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The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor
THE "ANTIPROGESTERONE RU486 STABILIZES THE HETEROOLIGOMERIC, NON-DNA-BINDING, 8S-FORM OF THE RABBIT UTERUS CYTOSOL PROGESTERONE RECEPTOR Jack-Michel Renoir, Christine Radanyi, and Etienne-Emile Baulieu INSFRM U 33, Lab. Hormones, 94275, Le Kremlin-Bic~tre, France Corresponding author: Etienne-Emile Baulieu, Lab. Hormones, 94275 Bic~tre Cedex, France Received November 30, 1988 Revised February 7, 1989
ABSTRACT The salt-induced (0.3 M KCI) transformation of the nontransformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor ( P R ) was analyzed by density gradient ultracentrifugation (8S ~ 4S conversion) and DNAcellulose chromatography (non-binding ~ binding forms). After 1 h treatment at 2 C, > 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist-and antagonist-bound non-transformed receptors were similar and the calculated Mr were ~ 283,000 and 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.
STEROIDS 53/1-2
January-Eebruary 1989 (1-20)
1
2
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
INTRODUCTION Steroid target
cell
form :
they
receptor
receptors, cytosol are
in
in a
absence
hormone,
non-transformed,
heterooligomers
molecule
of
and the heat
associated
with
hspg0
including
a
shock protein
(4-6).
found
non-DNA-binding
(1-3). The rabbit uterus cytosol progesterone also
are
Steroid
in 8S-
hormone-binding
of
90 kD
receptor
hormones
(hsp90) (PR) t is
and
their
synthetic agonists accelerate the temperature-dependent dissociation of the heterooligomer hormone
complexes
regulatory
can
elements
(7-9), and the released 4S receptor-
then
bind
(i0,Ii
for
to
appropriate
review)
and
DNA
trigger
hormone
transcrip-
tional events. The rabbit progesterone receptor, showing structurally much homology with the human progesterone receptor
(12), is
an excellent system to study the mechanism of action of RU486, an antiprogesterone
already
control
RU486 binds
(13,14).
successfully
receptor with high affinity gesterone
(15,16).
Already,
antiglucocorticosteroid glucocorticosteroid
to the
tested
in human
rabbit uterus
fertility
progesterone
(Kd ~ 0.2 nM), and it is an antiproRU486
(14,17)
8S receptor
which
has been (GR) form
is
also
a
powerful
shown to stabilize in the
chick
the
oviduct
(18). Experiments with the powerful progesterone agonists, R5020 and Org2058,
and the antagonist RU486 were performed in order to
compare the stability of the rabbit uterus 8S PR when binding an agonist or an antagonist. Ultracentrifugation in sucrose gradient
STEROIDS 53/1-2
January-February 1989
Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
and DNA-cellulose transformation of hsp90
3
chromatography were used in order to assess the
of the 8S to 4S form, which implies the separation
from the receptor protein,
and the DNA binding
ability
of the latter whether binding or not an agonist or an antagonist steroid.
MATERIALS AND METHODS Chemicals. [2,4,6,7-3H]~ogesterone ([3H]p, 82.7 Ci/mmol) and the synthetic progestin [-H]Org2058 (45 Ci/mmol) _ were from the Radiochemica~ Centre (Amersham, Bucks, UK). [~H]R5020 (50 Ci/ mmol) and ['H]RU486 (50 Ci/mmol) were a gift from Roussel-Uclaf (Romainville, France). Non-radioactive cortisol and progesterone (P) were obtained from Roussel-Uclaf. DEAE-Sephacel was from Pharmacia (Uppsala, Sweden), and DNA-cellulose (1.38 mg DNA/mL packed gel) and cellulose were obtained from P.L. Biochemicals, Inc. (Milwaukee, wi). All other reactants were from Merck (Darmstadt, West Germany), except when noted. Buffers. Buffer A included I0 mM potassium phosphate, 1.5 mM EDTA, 10% (v/v) glycerol, and 12 mM l-thioglycerol, pH 7.8; Na2MoO 4 (20 mM) was added to buffer A to make buffer B. Rabbit uterus cytosol. I~mature New Zealand rabbits were injected with diethylstilbestrol and the uterine cytosol was prepared as described (6), with the addition of PMSF (phenylmethylsulfonyl fluoride, Calbiochem, 0.3 mM) and leupeptin (Sigma, 20 ~M final concentration) as protease inhibitors. Non-radioactive cortisol (2 ~LM) was systematically added to cytosol to prevent the binding of progestins to contaminating transcortin and glucocorticosteroid receptor. P~ogesterone receptor binding assa[. Incubations of cytosol with [-H]steroid (20 ruM) were performed during 2.5 h at 0-2 C, in the presence or absence of molybdate. Non-specific binding was measured in parallel after addition of 2 ~M of non-radioactive progesterone. Dextran(0.025%)-charcoal (0.25%) adsorption (10 min at 0 C) was used to assay protein-bound steroid (see 19). Receptor-bound ligand was determined by subtraction of nonspecific from total binding.
STEROIDS 53/1-2
January-February 1989
4
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
DNA-cellulose assay. DNA-cellulose or unsubstituted cellulose was first pelleted in 0.2 mL aliquots in 5 mL polypropylen~ tubes after equilibration in buffer A. Triplicate samples of [-H]steroid-labeled cytosol were incubated 1 h at 0 C with DNA-cellulose slurry under gentle shaking. It was verified that the equilibrium was attained for each agonist-or antagonist-PR complex. The suspension was then centrifuged 5 min at 700xg, the supernatant discarded, and the pellet washed with 3 x 1 mL of buffer A. The pellets were resuspended in 7 mL of scintillation mixture (Readysolv, Beckman Instruments), and counted. Specific binding to DNA-cellulose was calculated after subtraction of radioactivity bound to cellulose. Density gradient ultracentrifugation. Samples (0.2 mL) were layered onto preformed discontinuous sucrose gradients (5-20%) made in buffer A or B. Centrifugation was performed during 16 h x 46,000 rpm at 2 C, in an SW60 Beckman rotor, with internal markers (glucose oxidase, S20,W = 7.9 and peroxidase S20,W = 3.6). High performance liquid chromato@raphy (HPLC) analysis. analyses were performed as previously described (6).
HPLC
SDS-slab gel electrophoresis and staining. Proteins were resolved in 7.5% SDS polyacrylamide gels according to the method of Laemmli (20). The protein bands were stained with silver nitrate as described in (21). Purification of PR. The protocol described earlier (6) was used. Briefly, cytosol from estrogen stimulated uteri was prepared in molybdate ions containing buffer to stabilize the non-transformed PR, and transferred at 4 C on the top of NADAc-Sepharose affinity g~l (22). Afte{ washing and biospecific el~tion with either [ H]R5020 or [-H]RU486 (i ~M), the eluted [ H]steroid-PR complexes were loaded onto two DEAE-Sephacel columns (I mL) equilibrated in buffer B. Non-transformed PR complexes bound to the ion exchange gel, whereas transformed PR complexes did not. Under these conditions the former elutes at 0.15 M KCI during the salt gradient, and with ~ 20% purity (calculated on the basis of specific activity, assuming one steroid binding site per protein molecule of i00 kD), whether the receptor was labeled with agonist or antagonist. Each flowthrough was applied to DNAcellulose columns (25 mL). The transformed PR was eluted at 0.32 M KCI with ~ 65% purity. Transformation studies of purified 8S-PR by DNA-ce!lulose chromatography. The DEAE-Sephacel eluates were diluted 5-fold in buffer
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
5
A and loaded on i0 mL DNA-cellulose column equilibrated with the same buffer (flow-rate 30-35 mL/h). After washing with 40 mL of buffer A, a 50 mL linear 0-0.5 M KCI gradient was applied. Fractions of 9 mL were collected during sample adsorption to the column, 4 mL during the washing, and 1 mL during the 0-0.5 M KCI gradient. Aliquots of each fraction (200 ~L) were counted for radioactivity and 0.5-mL aliquots were boiled in 1% SDS for analysis by SDS-PAGE. Protein determination and radioactivity counting. Protein concentration of PR containing samples was determined according to the method described (23) and radioactivity was measured in a Minaxi Scintillation counter (Packard) after addition of 7 mL Toluene scintillator (Packard) to radioactive samples.
RESULTS
Comparison
of
the
rate
of
salt-induced
transformation
of
[3H]-
RU486-PR and [3H]a~onist-PR complexes In
the
agonist-PR cytosol
absence
of
molybdate,
transformation
of
[3H]-
complexes was almost complete after 1 h treatment of
with
0.3 M
KCl
(at
2
C).
This
was
indicated
radioactivity in the 4-5S region of the density res IA and 2A, see also Table
gradient
by
the
(Figu-
I). After longer exposure to salt
(> 2 h), the sedimentation coefficient of the transformed PR was 3S
(not shown), suggesting a cleavage of the receptor in spite
of the presence of protease inhibitors. When RU486 was employed, exposure initial
to
0.3 M
8S peak
KCI
during
(Figure
IB).
1 h
at
I0 C,
Even after
did
not
effect
72 h salt treatment
the at
2 C, ~ 90% of [3H]RU486-PR complexes still sedimented in 8S-form (Figure 2B). To achieve complete transformation, it was then
STEROIDS 53/1-2
January-February 1989
6
Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
GO
pO
G O 1C-
pO
B o
b
u
8 ~
c3
5
Z
10 Fraction
20 Fraction
No
N~
Figure i. Salt-induced t r a n s f o r m a t i o n of the rabbit uterine PR. Effect of the ligand. Cytosol was p r e p a r e d in b u f f e r A and one p~rt was incubated for 2 h at 2 C with 20 nM [ H]Org2058 or [-H]RU486; another p a r t was kept at 2 C w i t h o u t ligand. 0.3 M KCI was added to each cytosol at 2 C. The t r a n s f o r m a t i o n p r o c e s s was b l o c k e d by adding 20 m M sodium m o l y b d a t e to each sample after 1 or 2 h panel C KCl treatment, and aliquots (200 ~L) were immediately loaded on top of p r e f o r m e d 5-20% sucrose gradients in b u f f e r B containing 0.3 M KCI. P o s i t i o n of internal standards is indicated by arrows. IA: sedimentation profile of [ H]Org2058-PR treated (e---O) o ~ not (o o) for 1 h with salt. IB: sedimentation profile of [ H ] R U 4 8 6 - P R treated (~ &) or not (o o) for 1 h, or treated 6 h (x x), with 0.3 M KCI at I0 C. necessary
to
treatment.
For
was
necessary
transformation exposure previously
to
increase
both
example,
exposure
to
effect
was
therefore
0.3 M
exposed
KCI
the
temperature to
0.3 M KCI
transformation obtained
(Table
I)
or
at 6 h
and
the
for
6 h
(Figure I0
C
time at
IB).
either
treatment
i0 C,
Complete after
of
8 h
cytosol
to 0.3 M KCl for one night at 2 C (Figure
STEROIDS 53/1-2
of
2C).
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
B
A
IO0
0 " - - - 0 ~
~o
30
60
C
~,,,~u486
1
90
T,me(mln)
RU 486
2C
0
50
30
°
70
T~me( hours)
D 1000~,~
1~
=~
7
O ~
10 C
°
~
~~ N
2
4 Time (hours)
RU486
O~
o 10 20 Temperature (C)
Figure 2. R a t e of transformation of the rabbit uterine PR. Cytosol was prepared in buffer A and PR w a ~ i n c u b a t e d for 2 h at 2 C with 20 nM [~H]R5020 (Figure 2A) or [ H]RU486 (Figure 2B). Transformation was induced by adding 0.3 M KCI at 2 C. At different times, 20 mM molybdate was added to prevent further transformation, and aliquots (i00 ~L) were loaded on the top of 5-20% sucrose gradients containing 20 mM molybdate and 0.3 M KCI. At zero time, all the radioactivity migrated at ~ 8S. The percent of PR recovered as non-transformed, 8S (o o), or transformed, 4S (e----e), was plotted versus the time of KCl treatment prior to molybdate addition. Two experiments are p r e s e n t ~ for each time studied (Figure 2A). 2B: Same experiment with [-H]RU486-PR. 2C: Same experiment as in Figure 2B, but the sample in which transformation was induced with salt at 2 C, was kept as such overnight and then set at i0 C instead of 2 C. The loss of RU486 binding activity after 6 h was 15% of the initial qtime zero) binding activity. 2D: Percent of remaining 8S [-H]RU486-PR obtained after 2h exposure to 0.3 M KCl at different temperatures. The loss of RU486 binding activity was 15, 18, and 22% for i0, 20, and 25 C incubation temperatures, respectively.
STEROIDS 53/1-2
January-February 1989
8
At
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
25°C,
the 8S peak was
(Figure
2D),
but
loss
lost
of
after
2 h treatment
hormone
binding
we deduced
that
with
(~ ].5%) was
0.3 M KCI concomi-
tantly observed. From of
the
8S
conditions
these PR
results, into
a
for both
transformation
4-5S
form
can
agonist-and
occurs m u c h more
TABLE i. H y d r o d y n a m i c complexes.
be
complete obtained
[ 3H ]R5020
s, nm
7.7 + 0.3 n=3
[3H]Org2058
7.7 + 0.3 n=3
[ 3H ]RU486
7.8 + 0.2 n=4
under
antagonist-labeled
PR,
parameters
$20,
of
agonist-and
vitro
but
that
antagonist-
PR
Transformed R
W
8.7 + 0.3
s,nm
n.d.
n=5
S20,w
4.5 + 0.5 a n=5
8.7 + 0.3
n.d.
n=8
4.5 + 0.4 a n=8
8.9 + 0.3 n=4
in
slowly w i t h the latter.
Non-transformed R
transformation
5.6 + 0.5 b n=4
5.0 + 0.5 b n=4
n.d.: not determined due to loss of binding during HPLC. a . . . . . T r a n ~ f o r m a t l o n obtalned by i n c u b a t l o n for 2 h wlth 0.3 M KCI at 2 C. T r a n s f o r m a t i o n obtained after 8 h exposure to 0.3 M KCI at i0 C.
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
9
Effect of steroids on the thermal inactivation of
PR
Both molybdate-stabilized PR and salt-transformed at 0 C) PR were
labeled with either
[3H]R5020
or
(overnight
[3H]RU486
and
kept at 0, I0, and 25 C during 24 h and 18 h, respectively.
The
results show that [3H]RU486-PR complexes are more stable, presence
of
R5020-PR
complexes
were
less
either
stable
molybdate (Figure
than
3).
or
KCI,
than
the
Salt-treated
molybdate-stabilized
in the
corresponding
RU486-PR RU486-PR
complexes complexes,
but more than KCl-treated R5020-PR species.
Plus KCI 18h
Plus Molybdate 24h
[] [~R~o
100
m ~u4e6
OC
10C
1 25C
C
10C
25C
Figure 3. Thermal stability of non-transformed and transformed PR-steroid complexes. Cytosol was prepared in buffer A and divided into two parts. One part was supplemented with 20 m M sodium molybdate, the other one with 0.3 M KCI. The cytosols were kept o v e r n i g ~ at 0 C and ~-mL aliquots were then incubated 2.5 h w~th 20 nM [-H]R5020 or [--H]RU486. The percentage of remaining [-H]steroid binding was measured after 2.5 h incubation with each ligand (time 0, 100%), and after storage for 24 h (plus molybdate) or 18 h (plus KCl), at 0, i0, and 25 C, as indicated on the abscissa.
STEROIDS 53/1-2
January-February 1989
10
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
Hydrodynamic
parameters
of non-transforme d and transformed
[3H]-
steroid-PR complexes Sedimentation non-transformed agonists
in Table i. An
non-transformed
parameters.
values
and
Stokes
radii
of both
and transformed PR complexes liganded with either
(R5020 or Org2058)
summarized for
coefficient
The
or antagonist apparent
[3H]RU486-PR
apparent
Mr
of
Mr
(RU486)
~ 293,000
complexes
steroids, was
from
[3H]agonist-PR
are
calculated
hydrodynamic complexes
was
283,000. After
salt-induced
the calculated previous
analysis, s
obtained
after
(see legend 118,000
SDS-PAGE
of Table
in agreement
analysis
(4,6,24)
I), with and
(25). It was not possible to measure the apparent Mr
of transformed
with R
Mr of [3H]RU486-PR was
values
cloning data
transformation
[3H]agonist-PR
due to loss of binding during HPLC
the latter giving a broad radioactive peak
(not shown)
smaller than 4 nm.
DNA-cellulose binding of transformed PR Agonist-labeled formed
PR complexes
PR was assayed for DNA binding. were
other ligand-transformed may
reflect
assay,
since
the
found
PR complexes
dissociation
kinetic
to bind
DNA-cellulose
less
than
(Table 2). This difference
of
[3H]P-PR
complexes
experiments
revealed
that
was shorter than that of either
[3H]P-trans-
synthetic
during
their
[3H]agonists
STEROIDS 53/I-2
the
half-life or [3H]-
January-February 1989
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
RU486-PR reason, nists.
complexes, most
even
at
experiments
Antagonist
0-2
were
C
(data
carried
[3H]RU486-transformed
11
not
out
shown).
with
PR
For
this
synthetic
ago-
complexes
bound
to
DNA-cellulose to the same extent as PR liganded with the agonist. We concluded that transformed PR binds similarly to DNA, whatever it is liganded with an agonist or the RU486 antagonist. tested
the
stabilized
binding
to
DNA-cellulose
non-transformed
PR,
but,
at
20
whatever
C the
of
We also
molybdate-
nature
of the
ligand, no binding was observed.
TABLE 2. DNA-cellulose binding of transformed PR bound to either agonist or RU486
ligand
incubated 4S-PR cpm
bound 4S-PR cpm (mean + SD)
% binding
P
22,750
8,645 + 907
38
R5020
13,748
5,534 + 638
40
Org2058
12,380
6,716 + 298
54
RU486
13,750
7,837 + 417
57
Rabbit uterus cytosol was prepared in buffer A, then supplemented with 0.3 M KCI final concentration for 2 h at ~ C. A%iquots were incubated fo~ 2 h at 0 C with 20 nM [~H]P, [~H]R5020, [~H]Org2058, or [ H]RU486. Aliquots of each incubate were then diluted 6-fold in buffer A ~and added to 200 ~L of pelleted DNA-cellulose (0.~5 pmol of [JH]ligand-PR complex) in triplicate. DNA-cellulose-[ H]ligand-PR complexes were treated as mentioned in Materials and Methods.
STEROIDS 5311-2
January-February 1989
!2
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
Transformation of purified 8S PR complexes Molybdate-stabilized, complexes
were
complexes
analyzed
purified
agonist-labeled
PR
bioaffinity in
parallel
similarly. contained,
with
The in
purified
[3H]RU486-PR
[3H]R5020-1abeled
purified
addition
to
PR
non-transformed hormone
binding
units of ~ 120 and ~ 85 kD, an abundant non-hormone binding 90-kD protein kD)
(Figure 4A,
lane 2). Other protein bands
represent contaminants as already described
purified non-transformed retic pattern
(at 66 and 45
(6). Analysis of
[3H]RU486-PR gave a similar electropho-
(Figure 4A,
lane I). During purification,
the continuous presence of molybdate,
even in
a portion of PR is trans-
formed to a DNA-binding form unable to bind to DEAE-Sephacel During
purification
of
[3H]RU486-1abeled
8S
PR,
the
(6).
amount
of
transformed receptor represented only one-tenth of that obtained in the case of the [3H]R5020-1abeled different purifications),
8S PR
(data obtained
in 5
as visualized on Figure 4A, lanes 3 and
4. This appears to reflect an intrinsic difference in the rate of transformation after labeling the receptor with agonist or RU486, and thus confirms the results obtained with cytosol preparations (see Figures 1 and 2). In one
series
of
experiments,
DEAE-Sephacel
transformed [3H]R5020 and [3H]RU486-PR complexes
purified
non-
(purity ~ 22% in
both cases) were diluted with buffer A, incuba~ted for 20 min and 2 h, respectively at 20 C (optimum conditions to achieve complete
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONERECEPTORAND RU486
A 212
1 2
3
13
B
4
1 2
~
3
/
i
130 ~ _
//. /
_130
~ o
~-
97__ ~ W o
66 ~
-4s
////
_3o
_97 .3-
/////
-6e
45~
//"
4
--212 --212
3 o
66
//
--45
2
O0
20
40
60
80
Fraction number
Figure 4. Dissociation of hsp90 from the hormone binding protein upon activation of PR. 4A: SDS-PAGE of purified PR. 3 Lane % and 2: non-transformed [ H]RU486-PR (1.4 ~g of protein) and [-H]R5020-PR (1.4 ~g of protein), respectively eluted from the DEAE-Sephacel column, purity ~ 22% in both cases (6), calculated on specific activity basis according to one m o l e c ~ e of steroid bound per I00,000 Mr protein; lane 3: activated [ H]RU486-PR (i00 ng of protein) eluted from the DNA-cellulose column loaded with 3the DEAE-Sephacel flow-through fractions; lane 4: activated [ H]R5020-PR (300 ng of protein) eluted from the DNA-cellulose column. The arrows indicate the migration of marker proteins. 4B: Activation of purified non-transformed PR. Purified rabbit uterine 8S PR labeled either with [[H]R5020 (o o; 150 ~g of protein, 32 pmol of bound steroid) or [~H]RU486 (e----o; 155 ~g protein, 38 pmol of bound steroid) was diluted in buffer A, treated as indicated in the text and loaded in parallel onto two i0 mL columns of DNA-cellulose equilibrated in buffer A. Lanes 1 and 2: 400 ng of protein o~ the flow through fractions of the DNA-cellulose columns for [--H]R5020- and [ H]RU486-bound receptors, respectively; lane 3 : 1 5 0 ng of protein from the top of the [-H]R5020 peak of the DNA-cel~ulose column; lane 4: 200 ng of protein from the top of the [ H]RU486 peak. Position of marker proteins is indicated by arrows. transformation of purified PR), and then loaded on the top of two DNA-cellulose proteins
STEROIDS 53/1-2
columns. As shown in Figure 4B, the hormone binding
(~ 120
and
~ 85 kD)
January-February1989
were
together
eluted
as a
single
14
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
radioactive DNA
p e a k at 0.32 M KCI,
of both
sucrose
RU486
gradient
DNA-cellulose with
or
protein,
R5020-PR
migrated sample
originally
columns,
at
~
was
in
the
for
Ultracentrifugation
in
R5020
both flow
affinities
similar
the run
in
similar
[3H]RU486-PR
but
with
during
present
found
indicating
4.5S,
labeled
PR inactivation
structures,
complexes.
of the top of p u r i f i e d
the parallel
to complete
suggesting
eluted
experience
was
made
unsuccessful
(not shown).
purified through
from
due
The 90-kD
non-transformed fractions
of
the
that it does not bind to DNA.
DISCUSSION The
present
report
vitro,
the
salt-induced
(from
the
non-DNA
DNA-binding,
indicates
transformation
binding,
monomeric
4S
In
RU486-PR
cell-free
complexes
form)
R5020
obtained
a
after
binds
gonist-PR ionic
to
non-specific
complexes
strength
are
eluted
as
agonist-PR
identical
apparent
affinities
rat
4S-GR
liver
labeled
DNA
with
to
the
slowly
than
2) or 0rg2058
(not
transformation to
0.3 M KCI
antagonist-bound
DNA-cellulose (Figure
for
Anta-
the
same
4B),
suggesting
DNA.
Similarly,
triamcinolone
STEROIDS 53/I-2
at
of
receptor
DNA-cellulose).
non-specific
either
in
complexes
form
more
exposure
complexes for
8S
much
complete
(i.e.,
from
temperature
RU486-PR
(Figure
8 h at i0 C or 2 h at 25 C. T r a n s f o r m e d readily
low
of the
occurred
experiments,
is
at
heterooligomeric
with PR labeled with the agonist shown).
that,
acetonide
or
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
15
RU486 binds with close affinities to DNA-cellulose
(27). However,
affinity for DNA-cellulose of the rabbit uterine PR complexed to RU486 is lower than that of R5020-PR complexes
(28) after activa-
tion for 15 min at 20 C. From
our
results,
such
conditions
certainly
activate
agonist-PR complexes, but they lead to only partial activation of antagonist-PR
complexes
(~ 15%
of
the
RU486-PR
complexes
are
still in 8S form after 2 h exposure to 0.3 M KCI at 20 C; Figure 2D). Insufficient activation also might explain results obtained with RU486 GR-complexes to
4S
forms
needs
(29). Transformation of receptor from 8S
to be
carefully
checked before
carrying
out
further studies with transformed species. Indeed, our results are in good agreement with recent findings, indicating that GR and PR bind with similar affinity to HRE-DNA of MMTV-LTR and uteroglobin genes, respectively, whatever they are liganded with an agonist, an antagonist or hormone-free We
have
biological labeled
compared
properties
with
0rg2058.
also
potent
The
PR-steroid
(15,30).
of
PR
were
of
the
labeled with
agonists,
hydrodynamic
complexes
some
mainly
parameters similar,
the of
physicochemical RU486
to those
synthetic the
regardless
s
~ 7.7 nm).
coefficient
STEROIDS 53/1-2
The
small
values
of
difference
the
agonist-
January-February 1989
concerning
of PR
R5020
and
non-transformed of
receptor was bound with agonists or the antagonist R
and
the
whether
the
(S20 ' w ~ 8.7; sedimentation
or antagonist-labeled
4S PR
16
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
complexes does not appear significatANT. Hormone binding was more stable
after
thermal
challenge
when
RU486 than when it bound an agonist
the
site
was
occupied
by
(Figure 3). Such stabiliza-
tion effect has been recently noted for the RU486-GR complexes in rat thymus cytosol
(31).
Rabbit uterine PR has been shown to form a 8S heterooligomer complex
in association with a 90-kD non-hormone-binding
protein
(4-6). This protein is likely identical to that identified in the non-transformed forms of the chick oviduct steroid receptors i.e., a non-DNA binding In
conclusion,
aspects
about
receptor.
the
First,
(32) heat shock protein hsp90
our
studies
interaction
point
between
out an
(I),
(2,3).
several
important
antagonist
and
the
RU486 is the ligand of choice to stabilize the
hormone binding site of purified preparations of non-transformed and/or transformed PR; second, in vitro experiments at 2 C and i0 C,
RU486
slows down the dissociation
of the 90-kD protein
from
the receptor protein, thus impairing DNA binding of the receptor. Consequently, promote unit,
contrary
receptor in
the
the
agonists
transformation
rabbit
non-DNA-binding,
to
uterus
and
system,
non-transformed
(R5020,
release RU486
of
0rg2058), the
which
120-kD
stabilizes
the
that hsp 90 may be a protein
modulator,
8S
form of the receptor and could
prevent its interaction with the hormone-responsive machinery. follows
PR
It
acting
as a transcription
which itself does not bind to DNA
(Figure 4B), masks
STEROIDS 53/1-2
January-February 1989
Re'noir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
the DNA-binding tor form the
is
not
progesterone
Whether
this
mechanism
proven.
Work
with
receptor
and
a
elements, DNA.
We
RU486-PR but are
receptor-hsp
complexes
fail
to
reporter
currently 90
which
regulate
at
gene
in vivo,
co-transfected (33?)
was
at
with
published
RU486 favors the formation of binding
to
transcription
examining
complexes
is operative
COS-cells
during the revision of this paper; abortive
unit in the 8S-recep-
(see review in (II)), thus precluding the DNA binding of
receptor.
37 C,
site of the hormone-binding
17
the
hormone after
properties
physiological
ionic
of
response
binding the
to
RU486-
strenght
and
temperature.
ACKNOWLEDGMENTS We thank F. Boussac, C. Legris, L. Outin, and J.C. Lambert for the preparation of the manuscript. We also thank D. Philibert and Roussel-Uclaf for the generous gift of radioactive R5020 and RU486. This work was partly supported by INSERM, CNRS, and Universit~ Paris-Sud (AI 86-8607). NOTES For reading convenience, "8S" although sedimentation coefficient been attributed to this form.
is the non-transformed PR, values between 7 and I0 have
%/~bbreviations RU486: [17~-hydroxy-ll~-~4-dimethylaminophenyl)-17a-(propynyl-l)estra~4,9-dien-3-one]; [ H]Org205~: 16u-ethyl-2l-hydro~y-19-nor[6,7--H]pregn-4-ene-3,20-dione; [-H]R5020: 17,21-([17--H]methyl)dimethylpregna-4,9(10)-diene-3,20-dione; NADAc-Sepharose: N-12amino-3-oxo-4-androstene-17~-carboxamide; SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis; P: progesterone; PR: progesterone receptor; GR: glucocorticosteroid receptor.
STEROIDS 53/1-2
January-February 1989
"~:
~
F,~IFERENCES ].
2.
3.
4.
5.
6.
7.
8.
9.
I0.
ii.
Joab I, Radanyz C, Renoi~ J~l, Buchou T, Catelli MG, Binart N, Mester J, and Baulieu EE (1984). Common non-hormone binding component in non-transformed chick oviduct receptors of four steroid hormones. NATURE 308: 850-853. Catelli MG, Binart N, Jung-Testas I, Renoir JM, Baulieu EE, Feramisco JP, and Welch WJ (1985). The common 90-kd protein component of non-transformed "8S" steroid receptors is a heat-shock protein. EMBO J 4: 3131-3135. Sanchez ER, Toft DO, Schlesinger MJ, and Pratt WB (1985). Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein. J BIOL CHEM 260: 12398-12401. Tai PKK and Faber LE (1985). Isolation of dissimilar components of the 8.5S nonactivated uterine progestin receptor. CAN J BIOCHEM CELL BIOL 63: 41-49. Tai PKK, Maeda Y, Nakao K, Wakim, NG, Duhring JL, and Faber LE (1986). A 59-kilodalton protein associated with progestin, estrogen, androgen and glucocorticoid receptors. BIOCHEMISTRY 25: 5269-5275. Renoir JM, Buchou T, and Baulieu, EE (1986). Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor. BIOCHEMISTRY 25: 6405-6413. Moudgil VK, Eessalu TE, Buchou T, Renoir JM, Mester J, and Baulieu EE (1985). Transformation of chick oviduct progesterone receptor in vitro: effects of hormone, salt, heat, and adenosine triphosphate. ENDOCRINOLOGY 116: 1267-1274. Yang CR, Mester J, Wolfson A, Renoir JM, and Baulieu EE (1982). Activation of the chick oviduct progesterone receptor by heparin in the presence or absence of hormone. BIOCHEM J 208: 399-406. Yang CR, Ye XM, and Zhang YD (1986). Transformation of chick oviduct progesterone receptor in the absence of hormone. J STER BIOCHEM 24: 215-218. Higgins SJ, Rousseau GG, Baxter JD, and Tomkins GM (1973). Early events in glucocorticoid action. Activation of steroid receptor and its subsequent specific nuclear binding studied in a cell free system. J BIOL CHEM 248: 5866-5872. Baulieu EE (1987). Steroid hormone antagonists at the receptor level: a role for the heat-shock protein MW 90,000 (hsp 90). J CELL BIOCHEM 35: 161-174.
STEROIDS 53/%2
January-February 1989
Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
~2.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
19
Mi~rahi M, Atger M, d'Auriol L, Loosfelt H, Meriel C, Fridlansky F, Guiochon-Mantel A, Galibert F, and Milgrom E (1987). Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA. BIOCHEM BIOPHYS RES COMMUN 143: 740-748. Herrmann W, Wyss R, Riondel A, Philibert D, Teutsch G, Sakiz E, and Baulieu EE (1982). Effet d'un steroide anti-progesterone chez la femme: interruption du cycle menstruel et de la grossesse au debut. CR ACAD SCI (PARIS) 294: 933-938. Baulieu EE and Segal SJ (1985). The Anti~rogestin Steroid RU 486 and Human Fertility Control (Baulieu EE and Segal SJ, eds), Plenum Press, New York. Bailly A, LePage C, Rauch M, and Milgrom E (1986). Sequencespecific DNA binding of the progesterone receptor to the uteroglobin gene: effects of hormone, antihormone and receptor phosphorylation. EMBO J 5: 3235-3241. Chen CLC, Chang CC, Bardin CW, and J~nne OA (1984). Inhibition of ~ t e r o g l o b i n gene expression by an antiprogestin, ~n RU486. 7 International Congress of Endocrinology, Quebec, Canada, abstr. 519, Excerpta Medica, Amsterdam. Gaillard RC, Riondel A, Muller MF, Herrmann W and Baulieu EE (1984). RU 486: a steroid with antiglucocorticosteroid activity that only disinhibits the human pituitary adrenal system at a specific time of day. PROC NATL ACAD SCI USA 81: 3879-3882. Groyer A, Schweizer-Groyer G, Cadepond F, Mariller M, and Baulieu EE (1987). Antiglucocorticosteroid effects suggest why steroid hormone is required for receptors to bind DNA in vivo but not in vitro. NATURE 328: 624-626. Renoir JM, Buchou T, Mester J, Radanyi C, and Baulieu EE (1984). Oligomeric structure of the molybdate-stabilized, non- transformed 8S progesterone receptor from chicken oviduct cytosol. BIOCHEMISTRY 23: 6016-6023. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. NATURE 227: 680-685. Wray W, Boulikas T, Wray VP, and Hancock R (1981). Silver staining of proteins in polyacrylamide gels. ANAL BIOCHEM 118: 197-203. Renoir JM, Yang CR, Formstecher P, Lustenberger P, Wolfson A, Redeuilh G, Mester J, Richard-Foy H, and Baulieu EE (1982). Progesterone receptor from chick oviduct: purification of molybdate-stabilized form and preliminary characterization. EUR J BIOCHEM 127: 71-79. Schaffner W, and Weissmann C (1973). A rapid, sensitive and specific method for the determination of protein in dilute solution. ANAL BIOCHEM 56: 502-514.
STEROIDS 53/1-2
January-February 1989
20
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
Logeat F, Pamphile R, Loosfelt H, Jolivet A, Fournier A, a n ~ Milgrom E (1985). One step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit. BIOCHEMISTRY 24: 1029-1035. Loosfelt H, Atger M, Misrahi M, Guiochon-Mantel A, Meriel C, Logeat F, Benarous R, and Milgrom E (1986). Cloning and sequence analysis of rabbit progesterone-receptor complementary DNA. PROC NATL ACAD SCI USA 83: 9045-9049. Moudgil VK and Hurd C (1987). Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486. BIOCHEMISTRY 26: 4993-5001. Sablonniere B, Danze PM, Formstecher P, Lefebvre P, and Dautrevaux M (1986). Physical characterization of the activated and non-activated forms of the gluco~orticoidreceptor complex bound to the steroid antagonist [~H] RU486. J STER BIOCHEM 25: 605-614. Rauch M, Loosfelt H, Philibert D, and Milgrom E (1985). Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effets of RU486 on the progesterone receptor and on the expression of the uteroglobin gene. EUR J BIOCHEM 148: 213-218. Bourgeois S, Pfahl M, and Baulieu EE (1984). DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU 486. EMBO J 3: 751-755. Willmann T and Beato M (1986). Steroid-free glucocorticoid receptor binds specifically to mouse mammary tumor virus DNA. NATURE 324: 688-691. Bell PA and Weatherill PJ (1986). Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU 38486 with glucocorticoid receptors in vitro and the role of molybdate. J STER BIOCHEM 25: 473-481. Renoir JM and Baulieu EE (1985). Structure du recepteur de la progesterone de l'oviducte de poule. La proteine B de ii0 kDa liant l'hormone a de l'affinite pour I'ADN. CR ACAD SCI (PARIS) 301: 859-864. Guiochon-Mantel A, Loosfelt H, Ragot H, Bailly A, Atger M, Misrahi M, Perricaudet M, and Milgrom E (1988) Receptors bound to antiprogestin form abortive complexes with hormone responsive elements. NATURE 336: 695-698.
STEROIDS 53/1-2
January-February 1989
ABSTRACT The salt-induced (0.3 M KCI) transformation of the nontransformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor ( P R ) was analyzed by density gradient ultracentrifugation (8S ~ 4S conversion) and DNAcellulose chromatography (non-binding ~ binding forms). After 1 h treatment at 2 C, > 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist-and antagonist-bound non-transformed receptors were similar and the calculated Mr were ~ 283,000 and 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.
STEROIDS 53/1-2
January-Eebruary 1989 (1-20)
1
2
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
INTRODUCTION Steroid target
cell
form :
they
receptor
receptors, cytosol are
in
in a
absence
hormone,
non-transformed,
heterooligomers
molecule
of
and the heat
associated
with
hspg0
including
a
shock protein
(4-6).
found
non-DNA-binding
(1-3). The rabbit uterus cytosol progesterone also
are
Steroid
in 8S-
hormone-binding
of
90 kD
receptor
hormones
(hsp90) (PR) t is
and
their
synthetic agonists accelerate the temperature-dependent dissociation of the heterooligomer hormone
complexes
regulatory
can
elements
(7-9), and the released 4S receptor-
then
bind
(i0,Ii
for
to
appropriate
review)
and
DNA
trigger
hormone
transcrip-
tional events. The rabbit progesterone receptor, showing structurally much homology with the human progesterone receptor
(12), is
an excellent system to study the mechanism of action of RU486, an antiprogesterone
already
control
RU486 binds
(13,14).
successfully
receptor with high affinity gesterone
(15,16).
Already,
antiglucocorticosteroid glucocorticosteroid
to the
tested
in human
rabbit uterus
fertility
progesterone
(Kd ~ 0.2 nM), and it is an antiproRU486
(14,17)
8S receptor
which
has been (GR) form
is
also
a
powerful
shown to stabilize in the
chick
the
oviduct
(18). Experiments with the powerful progesterone agonists, R5020 and Org2058,
and the antagonist RU486 were performed in order to
compare the stability of the rabbit uterus 8S PR when binding an agonist or an antagonist. Ultracentrifugation in sucrose gradient
STEROIDS 53/1-2
January-February 1989
Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
and DNA-cellulose transformation of hsp90
3
chromatography were used in order to assess the
of the 8S to 4S form, which implies the separation
from the receptor protein,
and the DNA binding
ability
of the latter whether binding or not an agonist or an antagonist steroid.
MATERIALS AND METHODS Chemicals. [2,4,6,7-3H]~ogesterone ([3H]p, 82.7 Ci/mmol) and the synthetic progestin [-H]Org2058 (45 Ci/mmol) _ were from the Radiochemica~ Centre (Amersham, Bucks, UK). [~H]R5020 (50 Ci/ mmol) and ['H]RU486 (50 Ci/mmol) were a gift from Roussel-Uclaf (Romainville, France). Non-radioactive cortisol and progesterone (P) were obtained from Roussel-Uclaf. DEAE-Sephacel was from Pharmacia (Uppsala, Sweden), and DNA-cellulose (1.38 mg DNA/mL packed gel) and cellulose were obtained from P.L. Biochemicals, Inc. (Milwaukee, wi). All other reactants were from Merck (Darmstadt, West Germany), except when noted. Buffers. Buffer A included I0 mM potassium phosphate, 1.5 mM EDTA, 10% (v/v) glycerol, and 12 mM l-thioglycerol, pH 7.8; Na2MoO 4 (20 mM) was added to buffer A to make buffer B. Rabbit uterus cytosol. I~mature New Zealand rabbits were injected with diethylstilbestrol and the uterine cytosol was prepared as described (6), with the addition of PMSF (phenylmethylsulfonyl fluoride, Calbiochem, 0.3 mM) and leupeptin (Sigma, 20 ~M final concentration) as protease inhibitors. Non-radioactive cortisol (2 ~LM) was systematically added to cytosol to prevent the binding of progestins to contaminating transcortin and glucocorticosteroid receptor. P~ogesterone receptor binding assa[. Incubations of cytosol with [-H]steroid (20 ruM) were performed during 2.5 h at 0-2 C, in the presence or absence of molybdate. Non-specific binding was measured in parallel after addition of 2 ~M of non-radioactive progesterone. Dextran(0.025%)-charcoal (0.25%) adsorption (10 min at 0 C) was used to assay protein-bound steroid (see 19). Receptor-bound ligand was determined by subtraction of nonspecific from total binding.
STEROIDS 53/1-2
January-February 1989
4
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
DNA-cellulose assay. DNA-cellulose or unsubstituted cellulose was first pelleted in 0.2 mL aliquots in 5 mL polypropylen~ tubes after equilibration in buffer A. Triplicate samples of [-H]steroid-labeled cytosol were incubated 1 h at 0 C with DNA-cellulose slurry under gentle shaking. It was verified that the equilibrium was attained for each agonist-or antagonist-PR complex. The suspension was then centrifuged 5 min at 700xg, the supernatant discarded, and the pellet washed with 3 x 1 mL of buffer A. The pellets were resuspended in 7 mL of scintillation mixture (Readysolv, Beckman Instruments), and counted. Specific binding to DNA-cellulose was calculated after subtraction of radioactivity bound to cellulose. Density gradient ultracentrifugation. Samples (0.2 mL) were layered onto preformed discontinuous sucrose gradients (5-20%) made in buffer A or B. Centrifugation was performed during 16 h x 46,000 rpm at 2 C, in an SW60 Beckman rotor, with internal markers (glucose oxidase, S20,W = 7.9 and peroxidase S20,W = 3.6). High performance liquid chromato@raphy (HPLC) analysis. analyses were performed as previously described (6).
HPLC
SDS-slab gel electrophoresis and staining. Proteins were resolved in 7.5% SDS polyacrylamide gels according to the method of Laemmli (20). The protein bands were stained with silver nitrate as described in (21). Purification of PR. The protocol described earlier (6) was used. Briefly, cytosol from estrogen stimulated uteri was prepared in molybdate ions containing buffer to stabilize the non-transformed PR, and transferred at 4 C on the top of NADAc-Sepharose affinity g~l (22). Afte{ washing and biospecific el~tion with either [ H]R5020 or [-H]RU486 (i ~M), the eluted [ H]steroid-PR complexes were loaded onto two DEAE-Sephacel columns (I mL) equilibrated in buffer B. Non-transformed PR complexes bound to the ion exchange gel, whereas transformed PR complexes did not. Under these conditions the former elutes at 0.15 M KCI during the salt gradient, and with ~ 20% purity (calculated on the basis of specific activity, assuming one steroid binding site per protein molecule of i00 kD), whether the receptor was labeled with agonist or antagonist. Each flowthrough was applied to DNAcellulose columns (25 mL). The transformed PR was eluted at 0.32 M KCI with ~ 65% purity. Transformation studies of purified 8S-PR by DNA-ce!lulose chromatography. The DEAE-Sephacel eluates were diluted 5-fold in buffer
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
5
A and loaded on i0 mL DNA-cellulose column equilibrated with the same buffer (flow-rate 30-35 mL/h). After washing with 40 mL of buffer A, a 50 mL linear 0-0.5 M KCI gradient was applied. Fractions of 9 mL were collected during sample adsorption to the column, 4 mL during the washing, and 1 mL during the 0-0.5 M KCI gradient. Aliquots of each fraction (200 ~L) were counted for radioactivity and 0.5-mL aliquots were boiled in 1% SDS for analysis by SDS-PAGE. Protein determination and radioactivity counting. Protein concentration of PR containing samples was determined according to the method described (23) and radioactivity was measured in a Minaxi Scintillation counter (Packard) after addition of 7 mL Toluene scintillator (Packard) to radioactive samples.
RESULTS
Comparison
of
the
rate
of
salt-induced
transformation
of
[3H]-
RU486-PR and [3H]a~onist-PR complexes In
the
agonist-PR cytosol
absence
of
molybdate,
transformation
of
[3H]-
complexes was almost complete after 1 h treatment of
with
0.3 M
KCl
(at
2
C).
This
was
indicated
radioactivity in the 4-5S region of the density res IA and 2A, see also Table
gradient
by
the
(Figu-
I). After longer exposure to salt
(> 2 h), the sedimentation coefficient of the transformed PR was 3S
(not shown), suggesting a cleavage of the receptor in spite
of the presence of protease inhibitors. When RU486 was employed, exposure initial
to
0.3 M
8S peak
KCI
during
(Figure
IB).
1 h
at
I0 C,
Even after
did
not
effect
72 h salt treatment
the at
2 C, ~ 90% of [3H]RU486-PR complexes still sedimented in 8S-form (Figure 2B). To achieve complete transformation, it was then
STEROIDS 53/1-2
January-February 1989
6
Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
GO
pO
G O 1C-
pO
B o
b
u
8 ~
c3
5
Z
10 Fraction
20 Fraction
No
N~
Figure i. Salt-induced t r a n s f o r m a t i o n of the rabbit uterine PR. Effect of the ligand. Cytosol was p r e p a r e d in b u f f e r A and one p~rt was incubated for 2 h at 2 C with 20 nM [ H]Org2058 or [-H]RU486; another p a r t was kept at 2 C w i t h o u t ligand. 0.3 M KCI was added to each cytosol at 2 C. The t r a n s f o r m a t i o n p r o c e s s was b l o c k e d by adding 20 m M sodium m o l y b d a t e to each sample after 1 or 2 h panel C KCl treatment, and aliquots (200 ~L) were immediately loaded on top of p r e f o r m e d 5-20% sucrose gradients in b u f f e r B containing 0.3 M KCI. P o s i t i o n of internal standards is indicated by arrows. IA: sedimentation profile of [ H]Org2058-PR treated (e---O) o ~ not (o o) for 1 h with salt. IB: sedimentation profile of [ H ] R U 4 8 6 - P R treated (~ &) or not (o o) for 1 h, or treated 6 h (x x), with 0.3 M KCI at I0 C. necessary
to
treatment.
For
was
necessary
transformation exposure previously
to
increase
both
example,
exposure
to
effect
was
therefore
0.3 M
exposed
KCI
the
temperature to
0.3 M KCI
transformation obtained
(Table
I)
or
at 6 h
and
the
for
6 h
(Figure I0
C
time at
IB).
either
treatment
i0 C,
Complete after
of
8 h
cytosol
to 0.3 M KCl for one night at 2 C (Figure
STEROIDS 53/1-2
of
2C).
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
B
A
IO0
0 " - - - 0 ~
~o
30
60
C
~,,,~u486
1
90
T,me(mln)
RU 486
2C
0
50
30
°
70
T~me( hours)
D 1000~,~
1~
=~
7
O ~
10 C
°
~
~~ N
2
4 Time (hours)
RU486
O~
o 10 20 Temperature (C)
Figure 2. R a t e of transformation of the rabbit uterine PR. Cytosol was prepared in buffer A and PR w a ~ i n c u b a t e d for 2 h at 2 C with 20 nM [~H]R5020 (Figure 2A) or [ H]RU486 (Figure 2B). Transformation was induced by adding 0.3 M KCI at 2 C. At different times, 20 mM molybdate was added to prevent further transformation, and aliquots (i00 ~L) were loaded on the top of 5-20% sucrose gradients containing 20 mM molybdate and 0.3 M KCI. At zero time, all the radioactivity migrated at ~ 8S. The percent of PR recovered as non-transformed, 8S (o o), or transformed, 4S (e----e), was plotted versus the time of KCl treatment prior to molybdate addition. Two experiments are p r e s e n t ~ for each time studied (Figure 2A). 2B: Same experiment with [-H]RU486-PR. 2C: Same experiment as in Figure 2B, but the sample in which transformation was induced with salt at 2 C, was kept as such overnight and then set at i0 C instead of 2 C. The loss of RU486 binding activity after 6 h was 15% of the initial qtime zero) binding activity. 2D: Percent of remaining 8S [-H]RU486-PR obtained after 2h exposure to 0.3 M KCl at different temperatures. The loss of RU486 binding activity was 15, 18, and 22% for i0, 20, and 25 C incubation temperatures, respectively.
STEROIDS 53/1-2
January-February 1989
8
At
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
25°C,
the 8S peak was
(Figure
2D),
but
loss
lost
of
after
2 h treatment
hormone
binding
we deduced
that
with
(~ ].5%) was
0.3 M KCI concomi-
tantly observed. From of
the
8S
conditions
these PR
results, into
a
for both
transformation
4-5S
form
can
agonist-and
occurs m u c h more
TABLE i. H y d r o d y n a m i c complexes.
be
complete obtained
[ 3H ]R5020
s, nm
7.7 + 0.3 n=3
[3H]Org2058
7.7 + 0.3 n=3
[ 3H ]RU486
7.8 + 0.2 n=4
under
antagonist-labeled
PR,
parameters
$20,
of
agonist-and
vitro
but
that
antagonist-
PR
Transformed R
W
8.7 + 0.3
s,nm
n.d.
n=5
S20,w
4.5 + 0.5 a n=5
8.7 + 0.3
n.d.
n=8
4.5 + 0.4 a n=8
8.9 + 0.3 n=4
in
slowly w i t h the latter.
Non-transformed R
transformation
5.6 + 0.5 b n=4
5.0 + 0.5 b n=4
n.d.: not determined due to loss of binding during HPLC. a . . . . . T r a n ~ f o r m a t l o n obtalned by i n c u b a t l o n for 2 h wlth 0.3 M KCI at 2 C. T r a n s f o r m a t i o n obtained after 8 h exposure to 0.3 M KCI at i0 C.
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
9
Effect of steroids on the thermal inactivation of
PR
Both molybdate-stabilized PR and salt-transformed at 0 C) PR were
labeled with either
[3H]R5020
or
(overnight
[3H]RU486
and
kept at 0, I0, and 25 C during 24 h and 18 h, respectively.
The
results show that [3H]RU486-PR complexes are more stable, presence
of
R5020-PR
complexes
were
less
either
stable
molybdate (Figure
than
3).
or
KCI,
than
the
Salt-treated
molybdate-stabilized
in the
corresponding
RU486-PR RU486-PR
complexes complexes,
but more than KCl-treated R5020-PR species.
Plus KCI 18h
Plus Molybdate 24h
[] [~R~o
100
m ~u4e6
OC
10C
1 25C
C
10C
25C
Figure 3. Thermal stability of non-transformed and transformed PR-steroid complexes. Cytosol was prepared in buffer A and divided into two parts. One part was supplemented with 20 m M sodium molybdate, the other one with 0.3 M KCI. The cytosols were kept o v e r n i g ~ at 0 C and ~-mL aliquots were then incubated 2.5 h w~th 20 nM [-H]R5020 or [--H]RU486. The percentage of remaining [-H]steroid binding was measured after 2.5 h incubation with each ligand (time 0, 100%), and after storage for 24 h (plus molybdate) or 18 h (plus KCl), at 0, i0, and 25 C, as indicated on the abscissa.
STEROIDS 53/1-2
January-February 1989
10
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
Hydrodynamic
parameters
of non-transforme d and transformed
[3H]-
steroid-PR complexes Sedimentation non-transformed agonists
in Table i. An
non-transformed
parameters.
values
and
Stokes
radii
of both
and transformed PR complexes liganded with either
(R5020 or Org2058)
summarized for
coefficient
The
or antagonist apparent
[3H]RU486-PR
apparent
Mr
of
Mr
(RU486)
~ 293,000
complexes
steroids, was
from
[3H]agonist-PR
are
calculated
hydrodynamic complexes
was
283,000. After
salt-induced
the calculated previous
analysis, s
obtained
after
(see legend 118,000
SDS-PAGE
of Table
in agreement
analysis
(4,6,24)
I), with and
(25). It was not possible to measure the apparent Mr
of transformed
with R
Mr of [3H]RU486-PR was
values
cloning data
transformation
[3H]agonist-PR
due to loss of binding during HPLC
the latter giving a broad radioactive peak
(not shown)
smaller than 4 nm.
DNA-cellulose binding of transformed PR Agonist-labeled formed
PR complexes
PR was assayed for DNA binding. were
other ligand-transformed may
reflect
assay,
since
the
found
PR complexes
dissociation
kinetic
to bind
DNA-cellulose
less
than
(Table 2). This difference
of
[3H]P-PR
complexes
experiments
revealed
that
was shorter than that of either
[3H]P-trans-
synthetic
during
their
[3H]agonists
STEROIDS 53/I-2
the
half-life or [3H]-
January-February 1989
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
RU486-PR reason, nists.
complexes, most
even
at
experiments
Antagonist
0-2
were
C
(data
carried
[3H]RU486-transformed
11
not
out
shown).
with
PR
For
this
synthetic
ago-
complexes
bound
to
DNA-cellulose to the same extent as PR liganded with the agonist. We concluded that transformed PR binds similarly to DNA, whatever it is liganded with an agonist or the RU486 antagonist. tested
the
stabilized
binding
to
DNA-cellulose
non-transformed
PR,
but,
at
20
whatever
C the
of
We also
molybdate-
nature
of the
ligand, no binding was observed.
TABLE 2. DNA-cellulose binding of transformed PR bound to either agonist or RU486
ligand
incubated 4S-PR cpm
bound 4S-PR cpm (mean + SD)
% binding
P
22,750
8,645 + 907
38
R5020
13,748
5,534 + 638
40
Org2058
12,380
6,716 + 298
54
RU486
13,750
7,837 + 417
57
Rabbit uterus cytosol was prepared in buffer A, then supplemented with 0.3 M KCI final concentration for 2 h at ~ C. A%iquots were incubated fo~ 2 h at 0 C with 20 nM [~H]P, [~H]R5020, [~H]Org2058, or [ H]RU486. Aliquots of each incubate were then diluted 6-fold in buffer A ~and added to 200 ~L of pelleted DNA-cellulose (0.~5 pmol of [JH]ligand-PR complex) in triplicate. DNA-cellulose-[ H]ligand-PR complexes were treated as mentioned in Materials and Methods.
STEROIDS 5311-2
January-February 1989
!2
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
Transformation of purified 8S PR complexes Molybdate-stabilized, complexes
were
complexes
analyzed
purified
agonist-labeled
PR
bioaffinity in
parallel
similarly. contained,
with
The in
purified
[3H]RU486-PR
[3H]R5020-1abeled
purified
addition
to
PR
non-transformed hormone
binding
units of ~ 120 and ~ 85 kD, an abundant non-hormone binding 90-kD protein kD)
(Figure 4A,
lane 2). Other protein bands
represent contaminants as already described
purified non-transformed retic pattern
(at 66 and 45
(6). Analysis of
[3H]RU486-PR gave a similar electropho-
(Figure 4A,
lane I). During purification,
the continuous presence of molybdate,
even in
a portion of PR is trans-
formed to a DNA-binding form unable to bind to DEAE-Sephacel During
purification
of
[3H]RU486-1abeled
8S
PR,
the
(6).
amount
of
transformed receptor represented only one-tenth of that obtained in the case of the [3H]R5020-1abeled different purifications),
8S PR
(data obtained
in 5
as visualized on Figure 4A, lanes 3 and
4. This appears to reflect an intrinsic difference in the rate of transformation after labeling the receptor with agonist or RU486, and thus confirms the results obtained with cytosol preparations (see Figures 1 and 2). In one
series
of
experiments,
DEAE-Sephacel
transformed [3H]R5020 and [3H]RU486-PR complexes
purified
non-
(purity ~ 22% in
both cases) were diluted with buffer A, incuba~ted for 20 min and 2 h, respectively at 20 C (optimum conditions to achieve complete
STEROIDS 53/1-2
January-February 1989
Renoir et al: RABBIT PROGESTERONERECEPTORAND RU486
A 212
1 2
3
13
B
4
1 2
~
3
/
i
130 ~ _
//. /
_130
~ o
~-
97__ ~ W o
66 ~
-4s
////
_3o
_97 .3-
/////
-6e
45~
//"
4
--212 --212
3 o
66
//
--45
2
O0
20
40
60
80
Fraction number
Figure 4. Dissociation of hsp90 from the hormone binding protein upon activation of PR. 4A: SDS-PAGE of purified PR. 3 Lane % and 2: non-transformed [ H]RU486-PR (1.4 ~g of protein) and [-H]R5020-PR (1.4 ~g of protein), respectively eluted from the DEAE-Sephacel column, purity ~ 22% in both cases (6), calculated on specific activity basis according to one m o l e c ~ e of steroid bound per I00,000 Mr protein; lane 3: activated [ H]RU486-PR (i00 ng of protein) eluted from the DNA-cellulose column loaded with 3the DEAE-Sephacel flow-through fractions; lane 4: activated [ H]R5020-PR (300 ng of protein) eluted from the DNA-cellulose column. The arrows indicate the migration of marker proteins. 4B: Activation of purified non-transformed PR. Purified rabbit uterine 8S PR labeled either with [[H]R5020 (o o; 150 ~g of protein, 32 pmol of bound steroid) or [~H]RU486 (e----o; 155 ~g protein, 38 pmol of bound steroid) was diluted in buffer A, treated as indicated in the text and loaded in parallel onto two i0 mL columns of DNA-cellulose equilibrated in buffer A. Lanes 1 and 2: 400 ng of protein o~ the flow through fractions of the DNA-cellulose columns for [--H]R5020- and [ H]RU486-bound receptors, respectively; lane 3 : 1 5 0 ng of protein from the top of the [-H]R5020 peak of the DNA-cel~ulose column; lane 4: 200 ng of protein from the top of the [ H]RU486 peak. Position of marker proteins is indicated by arrows. transformation of purified PR), and then loaded on the top of two DNA-cellulose proteins
STEROIDS 53/1-2
columns. As shown in Figure 4B, the hormone binding
(~ 120
and
~ 85 kD)
January-February1989
were
together
eluted
as a
single
14
Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
radioactive DNA
p e a k at 0.32 M KCI,
of both
sucrose
RU486
gradient
DNA-cellulose with
or
protein,
R5020-PR
migrated sample
originally
columns,
at
~
was
in
the
for
Ultracentrifugation
in
R5020
both flow
affinities
similar
the run
in
similar
[3H]RU486-PR
but
with
during
present
found
indicating
4.5S,
labeled
PR inactivation
structures,
complexes.
of the top of p u r i f i e d
the parallel
to complete
suggesting
eluted
experience
was
made
unsuccessful
(not shown).
purified through
from
due
The 90-kD
non-transformed fractions
of
the
that it does not bind to DNA.
DISCUSSION The
present
report
vitro,
the
salt-induced
(from
the
non-DNA
DNA-binding,
indicates
transformation
binding,
monomeric
4S
In
RU486-PR
cell-free
complexes
form)
R5020
obtained
a
after
binds
gonist-PR ionic
to
non-specific
complexes
strength
are
eluted
as
agonist-PR
identical
apparent
affinities
rat
4S-GR
liver
labeled
DNA
with
to
the
slowly
than
2) or 0rg2058
(not
transformation to
0.3 M KCI
antagonist-bound
DNA-cellulose (Figure
for
Anta-
the
same
4B),
suggesting
DNA.
Similarly,
triamcinolone
STEROIDS 53/I-2
at
of
receptor
DNA-cellulose).
non-specific
either
in
complexes
form
more
exposure
complexes for
8S
much
complete
(i.e.,
from
temperature
RU486-PR
(Figure
8 h at i0 C or 2 h at 25 C. T r a n s f o r m e d readily
low
of the
occurred
experiments,
is
at
heterooligomeric
with PR labeled with the agonist shown).
that,
acetonide
or
January-February 1989
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
15
RU486 binds with close affinities to DNA-cellulose
(27). However,
affinity for DNA-cellulose of the rabbit uterine PR complexed to RU486 is lower than that of R5020-PR complexes
(28) after activa-
tion for 15 min at 20 C. From
our
results,
such
conditions
certainly
activate
agonist-PR complexes, but they lead to only partial activation of antagonist-PR
complexes
(~ 15%
of
the
RU486-PR
complexes
are
still in 8S form after 2 h exposure to 0.3 M KCI at 20 C; Figure 2D). Insufficient activation also might explain results obtained with RU486 GR-complexes to
4S
forms
needs
(29). Transformation of receptor from 8S
to be
carefully
checked before
carrying
out
further studies with transformed species. Indeed, our results are in good agreement with recent findings, indicating that GR and PR bind with similar affinity to HRE-DNA of MMTV-LTR and uteroglobin genes, respectively, whatever they are liganded with an agonist, an antagonist or hormone-free We
have
biological labeled
compared
properties
with
0rg2058.
also
potent
The
PR-steroid
(15,30).
of
PR
were
of
the
labeled with
agonists,
hydrodynamic
complexes
some
mainly
parameters similar,
the of
physicochemical RU486
to those
synthetic the
regardless
s
~ 7.7 nm).
coefficient
STEROIDS 53/1-2
The
small
values
of
difference
the
agonist-
January-February 1989
concerning
of PR
R5020
and
non-transformed of
receptor was bound with agonists or the antagonist R
and
the
whether
the
(S20 ' w ~ 8.7; sedimentation
or antagonist-labeled
4S PR
16
Renoir et al: RABBIT PROGESTERONE RECEPTOR AND RU486
complexes does not appear significatANT. Hormone binding was more stable
after
thermal
challenge
when
RU486 than when it bound an agonist
the
site
was
occupied
by
(Figure 3). Such stabiliza-
tion effect has been recently noted for the RU486-GR complexes in rat thymus cytosol
(31).
Rabbit uterine PR has been shown to form a 8S heterooligomer complex
in association with a 90-kD non-hormone-binding
protein
(4-6). This protein is likely identical to that identified in the non-transformed forms of the chick oviduct steroid receptors i.e., a non-DNA binding In
conclusion,
aspects
about
receptor.
the
First,
(32) heat shock protein hsp90
our
studies
interaction
point
between
out an
(I),
(2,3).
several
important
antagonist
and
the
RU486 is the ligand of choice to stabilize the
hormone binding site of purified preparations of non-transformed and/or transformed PR; second, in vitro experiments at 2 C and i0 C,
RU486
slows down the dissociation
of the 90-kD protein
from
the receptor protein, thus impairing DNA binding of the receptor. Consequently, promote unit,
contrary
receptor in
the
the
agonists
transformation
rabbit
non-DNA-binding,
to
uterus
and
system,
non-transformed
(R5020,
release RU486
of
0rg2058), the
which
120-kD
stabilizes
the
that hsp 90 may be a protein
modulator,
8S
form of the receptor and could
prevent its interaction with the hormone-responsive machinery. follows
PR
It
acting
as a transcription
which itself does not bind to DNA
(Figure 4B), masks
STEROIDS 53/1-2
January-February 1989
Re'noir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
the DNA-binding tor form the
is
not
progesterone
Whether
this
mechanism
proven.
Work
with
receptor
and
a
elements, DNA.
We
RU486-PR but are
receptor-hsp
complexes
fail
to
reporter
currently 90
which
regulate
at
gene
in vivo,
co-transfected (33?)
was
at
with
published
RU486 favors the formation of binding
to
transcription
examining
complexes
is operative
COS-cells
during the revision of this paper; abortive
unit in the 8S-recep-
(see review in (II)), thus precluding the DNA binding of
receptor.
37 C,
site of the hormone-binding
17
the
hormone after
properties
physiological
ionic
of
response
binding the
to
RU486-
strenght
and
temperature.
ACKNOWLEDGMENTS We thank F. Boussac, C. Legris, L. Outin, and J.C. Lambert for the preparation of the manuscript. We also thank D. Philibert and Roussel-Uclaf for the generous gift of radioactive R5020 and RU486. This work was partly supported by INSERM, CNRS, and Universit~ Paris-Sud (AI 86-8607). NOTES For reading convenience, "8S" although sedimentation coefficient been attributed to this form.
is the non-transformed PR, values between 7 and I0 have
%/~bbreviations RU486: [17~-hydroxy-ll~-~4-dimethylaminophenyl)-17a-(propynyl-l)estra~4,9-dien-3-one]; [ H]Org205~: 16u-ethyl-2l-hydro~y-19-nor[6,7--H]pregn-4-ene-3,20-dione; [-H]R5020: 17,21-([17--H]methyl)dimethylpregna-4,9(10)-diene-3,20-dione; NADAc-Sepharose: N-12amino-3-oxo-4-androstene-17~-carboxamide; SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis; P: progesterone; PR: progesterone receptor; GR: glucocorticosteroid receptor.
STEROIDS 53/1-2
January-February 1989
"~:
~
F,~IFERENCES ].
2.
3.
4.
5.
6.
7.
8.
9.
I0.
ii.
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STEROIDS 53/%2
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Renoir et ah RABBIT PROGESTERONE RECEPTORAND RU486
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13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
19
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Renoir et ah RABBIT PROGESTERONE RECEPTOR AND RU486
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