Biochem. J. (1985) 230, 203-210
Printed in Great Britain
203
Immunorecognition of the active form of the oestrogen receptor by using a
monoclonal antibody
Nora GIAMBIAGI and Jorge R. PASQUALINI*
C.N.R.S. Steroid Hormone Research Unit, Foundation for Hormone Research, 26 Boulevard Brune,
75014 Paris, France
(Received 12 February 1985/15 April 1985; accepted 24 April 1985)
In previous studies, two forms (a and IJ) of the oestrogen receptor, with different
immunological characteristics, were observed in the cytosol fraction of fetal guineapig uterus, by using a monoclonal antibody to the human oestrogen receptor
(D547Spy). Only the a form was recognized by the antibody, shifting its
sedimentation coefficient in high-salt sucrose gradients. The present work
investigated the effect of several factors (time, temperature, high salt concentrations
and Na2MoO4) on the interconversion of these two forms. Only the form was
observed when cytosol was incubated with oestradiol for only 2-3h, but 20h later,
40-60% of the total oestradiol-receptor complexes were found as the a form. The
transformation from the ,B to the a form was accelerated by temperature (25°C,
15min) and exposure to high salt concentrations (0.4M-KCI). On the other hand,
Na2MoO4 completely blocked the transformation induced by time and temperature,
but had little effect on that induced by KCI. The appearance of the a form always
correlated with an increase in receptor binding to nuclei and DNA-cellulose. Finally,
it was found that the isolated ,B form, recovered from the gradient, was transformed
into the a form after overnight dialysis under reduced pressure. The present data
suggest that the a form, which is recognized by the monoclonal antibody, is the
activated form of the oestrogen receptor.
It is generally accepted that the primary action
of steroid hormones is mediated by specific highaffinity receptor proteins. On binding to the
hormone, cytosol receptors translocate to the
nuclei (Jensen et al., 1968; Giannopoulos &
Gorski, 1971), where they interact with chromatin
to modulate the transcription of specific genes
(O'Malley et al., 1976). From observations in vitro,
translocation of the steroid-receptor complexes to
the nucleus requires a change in the properties of
the cytosol receptor, which increases its affinity for
nuclear components (Jensen et al., 1972; Grody et
al., 1982). This process, referred to as 'activation',
can be achieved by various procedures, including
heating (Jensen et al., 1971), exposure to high ionic
strength (Milgrom et al., 1973; Bailly et al., 1980),
gel filtration, dilution or dialysis (Goidl et al.,
1977; Sato et al., 1979).
Previous studies in our laboratory have demonstrated the presence of specific binding sites for
* To whom
correspondence and
should be addressed.
Vol. 230
requests for reprints
oestrogens in the fetal guinea-pig uterus (Pasqualini & Nguyen, 1976; Sumida & Pasqualini, 1979).
Using a monoclonal antibody developed against
the oestrogen receptor from a human breast-cancer
cell line (Greene et al., 1980b), we have shown that
the oestrogen receptor measured in fetal uterine
cytosol is composed of two forms (a and ,B), which
can be differentiated on high-salt sucrose gradients, since only one (the a form) is bound by the
antibody, shifting its sedimentation coefficient
from 4.5S to 7.4S (Giambiagi & Pasqualini, 1982).
The present paper examines the different factors
that influence the interconversion of these two
forms of the oestrogen receptor, and the relationship between receptor activation and its binding to
the monoclonal antibody.
Experimental
Biological material
Pregnant guinea pigs of the Hartley albino strain
(55-65days of gestation) were purchased .from a
commercial breeder (C.E.S.A.L., Vigneul-sous-
N.
204
Montmedy, Meuse, France). After anaesthesia of
the mothers with diethyl ether, the fetuses were
obtained by laparotomy and the uteri removed and
stripped of fat.
Chemicals
[6,7-3H]Oestradiol (sp. radioactivity 52 Ci/
mmol) was obtained from New England Nuclear
Corp. (Frankfurt, West Germany). Radioinert
oestradiol was purchased from Steraloids (Touzart
et Matignon, Vitry-sur-Seine, France). Doublestranded DNA (calf thymus)-cellulose (containing
3.8mg of DNA/g of DNA-cellulose) was obtained
from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
The monoclonal antibody D547Spy against the
oestrogen receptor from MCF-7 human breastcancer cells was given by Dr. E. V. Jensen (Zurich,
Switzerland) and Dr. G. Greene (Chicago, IL,
U.S.A.). Buffers were: 10mM-Tris/HCl, 1.5mMEDTA, 0.5 mM-dithiothreitol, pH 7.4 (TED);
TED+0.25M-sucrose, pH7.4 (TEDS); TED+
0.4M-KCI, pH7.4 (0.4M TKED); TED+0.6MKCI, pH7.4 (0.6M TKED).
Preparation of uterine cytosol and nuclear fractions
Fetal uteri were homogenized in TEDS buffer
with a Teflon/glass Potter-Elvehjem homogenizer.
The homogenate was centrifuged at 900g for
10min. The pellet was washed twice by resuspension in TEDS buffer and centrifugation at 900g for
10min. The supernatants were pooled and centrifuged at 200000g for 30min to obtain the cytosol
fraction. The 900g pellet was washed three times
with TEDS buffer and resuspended in the same
buffer to obtain the nuclear fraction. All procedures were carried out at 4°C.
Oestradiol-receptor complex
Portions of cytosol (containing 3-4mg of protein/ml) were incubated with lOnM-[3H]oestradiol
with or without a 100-fold excess of unlabelled
oestradiol for 2h at 4°C. Unbound [3H]oestradiol
was removed by 0.05% (w/v) dextran-coated 0.5%
(w/v) charcoal (Korenman & Rao, 1968) for 10min
at 4°C, and specific binding was calculated by the
difference between binding in the absence and in
the presence of unlabelled oestradiol.
Oestradiol-receptor-monoclonal-antibody complex
Portions (100 p1) of cytosol containing the
oestrogen-receptor complex (3-4pmol/ml) were
incubated with 10,pg of the monoclonal antibody
for I h or 20h at 4°C.
Nuclear and DNA-cellulose binding
Portions (0.5 ml) of cytosol containing the
oestradiol-receptor complex were added to the
nuclear pellet (100-150 pg of DNA) or to 55mg of
Giambiagi and J. R. Pasqualini
DNA-cellulose (containing 200 pg of DNA) swollen in TED buffer and incubated for 1 h at 4°C. At
the end of the incubation, the tubes were centrifuged at 900g for 10min. Unbound complexes
were measured in the supernatant after dextrancoated-charcoal treatment. The nuclear or DNAcellulose pellets were washed three times with
TEDS buffer. To measure the nuclear-bound
complexes, nuclei were extracted with I ml of 0.6M
TKED buffer for 1 h at 4°C, centrifuged at 15000g
for 30min, and samples of the supernatant were
removed for radioactivity counting. To determine
the complexes bound to DNA-cellulose, the pellets
were suspended in 1 ml of TED buffer and samples
were removed for radioactivity counting. Nonspecific binding to nuclei or DNA-cellulose was
determined with a cytosol which had been incubated with [3H]oestradiol in the presence of a 100-fold
excess of radioinert oestradiol.
Sucrose density gradients
Portions (00op1) of dextran-coated-charcoaltreated oestradiol-receptor or oestradiol-receptormonoclonal-antibody complexes were layered on
5 ml density gradients prepared with 10-30% (w/v)
sucrose solutions in 0.4M TKED buffer and
centrifuged at 400000g for 2h in a Beckman VTi
65 vertical rotor. Fractions (110-130p1) were
collected and counted for radioactivity.
Protein and DNA assays
Protein was measured by the method of Lowry et
al. (1951), and DNA was determined by that of
Burton (1956).
Results
Effect of time and temperature on cytosolic oestrogen
receptor binding to the monoclonal antibody
Previous results have shown that after overnight
incubation at 4°C of the cytosolic oestradiolreceptor complex with the antibody, 40-60% of the
total receptors was bound to it. This fraction was
called the a form, and the remaining fraction, not
recognized by the antibody, was called the P form.
The sedimentation coefficient in high-salt sucrose
density gradients of the a-form-antibody complex
is 7.4S and that of the /3 form 4.5S (Giambiagi &
Pasqualini, 1982). To determine if the existence of
these two forms was a result of a time-dependent
process, cytosol was incubated with [3H]oestradiol
at 4°C for different periods (1 h, 4 h and 19 h) before
being incubated for 1 h with the antibody. When
analysed through high-salt sucrose gradients (Fig.
I a), no binding between receptor and antibody was
detected after the 2 h incubation; all the specific
radioactivity sedimented in the /3-form zone (4.5 S).
1985
Immunorecognition of the active form of the oestrogen receptor
205
q
1-:
0.
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0
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Fraction no.
Fig. 1. Ejfect of time and temperature on the oestrogen receptor binding to the monoclonal antibody
(a) Samples of cytosol (0.5ml) were incubated with lOmM-[3H]oestradiol for 1 h (O), 4h (A) or 19h (-)at 4°C; then
the monoclonal antibody D547Spy (10 pg/100p1 of cytosol) was added and the incubation was continued for 1 h at
4°C. (b) Samples of cytosol were incubated with lOnM-[3Hloestradiol for 2h at 4°C; then one was heated at 25°C for
15 min (-) and the other was kept at 4°C (0). After cooling, samples were re-incubated with the antibody for 1 h at
4°C. Unbound radioactivity was adsorbed with dextran-coated charcoal, and 0.1 ml of the supernatant was layered
on sucrose gradients (10-30%, w/v; 0.4M-KCI), which were centrifuged for 120min at 400000g in a vertical rotor
(Beckman VTi 65). Non-specific binding was determined in parallel by adding a 100-fold excess of unlabelled
oestradiol (O).
After the 5 h incubation, some binding to the
antibody could be distinguished by a shoulder in
the f-form peak, and 40-60% of the total receptors
was bound to the antibody after the 20 h incubation; longer incubations did not increase these
values. These data suggest that at 4°C there is a
slow time-induced conversion from a form of the
receptor initially present in the cytosol that is not
recognized by the antibody (,B form) into a form
that it does recognize (a form).
The conversion into the a form was found to be
also a temperature-dependent process. When the
oestradiol-receptor complex was incubated at 4°C
for 2 h, then warmed at 25°C for 15 min and reincubated with the antibody for 1 h, the gradient
profiles showed the presence of the a form, but it
was absent from a similarly treated, but not
warmed, cytosol (Fig. lb).
Effect of high salt concentration on oestrogen receptor
binding to the monoclonal antibody
Since modifications in the characteristics of the
cytosol oestrogen receptor occur at high salt
concentrations, the effect of KCI on the conversion
of the P form into the a form was analysed. Fig.
2(a) shows that incubation of the oestradiolreceptor complex with 0.4M-KCI for 2h resulted in
a significant conversion of the # form into the a
form, which did not occur under similar experimental conditions but in the absence of KCI. The
Vol. 230
conversion was even higher after 20 h of incubation (Fig. 2b). As shown in Fig. 2(b), the
spontaneous conversion into the a form after 20h
in the absence of KCl was less than expected (see
Fig. la). This could be explained by the fact that all
samples were treated with dextran-coated charcoal, to remove unbound [3H]oestradiol, before the
addition of KCI, since the charcoal adsorption
procedure is not valid in high salt concentrations
(Peck & Clark, 1977).
Effect of sodium molybdate on oestrogen receptor
binding to the monoclonal antibody
Those factors that induce the conversion into the
a form are also known to induce receptor activation, suggesting that the cx form could be an activated form of the receptor. To explore this possibility further, we tested the effect of Na2MoO4,
a well-known stabilizer of receptors and inhibitor
of receptor activation.
Figs. 3(a) and 3(b) show that 10mM-Na2MoO4
completely blocked both the time- and temperature-induced appearance of the cx form; however,
when Na2MoO was added after 20h of incubation
at 4°C or warming for 15 min at 25°C, no effect was
observed. The effect of molybdate on the 0.4MKCl-induced conversion into the a form was
somewhat different. When the oestradiol-receptor
complex was prepared in a buffer containing
10mM-Na,MoO4, only a partial inhibition of the
N. Giambiagi and J. R. Pasqualini
206
-o
;:!C.)
C.
la
cqs
x
I
I0
Top
Top
Fraction no.
Fig. 2. Effect of 0.4M-KCI on the oestrogen receptor binding to the monoclonal antibody
Cytosol (2ml) was incubated with l0nM-[3H]oestradiol for 2h, and unbound radioactivity was adsorbed with
dextran-coated charcoal. (a) Samples (0.5ml) were incubated without (0) or with (0) 0.4M-KCI for 1 h, then the
antibody was added and the incubation was continued for 1 h. (b) Samples (0.5 ml) were incubated without (0) or
with (0) 0.4M-KCI for 19h, then the antibody was added and the incubation was continued for 1 h. All procedures
were carried out at 4°C. Samples were centrifuged through sucrose gradients as indicated in Fig. 1. Non-specific
binding was determined in parallel by adding a 100-fold excess of unlabelled oestradiol (El).
4
ci.
*s
C)
CIO
0
Cd
Cu
x
I0
40 0
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20
30
40
Top
Fraction no.
Fig. 3. Effect of Na,MoO4 on the oestrogen receptor binding to the monoclonal antibody
(a) A sample of cytosol (0.5ml) was incubated with lOnM-[3H]oestradiol+ 10mM-Na2MoO4 for 19h at 4°C (A);
another was incubated with lOnM-[3H]oestradiol for 19h at 4°C, and then 10mM-Na,MoO4 was added (0). Both
samples were re-incubated with the antibody for 1 h. Unbound [3H]oestradiol was removed with dextran-coated
charcoal, and samples were layered on sucrose gradients. (b) A samples of cytosol (0.5 ml) was incubated with 10 nM[3HJoestradiol + lOmM-Na,MoO4 for 2h at 4°C and heated for 15 min at 25°C (A); another was incubated with
lOnM-(3H]oestradiol for 2h at 4°C, heated for 15min at 25°C and then 10mM-Na2MoO4 was added (*). Both
samples were cooled and re-incubated with the antibody for 1 h at 4°C. Unbound [3H]oestradiol was removed with
dextran-coated charcoal and samples were layered on sucrose gradients. (c) A sample of cytosol (0.5 ml) was
incubated with l0nM-[3H]oestradiol+ 10mM-Na,MoO4 for 2h at 4°C, treated with dextran-coated charcoal to
remove the free [3H]oestradiol and re-incubated with 0.4M-KCI for 2h (A); another two samples were incubated
with [3H]oestradiol for 2 h at 4°C and treated with dextran-coated charcoal, then one was re-incubated with 10mMNa,MoO4+0.4M-KCI for 2h (A) and the other was re-incubated with 0.4M-KCI for 2h and then 10mM-Na,MoO4
was added (0). Both samples were re-incubated with the antibody for 1 h at 4°C and then layered on to sucrose
gradients, and centrifuged as indicated in Fig. 1. Non-specific binding was determined in parallel by adding a 100fold excess of unlabelled oestradiol (E).
1985
Immunorecognition of the active form of the oestrogen receptor
KCl-induced conversion into the a form was found
(Fig. 3c). No significant effect on this conversion
was observed when lOmM-Na,MoO4 was added at
the same time as, or after, the 2 h exposure to 0.4MKCI.
Activation of oestrogen receptor as determined by its
binding to nuclei or DNA-cellulose
Since the observations on the formation of the
immunorecognized a form seemed to indicate that
it was induced by the same factors that induce
receptor activation, this process itself was studied
in more detail and compared with the formation of
the a form. The extent of activation of the receptor
was estimated by the increase in its binding to
nuclei or DNA-cellulose at 4°C (Yamamoto &
Alberts, 1972).
Table 1 shows that warming the oestradiolreceptor complex for 15min at 25°C increases its
nuclear and DNA-cellulose binding; 10mMNa,MoO4 blocked this increase when added
before heating, but had no effect on the temperature-activated complex. The receptor was also
partially activated with time, as indicated by the
increase in DNA-cellulose binding from 8.6%
after 2h at 4°C to 18.6% after 20h; 10mMNa,MoO4 blocked the time-induced activation
when it was present during the 20 h incubation, but
had no effect when added afterwards. These results
can be compared with the observations shown in
Fig. 1 and Figs. 3(a) and 3(b), and a correlation can
be seen between the time- and temperatureinduced activation of the receptor and the appearance of the oa form.
Exposure to 0.4M-KCI for 2h also greatly
increased the binding of the receptor to nuclei and
DNA-cellulose (Table 2). To assess the nuclear
and DNA-cellulose binding of the KCl-activated
complexes, it was necessary to dilute the samples 4-
207
fold to lower the KCI concentration. The 4-fold
dilution itself never increased nuclear or DNAcellulose binding. After a 20h exposure to 0.4MKCI, although the DNA-cellulose binding was
increased, the values were less than those obtained
after 2 h, probably indicating some deactivation
occurring during the 20h incubation. Nuclear and
DNA-cellulose binding of the 0.4M-KCI-activated
oestradiol-receptor complexes was only partially
inhibited when they had been prepared in the
presence of 10mM-Na2MoO4. When 10mM-Na2MoO4 was added at the same time as, or after, the
2h exposure to 0.4M-KCI, it had relatively little
effect on the KCl-induced activation. These findings can be correlated with the observations shown
in Figs. 2 and 3(c). The KCl-activation of the
receptor corresponds to the KCl-induced appearance of the a-form peak in the gradient profiles.
Transformation ofthe fijorm ofthe oestrogen receptor
into the a form
In another series of experiments, the a and f
forms of the receptor were separately recovered
from the sucrose density gradient. Fractions 10-24
and 25-39 of the gradient were considered to
contain the a and ,B forms respectively. Each form
was recovered in 1.8ml of 0.4M-TKED/sucrose
buffer, and concentrated overnight by dialysis
under reduced pressure against TED buffer, with
a Micro-ProDiCon system (Bioblock Scientific,
Illkirch, France). A sample of this a form was resubmitted to sucrose-density-gradient analysis and
found to sediment with the same 7.4S coefficient,
indicating that it was not altered and remained
bound to the antibody (Fig. 4a). A sample of the #
form was re-incubated with the antibody for 1 h
and analysed on a sucrose density gradient. Fig.
4(a) indicates that most of the radioactivity
Table 1. Nuclear and DNA-cellulose binding of the time- and temperature-activated oestrogen receptor
Samples 1, 3, 4, 6 and 7 of cytosol (0.5ml) were incubated with lOnM-[3H]oestradiol, and samples 2, 5 and 8 with
lOnM-[3H]oestradiol+ lOmM-Na,MoO, ('+ Mo'), for 2h or 20h (as indicated below), at 4°C. Samples 3, 4 and 5
were warmed at 25°C for 15min, and the rest was kept at 4°C. At the end of the incubation, l0mM-Na2MoO4
was added to samples 4 and 7. Samples were assayed for nuclear and DNA-cellulose binding as indicated in
the Experimental section. The numbers of experiments are given in parentheses. The data are expressed as
means+S.E.M.; -, assays not performed.
DNA-cellulose binding
Nuclear translocation
(% of total receptor)
Experimental conditions
(% of total receptor)
I
40C 2h
2 + Mo 40C 2 h
3
4°C 2 h -25°C
4
40C 2h-+250C+Mo
5 +Mo 40C 2h- 250C
40C 20h
6
7
40C 20h- + Mo
8 +Mo 40C 20h
Vol. 230
10.2 + 1.9 (4)
5.8+ 1.5 (3)
20.8±4.4 (3)
21.7 ±2.5 (3)
6.5 + 1.2 (3)
8.6+ 1.3
6.0+0.6
17.8 + 3.4
22.2+3.1
6.6+0.5
18.6+2.7
24.0+ 3.5
6.5± 1.2
(3)
(3)
(3)
(3)
(3)
(5)
(3)
(3)
N. Giambiagi and J. R. Pasqualini
208
Table 2. Nuclear and DNA-cellulose binding of the 0.4M-KCl-activated oestrogen receptor
Samples 1, 3, 4, 5, 7 and 8 of cytosol (0.5ml) were incubated with l0nM-[3H]oestradiol, and samples 2 and 6 with
lOnM-[3H]oestradiol + l0mM-Na,MoO4 ('+ Mo'), for 2h and then treated with dextran-coated charcoal. Then 0.4MKCl was added to samples 3, 4, 6 and 8, and lOmM-Na,MoO4 +0.4M-KCI to sample 5; incubation was continued for
2h or 20h (as indicated below), and finally l0mM-Na,MoO, was added to sample 4. Samples were diluted 4-fold
with TEDS buffer or TEDS buffer+ l0mM-Na,MoO4 (samples 2, 4, 5, 6) and assayed for nuclear and DNAcellulose binding as indicated in the Experimental section. All procedures were carried out at 4°C. The numbers of
experiments are given in parentheses. The data are expressed as means+S.E.M.; -, assays not performed..
DNA-cellulose binding
Nuclear translocation
(% of total receptor)
(% of total receptor)
Experimental conditions
I
2+Mo
3
4
5
6 + Mo
7
8
5.8 ± 1.7
4.7+2.3
54.2+ 12.9
46.7+7.4
41.2+7.6
35.7+2.5
2h
2h
2h
2h-M+Mo
2h-++Mo+0.4M-KCI 2h
2 h+0.4M-KCI 2h
2h-4
20h
+
2h-,
+0.4M-KCI 20h
+
+
+0.4M-KCI
+0.4M-KC1
2h-l
2h-*
2h-*
2h-+
0
10
20
30
40 0
Top
10
(3)
(3)
(3)
(3)
(3)
(3)
20
5.8+ 1.1
5.0+0.3
61.6+4.2
62.4+4.5
57.5 + 2.4
45.2+7.6
5.8+3.7
38.2+ 11.8
30
(5)
(3)
(6)
(4)
(4)
(4)
(3)
(3)
40
Top
Fraction no.
Fig. 4. Isolation by density-gradient centrifugation of the ,B form and transformation to the a form
(a) A sample of cytosol (0.3ml) was incubated with 10nM-[3H]oestradiol and the antibody for 20h at 4°C, and after
dextran-coated-charcoal treatment 0.2ml of the supernatant was centrifuged through the sucrose gradient (see the
Experimental section). Fractions 10-24 (a) and 25-39 (,B) of the gradient were recovered separately and
concentrated (from 1.8 ml to 0.4ml) overnight by dialysis against TED buffer under reduced pressure at 4°C by using
a Micro ProDiCon system. Portions (0.2ml) of the recovered a form (@) and of the recovered /3 form, re-incubated
with the antibody for I h (A), were analysed through sucrose gradients. (b) Samples (200 Ml) of cytosol were
incubated with lOnM-[3H]oestradiol for 20h, treated with dextran-coated charcoal, diluted to 2ml with TED/sucrose
(El) or 0.4M TKED/sucrose buffers (M) and concentrated to 0.4ml by overnight dialysis against TED buffer under
reduced pressure at 4°C as in Fig. 4(a). Samples (200 p1) were incubated with the monoclonal antibody for 1 h and
analysed by centrifugation through sucrose gradients.
sedimented in the a zone, suggesting a complete
transformation of this ,B form into the a form.
To investigate if this conversion was spontaneous or induced by the treatment, 200 pl samples of
[3H]oestradiol-labelled cytosol were diluted to 2ml
with either TED/sucrose or 0.4M TKED/sucrose
buffers and concentrated overnight by dialysis
under reduced pressure against TED, incubated
for 1 h with the antibody and analysed on sucrose
density gradients. In both cases, a great conversion
into the a form was found, especially when 0.4M-
KCI was present in the dilution buffer (Fig. 4b),
indicating that the experimental conditions (dilution, exposure to KCI, time and dialysis) to which
the P form was submitted for isolation and analysis
could be responsible for its conversion into the a
form.
Discussion
Two forms (a and P) of the cytosol oestrogen
receptor from fetal guinea-pig uterus were
1985
Immunorecognition of the active form of the oestrogen receptor
differentiated on high salt sucrose gradients by
their selective binding to the monoclonal antibody
D547Spy. This IgG-class monoclonal antibody,
raised against a partially purified preparation from
MCF-7 human breast-cancer cells, reacts specifically with the oestrogen receptor from primates
and other mammals, such as rat and calf. Its
affinity is lower for rat and calf receptors than for
MCF-7 receptors, although, in the presence of an
excess of antibody, binding is complete, whereas
no interaction occurs with the hen oviduct receptor. These findings indicate the conservation of a
common antigenic determinant across the mammalian species (Greene et al., 1980b; Greene &
Jensen, 1982). This antigenic determinant, which
is recognized by the D547Spy antibody, appears to
be localized in an intermediate region of the MCF7 receptor, neither close to the steroid-binding
domain nor close to the DNA-binding domain
(Greene et al., 1984). As shown here and in
previous work (Giambiagi & Pasqualini, 1982;
Giambiagi et al., 1984), this monoclonal antibody
also recognized the oestrogen receptor from fetal
guinea-pig uterus; however, the cytosol receptor
was only partially bound, even in the presence of
an excess of antibody (Giambiagi & Pasqualini,
1982), revealing the existence of two forms of the
receptor with different immunological characteristics. This antibody did not recognize the progesterone receptor from fetal guinea-pig uterus
(Giambiagi & Pasqualini, 1982), confirming
the specificity studies described by Greene et al.
(1980b).
The first suggestion that the cx form could be an
activated form of the receptor appeared from the
different behaviour of the oa and P forms during the
translocation process. When cytosol was incubated
with nuclei at 25°C, the a form disappeared rapidly
from the cytosol, whereas the ,B form was only
slightly affected; the receptor extracted from the
nuclei increased with the time of incubation
and was always totally bound by the antibody
(Giambiagi et al., 1984).
The present data show that there is a spontaneous time-dependent partial transformation of the ,B
to the a form, suggesting that the two forms are not
independent, but that the a form originates from
the ,B form. Under these conditions a spontaneous
partial activation of the receptor was also shown by
the increase in its binding to DNA-cellulose. The
spontaneous activation of steroid receptors in long
incubations at 4°C has been reported by different
authors (Milgrom et al., 1973; Miiller et al., 1983).
The conversion into the a form was accelerated by
increasing the temperature, which also induced a
partial activation of the receptor. This temperature-induced activation of steroid receptors is a
well-described phenomenon (Grody et al., 1982).
Vol. 230
209
High salt concentrations also induced the transformation to the a form and, as was described for
several steroid receptor systems (Milgrom et al.,
1973; Bailly et al., 1980; Muller et al., 1983), had a
great effect on receptor activation. However, the
activation after 20h exposure to 0.4M-KCI was less
than after 2h, indicating a possible deactivation of
the activated complex which did not affect its
ability to bind to the antibody. Activation by highsalt treatment proved to be the most effective,
probably because this procedure avoids exposing
the receptor to elevated temperature or long
incubations. Thus the observations presented here
show a correlation between receptor activation
and the conversion of the P into the a form, which
is consistent with the hypothesis that the a form is
the activated form of the receptor.
Additional support for this proposal was provided by the use of Na2MoO4. In agreement with
the findings by other investigators (Leach et al.,
1979; Nishigori & Toft, 1980; Noma et al., 1980;
Shyamala & Leonard, 1980; Muller et al., 1983),
Na2MoO4 completely blocked the temperatureinduced activation, but had no effect once the
receptor was activated. The transformation from
the ,B to the cx form induced by brief heating was
also completely inhibited by Na,MoO4 only when
it was added before heating. Similarly, both
activation and transformation to the cx form
induced by long incubation at low temperature and
ionic strength were blocked when Na2MoO4 was
present from the beginning of incubation, but were
not affected when it was added afterwards.
On the other hand, we found only a weak
inhibition of the KCl-induced activation when
Na2MoO4 was present during preparation of the
oestradiol-receptor complex, and only a partial
diminution of the a form. This partial inhibitory
effect of Na2MoO4 on the high-salt-induced
activation was also observed by other authors
(Nishigori & Toft, 1980; Redevilh et al., 1981;
Mauck et al., 1982; Muller et al., 1983). The
different effect of Na2MoO4 on activation induced
by temperature or high ionic strength suggests that
these processes of activation are not identical. In
any case, the complete or partial inhibition of
activation was always accompanied by a complete
or partial inhibition of the transformation to the cx
form.
Finally, it was observed that the # form, isolated
and recovered from the sucrose gradient, could be
completely transformed into the ac form by the
dilution and dialysis to which the samples were
submitted. Dilution, in a time-dependent process
(Bailly et al., 1977; Goidl et al., 1977), and dialysis
(Sato et al., 1979, 1980) have been reported to
induce receptor activation by removing low-Mr
components of the cytosol. We have also observed
210
that overnight dialysis induced conversion of the /
into the a form and an increase in the receptor
binding to DNA-cellulose (N. Giambiagi & J. R.
Pasqualini, unpublished work). Low-Mr inhibitors
present in the cytosol could be responsible in part
for the incomplete nuclear and DNA-cellulose
binding and transformation to the a form.
The present work gives evidence that the form
recognized by the monoclonal antibody is the
activated form of the oestrogen receptor of fetal
guinea-pig uterus. The activation process involves
conformational changes in the receptor, which
apparently make the antigenic determinant recognized by the antibody more accessible. Differences
in reactivity of monoclonal antibodies with different forms of the oestrogen receptor have been
described for an IgM-class monoclonal antibody to
the calf nuclear oestrogen receptor, which has
more affinity for the nuclear than for the cytosol
form of this receptor (Greene et al., 1980a).
Subsequently, this monoclonal antibody was found
to interact more strongly with the activated than
with the non-activated cytosol form of the calf
oestrogen receptor, and also to have a different
affinity for the receptor bound to oestradiol or to 4hydroxytamoxifen (Borgna et al., 1984). On the
other hand, no differences have been reported in
the binding of the D547Spy monoclonal antibody
used in our studies to nuclear or activated and nonactivated cytosol forms of the human oestrogen
receptor, except for a slight difference in sedimentation coefficient between the nuclear and cytosol
receptor-antibody complex. However, the guineapig receptor must be different enough from the
human receptor so that the same antibody will
selectively bind to only the activated form. The
possibility of discriminating between activated
and non-activated forms of the oestrogen receptor
with a monoclonal antibody will be very useful for
further studies on the receptor-activation process.
Part of the expense of this work was defrayed by the
Centre National de la Recherche Scientifique, France
(Unite Associee du C.N.R.S. no. 548) and by the
Fondation pour la Recherche Medicale Franqaise. We
express our sincere thanks to Dr. E. V. Jensen (Zurich,
Switzerland) and Dr. G. L. Greene (Chicago, U.S.A.)
for the gift of monoclonal antibody D547Spy.
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1985
