-stabilized 10 S receptor oligomer does not dissociate during sedimentation (3). Peak positions for reference
proteins /?-amylase, 9.4 S (40), and BSA, 4.4 S (41), were
fractions 8-9 and 19-20, respectively. Throughout the text
the term "4 S" is used to describe a hormone- and DNAbinding form of the glucocorticoid receptor that sediments at
about 3.5 S under high salt conditions and in the region of 4
to 6 S under low salt conditions (3). The variable sedimentation
rate under low salt conditions probably results from reversible,
concentration-dependent interactions of the receptor protein
with itself or other proteins during sedimentation (3).
For dense amino acid labeling studies, discontinuous gradients consisted of 30%, 22.5%, and 15% sucrose solutions
(wt/vol) prepared in 95% D2O, 5% H2O and containing 20 mM
Tris-HCI, pH 7.4 at 25 C, 2 mM dithiothreitol, and 0.4 M KCI
(3, 14). Centrifugation was for 45 h at 5 C and 58,000 rpm in
the SW 60 Ti rotor.
Immunoblotting
MATERIALS AND METHODS
Cell Culture Conditions
GHi cells were grown in monolayer cultures at 37 C in 75-cm2
plastic flasks as described previously (3). Before each experiment, cells were cultured for 24 h in Ham's F-10 medium
supplemented to 10% (vol/vol) with calf serum that had been
treated with AG 1X-8 anion exchange resin and activated
charcoal to remove steroid and thyroid hormones (13). This
medium is designated steroid-depleted medium. Culture medium containing dense amino acids was prepared as described
previously (38).
Cell Harvesting and Analysis of Receptor Forms
After removing the culture medium, cells were washed three
times with isotonic saline at 4 C and drained thoroughly. The
washed cells were scraped off the culture flasks at 4 C in 0.25
ml buffer, pH 7.4 at 25 C, composed of 20 miui Tris-HCI, 5 mM
sodium molybdate, 2 mM dithiothreitol, and 0.02% Triton X100 (lysis buffer). After mixing for 5 s at the maximum setting
of a Vortex Genie, the soluble and paniculate fractions were
separated by centrifugation for 10 min at 12,800 x g in an
Eppendorf model 5412 microcentrifuge. The supernatant from
this centrifugation is referred to as cytosol.
For analysis and quantitation of glucocorticoid receptors
from cells previously incubated with [3H]triamcinolone acetonide, cytosol was applied directly to sucrose gradients as
described below. The particulate fraction from the 12,800 x g
centrifugation was washed three times by resuspending in 1
ml of lysis buffer followed by centrifugation as before. The
Cytosol and washed nuclei were prepared from GHi cells as
described above. A soluble nuclear extract was prepared by
resuspending the washed nuclei for 30 min at 4 C in 0.5 ml
lysis buffer containing 0.4 M KCI followed by centrifugation at
12,800 x g for 10 min. The procedure generally solubilizes
about 55% of receptor-bound [3H]triamcinolone acetonide
from GHT cell nuclei (14). The cytosol and nuclear extract were
adjusted to final concentrations of 200 mM KCI and 10 nM
triamcinolone acetonide. For immunoprecipitation, 0.01 ml ascites fluid containing monoclonal antibody BuGR2 (42) was
added to 1 ml adjusted cytosol or nuclear extract containing
approximately 10 or 2 mg protein, respectively. After incubation at 0 C for 16 h, 0.1 ml (wt/vol) suspension of protein Alinked Sepharose CL-4B in lysis buffer was added and the
resulting suspension was incubated at 4 C for 3 h with gentle
mixing by rotation. The protein A-linked Sepharose was then
sedimented by centrifugation at 12,800 x g for 10 min and
washed twice at 4 C using 1 ml lysis buffer. After extensive
washing at room temperature first using 50 mM Tris-HCI, pH
7.5, 5 mM EDTA, 0.75 M NaCI, and 0.05% NP-40 followed by
50 mM Tris-HCI, pH 7.5, 5 mM EDTA, and 0.2 M NaCI, the
protein A-linked Sepharose was resuspended in 0.075 ml 10
mM sodium phosphate, pH 7.0, 1 % 2-mercaptoethanol, and
1 % sodium dodecyl sulfate and incubated at 37 C for 30 min
to release adsorbed proteins.
Immunoadsorbed proteins were analyzed by electrophoresis in a 7.5% polyacrylamide gel in the presence of sodium
dodecyl sulfate (43). Proteins were then transferred by electroelution at 0.75 A for 3 h at room temperature. Transfer
buffer was 25 mM Tris base, 192 mM glycine, and 20% (vol/
vol) methanol (44). After transfer, the nitrocellulose filter was
incubated for 15 min in an aqueous solution containing 10%
(vol/vol) acetic acid and 10% (vol/vol) isopropanol. Unless
Vol 3 No. 2
MOL ENDO-1989
340
otherwise noted, this and all subsequent incubations were
performed at room temperature with gentle shaking on a
rotating platform. After rinsing briefly with water, the filter was
incubated for 10 min with buffer consisting of 20 mM Tris-HCI,
pH 7.5, and 200 mM NaCI (TBS) followed by a 60-min incubation in milk solution consisting of 20 mM Tris-HCI, pH 7.5,1
mM EDTA, 100 mM NaCI, 0.005% Antifoam A, and 5% (wt/
vol) Carnation nonfat dry milk (45). Ascites fluid containing
BuGR2 was then added to the milk solution at a dilution of
1:1000 and incubation was continued for 16 h at 4 C.
After incubation with BuGR2, the filter was washed for 15
min with TBS containing 0.05% Tween-20 and then for 15 min
in TBS. After a 60-min incubation in milk solution, purified
rabbit anti-mouse IgG was added at a 1:2000 dilution and
incubation was continued for 2 h. After 15-min washes first in
TBS containing 1 M NaCI and 0.05% Tween-20 and then
without Tween-20, the filter was incubated for 2 h in 50 ml of
milk solution containing 125l-Protein A at 150,000 cpm/ml. After
three 15-min washes in milk solution containing 1 M NaCI and
0.05% Tween-20, the filter was rinsed briefly in water prior to
autoradiography.
Chemicals and Media
[6,7-3H]Triamcinolone acetonide (44 Ci/mmol) was obtained
from New England Nuclear (Boston, MA). 125l-Protein A (30
mCi/mg) was from Amersham (Arlington Heights, IL) and rabbit
anti-mouse IgG was from Zymed Laboratories (South San
Francisco, CA). Steroids, protein A-Sepharose CL-4B, protamine sulfate, sodium molybdate, D2O, and other chemicals were
obtained from Sigma (St. Louis, MO). Cell culture media and
serum were supplied by Grand Island Biological Co. (Grand
Island, NY). Amino acids enriched in the dense isotopes 2 H,
13
C, and 15N to 98%, 80%, and 90% atom, respectively, was
obtained from Merck, Montreal, Qubec, Canada. Monoclonal
antibody BuGR2 which recognizes the hormone-binding subunit of glucocorticoid receptors (42) was generously provided
by Dr. Robert W. Harrison (University of Arkansas Medical
School, Little Rock, AR).
Acknowledgments
Received April 27,1988. Revision received October 31,1988.
Accepted November 11,1988.
Address requests for reprints to: Dr. Bruce M. Raaka,
Department of Medicine, UH Room 451, New York University
Medical Center, 550 First Avenue, New York, New York
10016.
This research was supported by Grant AM-21566 from the
NIH and Grant BC-550 from the American Cancer Society.
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