Differential inhibition of estrogen and antiestrogen binding to the estrogen receptor by diethylpyrocarbonate

J. steroid Biochem. Vol. 31, No. 4A, pp. 421-436, 1988 Printed in Great Britain. All rights reserved

0022-4731/88 53.00 + 0.00 Copyright 0 1988 PergamonPressplc

DIFFERENTIAL INHIBITION OF ESTROGEN AND ANTIESTROGEN BINDING TO THE ESTROGEN RECEPTOR BY DIETHYLPYROCARBONATE JEAN LOUIS BORGNA*and JACQUELINESCALI Unite d’Endocrinologie Cellulaire et Moleculaire, INSERM U 148, 60 Rue de Navacelles, 34090 Montpellier, France (Received 10 November 1987) Summary-Diethylpyrocarbonate differentially inhibited the specific binding, in lamb uterine cytosol, of estradiol (inhibition -90% with 4 mM reagent) and 4-hydroxytamoxifen (inhibition 6 50% with 4-16 mM reagent), a potent triphenylethylene antiestrogen. Saturation analysis experiments indicated that the effects of diethylpyrocarbonate were due to progressive but differing decreases in the concentration of binding sites for the two ligands, with no apparent change in the affinity constants. However, competitive binding and dissociation experiments evidenced that steroidal and nonsteroidal estrogens still bound, but with very low affinities, to diethylpyrocarbonate-modified receptor (> lOOO-folddecrease in affinity) whereas the affinities of triphenylethylene antiestrogens were much less affected (< IO-fold decrease). Both ligands prevented the inactivation of the estrogen receptor by diethylpyrocarbonate, estradiol being more efficient than Chydroxytamoxifen. These data indicate that the action of diethylpyrocarbonate results in the formation of two populations of estrogen receptor that are quantitatively nearly equivalent: the first does not bind estrogens or antiestrogens; the second does not bind estrogens significantly but still interacts with antiestrogens at a high affinity. The simplest interpretation is that these two populations arise from mutually exclusive modifications by diethylpyrocarbonate of at least two aminoacid residues located at or close to the ligand binding site; modification of one residue totally prevents the binding of estrogens and antiestrogens; the modification of the second impairs only the binding of estrogens. Considering that (i) hydroxylamine, which specifically reverses the diethylpyrocarbonate-induced modification of histidine and tyrosine residues, restored a large part (> 80%) of the estradiol- and 4hydroxytamoxifenbinding capacity of diethylpyrocarbonateinactivated cytosol, and that (ii) similar differential inhibition of estrogen and antiestrogen binding was observed following the action of tetranitromethane, it is likely that these residues are histidine(s) and/or tyrosine(s). These results evince a marked difference in the interaction of estrogens and triphenylethylene antiestrogens with the estrogen receptor, which could account for the altered activation of the receptor by triphenylethylene antiestrogens. Consequently, the screening of ligands with modified steroid receptors could be a useful method for distinguishing between potential hormone agonists and antagonists.

INTRODUCTION

antagonism according to the species, target organs, or estrogen responses studied. In the human species, Triphenylethylene-related antiestrogens are struc- they behave either as partial agonists/antagonists or tural analogs of estrogens used in human therapy, i.e. pure antagonists of estrogens. Their antiestrogenic for inducing ovulation in anovulatory women or activity is thought to result from (i) competition with treating estrogen-dependent breast cancer. The estrogens for binding to the estrogen receptor, and pharmacological properties of these compounds can (ii) their inability to activate the receptor properly. vary from full estrogen agonism to pure estrogen Studies carried out with estradiol and 4-hydroxytamoxifen, which is a potent triphenylethylene antiestrogen displaying high affinity for the estrogen *To whom correspondence should be addressed. receptor, evidenced a series of differences between Abbreviations: P50 buffer, 50 mM sodium phosphate pH 7.0; the properties of receptor-estrogen and receptorestradiol (E2), estra-1,3,5(10)-triene-3,17j-diol; estriol antiestrogen complexes related to: the dissociation (E,), estra-1,3,5(IO)-triene-3,16a, 178-trio]; diethylstilbestrol (DES), 3,4-bis(4-hydroxyphenyl)-hex-3-(E)-ene; rate [I], DNA binding [2], and immunoreactivity with a monoclonal antibody [3]. tamoxifen (?), 1-[4-@dimethyiamindethoxy) phenyll1.2-dinhenvlbut-1-(Z)-ene: 4-hvdroxvtamoxifen (OHT), However, none of the aminoacid residues involved ll(4 -hydrbxyphenyl )- I- (4 - ( 2- dimethylaminoethoxy ) - in the binding of an estrogen or an antiestrogen phenyll-2-phenyl-but-I-(Z)-ene; CI 628 (CI), 1-[4-(2by the estrogen receptor has yet been identified. pyrolidinoethoxy)phenyl]- l -(4-methoxyphenyl)-2-phenylAbundant data obtained from the use of electrophilic 2-nitro-ethyl(E)-ene; DEPC, diethylpyrocarbonate; R, estrogen receptor; RAC, relative affinity constant. affinity labels or reagents of nucleophilic aminoacids, 427

JEAN Louts BORGNA

428

suggest, and in some cases demonstrate, that various nucleophilic aminoacid residues are located at or near the catalytic or binding site of enzymes [4-101 and proteins [l l-291 interacting with steroid hormones. Diethylpyrocarbonate is one of the most frequently used reagents of nucleophilic aminoacids. Although it can react with a large variety of nucleophilic aminoacid residues of proteins including histidine, tyrosine, cysteine, methionine, lysine, arginine, tryptophane and serine [30], it shows a good selectivity for histidine at neutral pH. Moreover, only its modifications of the histidyl and tyrosyl groups are reversed by hydroxylamine [31]. Diethylpyrocarbonate has frequently been used to detect essential histidine residues in proteins, such as in recent studies suggesting that histidine residues play an important role in the ligand- and DNA-binding functions of the estrogen receptor [28,32]. In this study, we characterized the effect of this reagent on the binding of estrogens and triphenylethylene antiestrogens by the estrogen receptor. We report a differential inhibition of estrogen and antiestrogen binding by the estrogen receptor. We also present evidence that the integrity of at least two aminoacid residues of the receptor (probably histidine(s) and/or tyrosine(s)) is crucial for its estrogen binding function whereas the integrity of only one is important for its ability to bind triphenylethylene antiestrogens.

EXPERJMENTAL

Materials

act. [IV -methyl - 3H] 4 - hydroxytamoxifen (sp. 85 Ci/mmol; radiochemical purity > 95%) was purchased from the Radiochemical Centre (Amersham, England). [6,7 ‘Hlestradiol (sp. act. 60 Ci/mmol; radiochemical purity ~95%) was from the “Commissariat a 1’Energie Atomique” (Gif-sur-Yvette, France). Tamoxifen and 4-hydroxytamoxifen were from Imperial Chemical Industries (Macclesfield, England). CI 628 was provided by Parke-Davis (Detroit, MI, U.S.A.). Estrogens (estradiol, estriol, diethylstilbestrol), histidine, diethylpyrocarbonate, imidazole and hydroxylamine hydrochloride were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Preparation of lamb uterine cytosol

Cytosol (centrifuged at 180,000g for 45 min) was prepared from frozen (-80°C) uteri in P50 buffer and fresh cytosol was used in all studies. The protein concentration of cytosol was determined according to Layne [33]. Cytosol was diluted to 2mg protein/ml. Standard ligand binding assay

Aliquots of cytosol treated or not with diethylpyrocarbonate and hydroxylamine, as indicated in the table and figure legends, were incubated with

and

JACQUELINE SCALI

5 nM [‘Hlestradiol or [3H]4-hydroxytamoxifen without or with the corresponding radioinert ligand at 1 PM (stock solutions of ligands in ethanol and were added (l%, v/v) to cytosol to obtain the indicated concentrations) for at least 15 h at 0°C to determine the total (B,) and nonspecific (Br) binding of labeled ligands, respectively. Aliquots were then treated with an equal volume of charcoal suspension (0.5% charcoal, 0.05% dextran T70 in P50 buffer) for 0.5 h at 0°C. The charcoal was pelleted by centrifugation and the radioactivity of the supernatant was measured. Estrogen-specific binding of labeled ligands (Bs) was calculated from total (T) and bound concentrations (B, and B, measured in charcoal supematants, corresponding to cytosol incubated in the absence or presence of 1 PM radioinert ligand, respectively), according to Blondeau and Robe1 [34]:

B, = (B, - B,)T(T - B2)-’ Competitive binding assays-relative

a$inity constants

Aliquots of control and diethylpyrocarbonatetreated cytosol were incubated at 20°C with 5 nM [3H]4-hydroxytamoxifen with or without increasing concentrations of radioinert estrogens and antiestrogens. Binding of [3H]4-hydroxytamoxifen in the aliquots was determined after 4 h and 20 h of incubation by a charcoal assay for 0.5 h at 0°C. Apparent affinity constants of competitors (&) for the estrogen receptor, relative to those of’ Chydroxytamoxifen, were calculated according to Korenman [35] from the concentrations of 4-hydroxytamoxifen (H) and competitor (C) which inhibited 50% of the specific binding of [‘H]4-hydroxytamoxifen and from total (H:), charcoal-resistant bound (Hf), and specifically bound (H$) concentrations of [3H]4-hydroxytamoxifen at this 50% inhibition, using for the concentration of unbound [3H]4-hydroxytamoxifen (Hz) the approximation H,* NH: - H$ which gave: KA = H(Hj’ - H;)

[C(H:

- H$‘) + H$(C

-H)]-’

Dissociation kinetics

Aliquots of cytosol treated or not with diethylpyrocarbonate and labeled with 5 nM [‘Hlestradiol or [3H]4-hydroxytamoxifen in the absence or presence of the corresponding radioinert ligand at 1 PM, as indicated in the table and figure legends, were then incubated at 0 or 20°C and supplemented at time 0 with 1% ethanol (to measure the stability of complexes) or 1% ethanol containing 0.1 mM radioinert estradiol or 4-hydroxytamoxifen (to measure the dissociation rates of the complexes). At different times, aliquots were removed and treated with charcoal for 0.5 h or 1 h at 0°C to determine the binding of labeled ligands. The specific binding of [‘Hlestradiol and/or [3H]Chydroxytamoxifen was calculated as described above (standard ligand binding assay).

Differential inhibition of estrogen and antiestrogen binding Modzjication of the estrogen pyrocarbonate

receptor

429

by diethyl-

were Ethanol solutions of diethylpyrocarbonate, prepared from pure compound immediately before of diethylpyrocarbonate were use. Concentrations determined using imidazole as a substrate as described by Melchior and Fahrney [31]. Aliquots of cytosol preincubated or not with labeled hgands were supplemented with either 1% (v/v) ethanol or ethanol solutions of reagent, and agitated at 0°C for different periods of time. Aliquots were then added with at least a 3-fold excess of 0.3 M histidine in PSO buffer. [3H]estradiol and/or [‘HlChydroxytamoxifen binding was then determined as indicated in the table and figure legends. Hydroxylamine treatment of diethylpyrocarbonateinactivated estrogen receptor Aliquots of cytosol treated or not with diethylpyrocarbonate and then with histidine, were incubated for 20 h at 0°C with 10% (v/v) water or 1.1-4.4 M hydroxylamine hydrochloride solution adjusted to pH 7.0 with KOH. Aliquots were incubated with 3H-labeled ligands to measure the binding capacity of control and modified estrogen receptors or the dissociation rates of ligands from the different forms of the estrogen receptor.

TIME (min)

Fig. 1. Time-course of the inactivation of estradiol binding to the estrogen receptor by diethylpyrocarbonate. Lamb uterine cytosol prepared in PSO buffer and adjusted to 2 mg protein/ml was incubated at 0°C. At time 0, either ethanol, 0.152 M diethylpyrocarbonate or 0.303 M diethylpyrocarbonate in ethanol was added (l%, v/v). At the indicated times, aliquots were removed, complemented (IO%, v/v) with 0.3 M histidine and maintained at 0°C until time 90 min. Each sample was then incubated for 15 h at 0°C with 5 nM [3H]estradiol in the absence or presence of 1 PM radioinert estradiol. Specifically bound [‘Hlestradiol, determined by charcoal assay as described in Experimental, is plotted against the incubation time. (0) control cytosol incubated with ethanol; (a) cytosol incubated with 1.5 mM diethylpyrocarbonate; (m) cytosol incubated with 3 mM diethylpyrocarbonate.

RESULTS

The effects of increasing concentrations of diethylpyrocarbonate on cytosol binding of estradiol or 4-hydroxytamoxifen were determined after 30 min of reaction. As shown in Fig. 2, the variations in the Incubation at 0°C of lamb uterine cytosol in phos- specific binding of estradiol and 4-hydroxytamoxifen phate buffer, pH 7.0, with millimolar concentrations in cytosol differed according to the concentration of of diethylpyrocarbonate, resulted in a rapid and diethylpyrocarbonate used. In all the experiments concentration-dependent decrease in its estradiol and performed, the inhibition of estradiol binding, which 4-hydroxytamoxifen specific binding activity (using a varied slightly from experiment to experiment (cf. “saturating” 5 nM concentration in both cases). The Figs 2 and 3) was almost complete: i.e. ~-90% for kinetics of estradiol binding inactivation in cytosoi by 8 mM diethylpyrocarbonate; whereas the inhibition 1.5 or 3 mM diethylpyrocarbonate is shown in Fig. 1; of 4-hydroxytamoxifen binding was only partial: stabilized estradiol-binding values, respectively _ 45 from 4 to 16 mM diethylpyrocarbonate, a practically and N 15% of that of untreated cytosol, were ob- constant 4-hydroxytamoxifen binding was observed, served for incubation times 2 20 min (since the in- amounting to about 50% of the original binding hibition of estradiol binding by a given concentration (from 45 and 60% in the different experiments). It is of diethylpyrocarbonate was inversely correlated with noteworthy that the apparent charcoal-resistant nonspecific binding of 4-hydroxytamoxifen in the protein concentration in the cytosol preparation, the latter concentration was adjusted to 2mg diethylpyrocarbonate-treated cytosol varied accordprotein/ml in all experiments, prior to the addition of ing to whether the unlabeled ligand used for its diethylpyrocarbonate). determination, was estradiol or 4-hydroxytamoxifen; The diethylpyrocarbonate effect probably resulted higher values were measured when estradiol was used from a direct action of the reagent on the estrogen (Fig. 2 inset). This was not the case in the untreated receptor, since: (i) estradiol binding by diethylpyrocytosol or when measuring the nonspecific binding of carbonate-treated cytosol was not changed when estradiol in treated cytosol, which did not change treated cytosol was supplemented with an equal when unlabeled 4-hydroxytamoxifen was used involume of untreated cytosol, and (ii) estradiol bind- stead estradiol. The correct nonspecific binding of was in fact that determined ing by untreated cytosol was not modified when it Chydroxytamoxifen was supplemented with an equal volume of treated when 4-hydroxytamoxifen was also used as the uncytosol (not shown). labeled ligand (cf. Fig. 6 and Discussion). Diethylpyrocarbonate differentially inhibits the binding of estradiol and I-hydroxytamoxifen to the estrogen receptor

JEAN LOUIS E~ORGNAand

430

0

4

8

DIETHYLPYROCAREONATE

12

16

(mM)

JACQUELINE SCALI

I

0

1

2

I 4

DIETHYLPYROCARBONATE

1 8

I

6

(mM)

Fig. 2. Concentration-dependent inactivation of estradiol and 4-hydroxytamoxifen binding to the estrogen receptor by diethylpyrocarbonate. Lamb uterine cytosol was incubated with increasing concentrations of diethyIpyrocarbonate (O-16 mM) for 0.5 h at 0°C. Samples were then supplemented (20%, v/v) with 0.3 M histidine and agitated for 0.5 h at 0°C. Aliquots were then incubated for 15 h at 0°C with 5 nM [3H]estradiol or rH]4-hydroxytamoxifen either (i) in the absence of radioinert hgands, (ii) in the presence of 1 FM radioinert estradiol or (iii) in the presence of 1 PM radioinert 4-hydroxytamoxifen. Binding of IabeIed ligands in the various aliquots was determined after a 0.5 h charcoal assay. The speciftc binding of [sHIestradio and [‘H]4hydroxytamoxifen was calculated as described in Experimental and expressed as a percentage of the specific binding of the ligand in untreated cytosol. The specific binding of the two ligands in cytosoi is plotted vs the diethylpyrocarbonate concentration. (Es, 0) specific binding of [3~estradiol,

Fig. 3. Protection by estradiol and 4-hydroxytamoxifen of the estrogen receptor against diethylpyrocarbonate inactivation. Portions of uterine cytosol were incubated for 5 h at 0°C with 5 nM rH]estradiol or [3H]4-hydroxytamoxifen in the absence or presence of 1 PM radioinert ligand (prelabeled cytosol). Another portion was incubated without ligands. Radioinert estradiol or Chydroxytamoxifen (2 FM final concentration) was then added to labeled samples to occupy naked estrogen receptor and all samples were further incubated for 1 h at 0°C. Aliquots of labeled and unlabeled samples were incubated with increasing concentrations (O-g mM) of diethylpyrocarbonate at 0°C for 0.5 h. Aliquots were then supplemented (lo%, v/v) with 0.3 M histidine. Unlabeled aliquots were incubated with 5 nM 3H-labeled ligands in the absence or presence of the corresponding radioinert ligand (1~ M) for 15 h at 0°C (postlabeled cytosol) whereas labeled aliquots were maintained at 0°C. Binding of [%jestradiol or [3H]4-hydroxytamoxifen in the various (OHT, @) specific binding of [3H]4-hydroxytamoxifen. The aliquots was determined after a 0.5 h charcoal assay. The inset shows variations of the nonspecific binding of 13H]- specific binding of [‘H]estradiol and [3H)4-hydroxyestradiol in the presence of 1 PM &radio1 (O), the nontamoxifen was calculated as described in Experimental, and

specific binding of [3H]4-hydroxytamoxifen in the presence of I PM 4-hydroxytamoxifen (0) and the binding of TH]4hydroxytamoxifen in the presence of 1PM estradiol (R).

Preincubation of cytosol with estradiol or 4-hydroxytamoxifen markedly reduced the effect of diethylpyrocarbonate (Fig. 3). The receptor4-hydroxytamoxifen complex was more resistant than the receptor-estradiol complex to the action of diethylpyrocarbonate. However, the relative protective effect of the ligand, calculated as the ratio of residual binding of liganded receptor to that of unliganded receptor, was higher for estradiol than for 4-hydroxytamoxifen, at all concentrations of diethylpyrocarbonate used; at 8 mM diethylpyrocarbonate, the relative protective effects of the ligands were 4.5 and 1.2 respectively. The inactivation of receptorestradiol and receptor-4-hydroxytamoxifen complexes was not due to modification of the ligands by diethylpyrocarbonate since the mobilities of the ligands, analyzed by thin layer chromatography, did not change after incubation with diethylpyrocarbonate (not shown). The susceptibility of the receptor to inactivation by diethylpyrocarbonate, when either activated (30 min at 25°C) or stabilized

expressed as a percentage of the specific binding of the ligand in untreated cytosol. The specific binding of the two ligands in cytosol is plotted vs the diethylpyrocarbonate concentration: (R-E,, 0) cytosol prelabeled with [3H]eetradiol; (R-OHT, n ) cytosol prelabeled with [“H]4-hydroxytamoxifen; (R + E,, 0) cytosol postlabeled with [3Hjestradiol; (R --*OHT, a) cytosol postlabeled with [3H]4-hydroxytamoxifen.

by molybdate, was compared with that of the native receptor. Activation did not significantly change the effect of diethylpyrocarbonate on liganded receptor whereas molybdate slightly decreased the effect of both liganded and unliganded receptors (not shown). To determine whether the diethylpyrocarbonateinduced decrease in the cytosol binding of estrogen and antiestrogen resulted from a decrease in binding site concentration, from a decrease in the equilibrium affinity constant, or both, saturation experiments with estradiol and 4-hydroxytamoxifen were performed using cytosol treated with increasing concentrations (from 0 to 8 mM) of diethylpyrocarbonate. Scatchard plots of a typical experiment are shown in Fig. 4. In agreement with previous studies 1361, untreated cytosol appeared to contain a single class of binding sites for the two ligands, with very

Differential inhibition of estrogen and antiestrogen binding

ESTRADIOL

431

4-HYDROXYTAMOXIFEN 4 . t

LIGAND

BOUND

(nM)

Fig. 4. Saturation analysis of the binding of estradiol and Chydroxytamoxifen to control and diethylpyrocarbonate-inactivated estrogen receptor. Portions of the uterine cytosol prepared in PSO buffer and adjusted to 2 mg protein/ml were incubated with: 0, 1, 2, 4 or 8 mM diethylpyrocarbonate for 0.5 h at 0°C. They were then supplemented (lo%, v/v) with 0.3 M histidine and &ubated for 0.5 h at 0°C. Portions were diluted I-fold with P50 and incubated for 20 h at 0°C with increasing concentrations of [‘Hlestradiol or [‘H]4_hydroxytamoxifen (0.0410 nM) in the absence or presence or the corresponding radioinert ligand (2pM) for determination of total or nonspecific binding. Total concentrations of labeled ligands (_T)were determined by direct counting of aliquots whereas concentrations of charcoalresistant bound ligands (B, and B,, corresponding to cytosol labeled in the absence or presence of radioinert ligand, respectively) were determined by a 0.5 h charcoal assay after addition of 2 mg ovalbumin/ml. Concentrations of specifically bound ligands were calculated from the T, B,, and B, values as described in Experimental. Concentrations of unbound ligands were calculated as T - B,. These values were then used for Scatchard representation of the specific binding of [3H]estradiol (A) and [‘H]4_hydroxytamoxifen (B) in control (0) and diethylpyrocarbonate-treated (A, 1 mM; V, 2mM; n , 4 mM; 0, 8 mM) cytosol. similar binding parameters: N N 1.3 pmol/mg protein and KA N 1.2 x 10” M-’ for estradiol; N N 1.3 pmol/ mg protein and KA N 1.3 x 10” M-’ for 4-hydroxy-

tamoxifen. Diethylpyrocarbonate action led to a progressive decrease in the concentration of binding sites, which was more marked for estradiol (Fig. 4A) than for Chydroxytamoxifen (Fig. 4B) with no significant change in the apparent affinity constants, excepted for the binding of estradiol to 8 mhl-diethylpyrocarbonate-treated cytosol. A fraction of diethylpyrocarbonate-modjied estrogen receptor interacts with high afinity with triphenylethylene antiestrogens and with very low afinity with estrogens To determine the properties of the diethylpyrocarbonate-modified receptor, which still bound 4-hydroxytamoxifen with high affinity but did not significantly bind estradiol, the sedimentation in the dissociation kinetics of sucrose gradients, 4-hydroxytamoxifen and the ligand specificity of the modified receptor were compared to those determined with the intact receptor. Dissociation of 4-hydroxytamoxifen from the 4 mhl-diethylpyrocarbonate-modified receptor was a pseudo-first order process with a half-dissociation time of N 19 h at 0°C (Fig. SA) and -20 min at 20°C (Fig. 5B). Although, these values are -9- and _ 6-fold lower than those corresponding to the intact receptor (halfdissociation time of N 180 h and N 120 min respectively) they still reflect a strong interaction between the modified receptor and the ligand. Diethyl-

pyrocarbonate increased the dissociation rate of preformed receptor-4-hydroxytamoxifen complex to the same extent (not shown), suggesting that its action on the complex was qualitatively similar to that on the naked receptor. Diethylpyrocarbonate did not change the sedimentation coefficient of the receptor in high salt sucrose gradients, all the estradiol- and 4-hydroxytamoxifen-receptor complexes formed from control and treated cytosol, sedimented at 4.5 S (not shown). Our inability to obtain direct evidence of the binding of estradiol to the fraction of diethylpyrocarbonate-modified receptor which still bound 4-hydroxytamoxifen, might have been due to a low affinity of estradiol for this modified receptor. Since competitive binding experiments more readily reveal low affinity interactions than direct binding experiments [35], we compared the ability of different estrogens and antiestrogens to compete with [3H]4hydroxytamoxifen for binding to the native (Fig. 6A) and 4 mM-diethylpyrocarbonate-modified receptors (Fig. 6B). All the compounds studied totally inhibited the specific binding of [3H]4-hydroxytamoxifen to both the modified and the intact receptor, suggesting that they still interacted with the modified receptor. However, the relative affinity constants of the compounds for the two receptor states, calculated from concentrations inhibiting 50% of specific [‘H]4hydroxytamoxifen binding after 4 or 20 h of incubation at 20°C varied considerably according to the compound (Table 1). The relative affinity constants of estrogens were dramatically lower for the modified than for the intact receptor (by at least 200-fold for

JEAN LOUISBORGNA

432

and JACQUELINE SCALI

TIME (hours)

Fig. 5. Dissociation kinetics of 4-hydroxytamoxifen from intact and diethylpyrocarbonate-modified estrogen receptors. Cytosol treated or not with 4mM diethylpyrocarbonate for 0.5 h at 0°C was supplemented (lo%, v/v) with 0.3 M histidine. Samples were then incubated with 5 nM [3H]4-hydroxytamoxifen in the absence or presence of 1 PM radioinert 4-hydroxytamoxifen either for 20 h at 0°C (A) or for 1 h at 0°C then 1 h at 20°C (B). The stability and dissociation of complexes at 0°C (A) and 20°C (B) were then assayed at various times, by a 1 h of charcoal treatment at 0°C as described in Experimental. The saturable binding

of [3H]4-hydroxytamoxifen at 0 and 20°C in untreated (F& 0 and 0) and diethylpyrocarbonate-treated (RDEPC,H and 0) cytosol is plotted as a percentage of the corresponding value at time 0. 0 and n , saturable binding in the absence of radioinert Chydroxytamoxifen; 0 and 0, saturable binding in the presence of 1 PM radioinert Chydroxytamoxifen. estradiol, 600-fold for estriol and llO-fold for the nonsteroidal estrogen diethylstilhestrol). In contrast, the low affinity constants of the two triphenylethylene antiestrogens tested, i.e. tamoxifen and Cl 628, underwent little change when the receptor was modified (2- to 3-fold decrease for CI 628 and G 2-fold increase for tamoxifen). Characteristics of diethylpyrocarbonate inactivation of the estrogen receptor

The pH-dependence of receptor inactivation was studied in citrate (from pH 4 to pH 7) and phosphate

0

Lb

1

10-Q lb’

COMPETITOR

10.’

d

,a 16’

d

CONCENTRATION

IM)

Fig. 6. Inhibition of the binding of 4-hydroxytamoxifen to control and diethylpyrocarbonate-modified estrogen receptors by estrogens and antiestrogens. Cytosol, treated or not for 0.5 h at 0°C with 4mM diethylpyrocarbonate, was supplemented with 30 mM histidine and agitated for 0.5 h at 0°C. Aliquots of the two samples were then incubated at 20°C with 5nM [3H]4-hydroxytamoxifen with or without increasing concentrations of radioinert estrogens and antiestrogens. Binding of [3H]4-hydroxytamoxifen was determined after 4 h competition using a 0.5 h charcoal assay at 0°C. The binding in control (A) and diethylpyrocarbonatetreated cytosol (B) is plotted against the concentration of unlabeled competitor: (DES, 0) diethylstilbestrol; (E2, 0) estradiol, (E,, V) estriol, (OHT, 0) 4-hydroxytamoxifen, (CI, n ) CI 628, (T, V) tamoxifen.

buffer (from pH 7 to pH 9). Neutral and basic pH resulted in a more pronounced inactivation of recep tor than acidic pH (not shown). To gain additional information about the inactivation of the receptor by diethylpyrocarbonate, we tested the effect of hydroxylamine, which is a strong nucleophile capable of reversing the modification of histidyl and tyrosyl residues by the reagent [31].

Table 1. Relative affinity constants of estrogens and antiestrogens for intact and diethylpyrocarbonatemodified estroacn -tars 4-h competition RAC

4-Hydroxytamoxifen Diethylstilbestrol Estradiol Estriol CI 628 Tamoxifen

20-h competition RAC

Intact

Modified

RAC Ratio

IOil 270 100 7.1 1.2 0.43

100 2.5 0.49 0.012 0.49 0.49

110 200 590 2.4 0.88

1

Intact

100 >I000 220 II I.5 0.30

Modified

loo 4.0 0.93 0.015 0.58 0.58

RAC Ratio

1 > 250 240 730 2.6 0.52

Binding curves similar to those shown in Fig. 6, related to competition binding experiments, were used to determine the concentrations of competing estrogens and antiestrogens which inhibited 50% of the specific binding of [‘H]4_bydroxytamoxifen to intact and diethylpyrocarbonate-modified estrogen receptors after 4 and 20 h competition at 20°C. From these values the apparent relative affinity constants of competitors (related to those of Chydroxytamoxifen) were determined for intact and modified estrogen receptors as described in Experimental, taking as 100 the relative a&tity constants of 4hydroxytamoxifen for both forms of receptor. The ratio of relative affinity constants of each compound for intact and modified estrogen receptors was also calculated for both competition times. Data given are the mean of duplicate determinations in two separate experiments. Experimental variations were under 30%.

Differential inhibition of estrogen and antiestrogen binding

433

dissociation rate from the diethylpyrocarbonatemodified receptor (partial modification with 1.5 mM reagent, leaving 30% residual binding). In contrast, Chydroxytamoxifen dissociated from the rescued receptor with a half-dissociation time of -60 min, which is between that of the treated (h- 20 min) and untreated receptor (N 150 min) (not shown).

DISCUSSION

0 0.1

I

I

0.2

0.3

HVDROXVLAMlNE

0.4

(M)

Fig. 7. Reversal of diethylpyrocarbonate inactivation of estrogen receptor by hydroxylamine. Cytosol, treated or not with 8 mM diethylpyrocarbonate, was supplemented with 30mM histidine. Samples were then incubated with 10% (v/v) water or hydroxylamine solutions (1.14.4 M) for 20 h at 0°C. Aliquots were incubated for 20 h at 0°C with [‘Hlestradiol or [‘H]4-hydroxytamoxifen in the absence or presence of 1 yM radioinert ligand, and the specific binding of labeled ligands was determined as described in Experimental. (A) The specific binding of [3H]estradiol (E,, 0 and 0) and [3H]4-hydroxytamoxifen (OHT, n and 0) from control (&, 0 and n ) and diethylpyrocarbonate-treated cytosol (RoEpc, 0 and l ) is plotted against the concentration of hydroxylamine. (B) The specific binding of [‘Hlestradiol (0) and [3H]4-hydroxytamoxifen (0) in diethylpyrocarbonate-treated cytosol is expressed as a percentage of the corresponding binding in control cytosol.

As shown in Fig. ?‘A, increasing concentrations of hydroxylamine progressively decreased estradiol and hydroxytamoxifen binding in intact cytosol, whereas it increased binding in 8 mM-diethylpyrocarbonatetreated cytosol. When expressed as a percentage of binding measured after the action of hydroxylamine on intact cytosol, the binding of the two ligands in cytosol treated consecutively with diethylpyrocarbonate and hydroxylamine showed high values in both cases, reaching a plateau above 80% (estradiol) or 90% (Chydroxytamoxifen) at hydroxylamine concentrations 2 0.2 M (Fig. 7B). To characterize the properties of the receptor when treated consecutively with diethylpyrocarbonate and hydroxylamine (“rescued” receptor), the dissociation rates of estradiol and 4-hydroxytamoxifen from the treated receptor were compared to those from the intact receptor. Hydroxylamine alone did not change the dissociation rates of the ligands from the intact receptor. At 2O”C, estradiol dissociated from the rescued receptor (treated with 5 mM diethylpyrocarbonate and then 0.2 M hydroxylamine) with a half-dissociation time of = 35 min, which is IO-fold lower than that from the control receptor (_ 350 min) (not shown). This rate was equivalent to the

Titration of diethylpyrocarbonate-modified estrogen receptor with the two types of ligand revealed that the capacity of the receptor to bind estradiol was practically abolished by concentrations of diethylpyrocarbonate 2 4 mM whereas its capacity to bind Chydroxytamoxifen was decreased to only N 50% of that of the control. This suggests that the action of the reagent on native estrogen receptor results in two almost equivalent populations, the first of which does not bind estradiol and 4-hydroxytamoxifen significantly, and the second of which still interacts with high affinity with 4-hydroxytamoxifen, but not with estradiol. Experiments based on the competition of [3H]4-hydroxytamoxifen with unlabeled estradiol and 4-hydroxytamoxifen for binding to the receptor showed that estradiol does in fact bind to the second receptor population but with an affinity -200-fold lower than that of 4-hydroxytamoxifen even though the two ligands display very similar affinities for the intact receptor (Table 1). Taking into account the slight decrease in the affinity of 4-hydroxytamoxifen when the receptor is modified (reflected by the 6to 9-fold increase in the dissociation rate of the complex), the affinity of estradiol for the second receptor population appears to be at least lOOO-fold lower than its affinity for the native receptor. This dramatic loss in affinity when the receptor is modified explains our inability to obtain direct evidence of the binding of estradiol to modified receptor under the non-equilibrium conditions of the charcoal assay; taking into account the relative KA of estradiol (Table 1) and the half-dissociation time of 4-hydroxytamoxifen from the modified receptor (Fig. 5A) and assuming that the association rates of the two ligands for the modified receptor are similar, then the halfdissociation time of the modified receptor-estradiol complex would be <5 min at 0°C. The low affinity of estradiol for modified receptor also accounts for the discrepancy in the measurements of the nonspecific binding of 4-hydroxytamoxifen in diethylpyrocarbonate-treated cytosol, depending on whether estradiol or 4-hydroxytamoxifen is used as the unlabeled ligand, since estradiol, even at micromolar concentrations, was only partially effective in preventing the binding of 4-hydroxytamoxifen to the modified receptor (Fig. 6B). It is still not known whether the two quantitatively equivalent populations of modified receptor result from the action of diethylpyrocarbonate on two

434

JEAN LOUIS E%ORGNA

pre-existing forms of receptor or from its action on a single form of receptor. Under the first hypothesis, although the two forms of native receptor, bind both estrogens and antiestrogens (Fig. 4), they would have to differ at or near the ligand binding site since diethylpyrocarbonate would inhibit the binding of both ligands to the first form, but only the binding of estrogens to the second form. However, none of the physicochemical or immunological properties, or the ligand binding specificity of the native receptor suggest that it is composed of two quantitatively equivalent but qualitatively different forms. Consequently, the second hypothesis appears more plausible; this would imply that: (i) two types of modification of a single form of receptor occur upon treatment by diethylpyrocarbonate; and (ii) these two types of modification are mutually exclusive since increasing concentrations of reagent do not increase the proportion of the first population at the expense of the second. This could result from the mutually exclusive modification of at least two distinct aminoacid residues: modification of one residue would impair both estrogen and antiestrogen binding; modification of the other would dramatically decrease the binding of estradiol but not that of 4-hydroxytamoxifen. Protection of the ligand binding site by estradiol or 4-hydroxytamoxifen suggests that these aminoacid residues could be located at or close to the ligand binding site. However, ligand-induced conformational receptor changes could result in decreased accessibility or reactivity of these aminoacid residues to or with diethylpyrocarbonate. There is good agreement between the differential binding inhibition by diethylpyrocarbonate and protection of the binding site, since estradiol, whose binding was sensitive to the two types of modification, was more effective in protecting the ligand binding site than 4-hydroxytamoxifen, whose binding was sensitive to only one type of modification. The facts that stabilization of the receptor by molybdate or activation of the receptor-ligand complexes did not markedly change the effects of diethylpyrocarbonate, suggest that the accessibility or reactivity of target aminoacids was not altered under these conditions. Apart from its effect on these aminoacid residues that are essential for high affinity binding of estrogens and antiestrogens to the receptor, diethylpyrocarbonate appears to modify other residues whose integrity is not crucial for ligand binding, since (i) the dissociation rates of ligands from the hydroxylaminerescued receptor were higher than those from the control receptor, and (ii) although low concentrations of diethylpyrocarbonate left a fraction of receptor capable of binding with high affinity estradiol, dissociation rate of the latter from the treated receptor was increased. The results observed with estradiol and 4-hydroxytamoxifen were generalized using competition experiments. As with estradiol, both steroidal and

and JACQUELINE SCALI

nonsteroidal estrogens showed considerable losses in affinity when the receptor was modified, whereas the binding of nonsteroidal antiestrogens (displaying either high or low affinity for the intact receptor) was not markedly impaired by modification of the receptor (Fig. 6, Table 1). The differential inhibition of estrogen and antiestrogen binding to the estrogen receptor by diethylpyrocarbonate indicates that certain aminoacid residues of the ligand binding site that are involved in the binding of estrogens, are only slightly or not at all involved in the binding of triphenylethylene antiestrogens. However other aminoacid residues such as cysteyl groups also appear to be involved in both antiestrogen and estrogen binding, since Nethylmaleimide identically decreased the binding of estradiol and 4-hydroxytamoxifen to the receptor (unpublished results). Since the diethylpyrocarbonate effects were almost totally reversed by hydroxylamine, the two or more modified aminoacid residues crucial for ligand binding to the receptor are probably histidine(s) and/or tyrosine(s). Moreover, considering that modification of the estrogen receptor by tetranitromethane, which is usually highly selective for nitration of tyrosyl residues (although reaction with cysteyl, histidyl, methionyl and tryptophyl residues have also been reported [37]), produced results that were very similar to those caused by diethylpyrocarbonate (unpublished results) it is very likely that at least one of the two residues is tyrosine. Electrophilic affinity labels have been used in direct demonstrations of the presence of histidine at or close to the catalytic or binding site of several enzymes [4,6, 71 and binding proteins [26,29] interacting with steroid hormones. Other studies using reagents of nucleophilic aminoacids have either suggested [16,22,27,38] or demonstrated [23] the presence of tyrosine residues at or close to the catalytic or binding site of such proteins. Modification of the estrogen receptor by diethylpyrocarbonate, as well as the previously reported use of one monoclonal antireceptor antibody [3], make it possible to differentiate the binding of estrogens and antiestrogens to the receptor. This could also be an interesting method for probing ligands of steroid receptors; ligands that are found to behave like the hormone with the modified receptor, may be probably agonists of the hormone, and ligands that behave differently, such as triphenylethylene antiestrogens in this study, may be antagonists. Work is now in progress to check this hypothesis, using a wide variety of ligands of the estrogen receptor. Preliminary results agree with the hypothesis since only the 178 -hydroxy function of steroidal estrogens or their 4-hydroxy counterpart in diethylstilbestrol (unlike their counterpart in certain antiestrogen molecules), appears to be directly involved in decreasing ligand affinity for the diethylpyrocarbonate-modified receptor.

Differential inhibition of estrogen and antiestrogen binding Acknowledgements-This work was supported by the “Institut National de la Sante et de la Recherche Medicale”, the University of Montpellier I, the “Fondation pour la Recherche Medicale” and the “Association uour la Recherche sur le Cancer”. We thank M. Egea and E. Barrid for their excellent secretarial assistance.

REFERENCES

1. Rochefort H. and Borgna J. L.: Differences between oestrogen receptor activation by oestrogen and antioestrogen. Nature 292 (1981) 257-259. 2. Evans E., Baskevitch P. P. and Rochefort H.: Estrogen receptor-DNA interaction. Difference between activation by estrogen and antiestrogen. Eur. J. Biochem. 128 (1982) 185-191. 3. Fauque J., Borgna J. L. and Rochefort H.: A monoclonal antibody to the estrogen receptor inhibits in virro criteria of receptor activation by an estrogen and an antiestroaen. J. biol. Chem. 260 (1985) 15547-15553. 4. Ganguly-M. and Warren J. C.: Afhnity labeling of steroid binding sites. J. biol. Chem. 246, (1971) 36463652. 5. Chin C. C. and Warren J. C.: Synthesis of 2u-, 6a- and 68-bromo-progesterone and study of the binding site of 20/I-hydroxysteroid dehydrogenase. Biochemistry 11 I1972) 2720-2725. 6. Pons ‘M., Nicolas J. C., Boussioux A. M., Descomps B. and Crastes de Paulet A.: Affinity labelling of an histidine of the active site of the human placental 17/Ioestradiol dehydrogenase. FEBS Len. 36 (1973) 23-26. 7. Boussioux A. M., Pons M., Nicolas J. C., Descomps B. and Crastes de Paulet A.: Enhancement of the catalytic activity of the human placental 17/I-oestradiol dehydrogenase by carboxymethylation of an histidyl residue of the active site. FEBS Letf. 36, (1973) 27-30. 8. Onoda M., Haniu M., Yanagibashi K., Sweet F., Shivelv J. E. and Hall P. F.: AiBnitv alkvlation of the active site of C2 1 steroid side chain cleavage cytochrome P-450 from neonatal porcine testis. A unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone. Biochemistrv 25. (1987) 657462. 9. Sweet F. and ‘Samant B. R.: Bifunctional enzyme activity at the same active site. Study of 3a and 208 activity by affinity alkylation of 3a, 20/I-hydroxysteroid dehydrogenase with 17-(bromoacetoxy)steroids. Biochemistry 19 (1980) 978-986. 10. Thomas J. L. and Strickler R. C.: Human placental 17/l-estradiol dehydrogenase and 20a-hydroxysteroid dehydrogenase. Studies with 6/I-bromoacetoxyprogesterone. J. biol. Chem. 258 (1983) 1587-l 590. 11. Simons S. S. Jr and Thompson E. B.: Dexamethasone 21-mesylate. An affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells. Proc. natn. Acad. Sci. U.S.A. 78 (1981) 3541-3545. 12. Eisen H. J., Schleenbaker R. E. and Simons S. S. Jr: Affinity labeling of the rat liver glucocorticoid receptor with dexamethasone 21-mesylate. J. biol. Chem. 256 (1981) 12920-12925 13. Robertson D. W., Wei L. L., Hayes J. R., Carlson K. E., Katzenellenbogen J. A. and Katzenellenbogen B. S.: Tamoxifen aziridines. Effective inactivators ofthe estrogen receotor. Endocrinofoav 109 (1981) 1298-1300. 14. Katzenellenbogen J. A., Carl& K. E., H&man D. F., Robertson D. W., Wei L. L. and Katzenellenbogen B. S.: Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen axiridme. J. biol. Chem. 258 (1983) 3487-3495. 15 Le Gaillard F. and Dautrevaux M.: Affinity labelling of human transcortin. Biochim. biophys. Acfa 4% (1977) 312-323.

435

16. Westphal U., Stroupe S. D., Kute T. and Cheng S. L.: Steroid interactions with progesterone-binding globulin, J. steroid Biochem. 8 (1977) 367-374. 17. Saavedra A. and Beato M.: Influence of chemical modifications of amino acid side chains on the binding of progesterone to uteroglobin. J. steroid Biochem. 13 (1980) 1347-1353. 18. Coty W. A.: Reversible dissociation of steroid hormonereceptor complexes by mercurial agents. J. biol. Chem. 255 (1980) 8035-8037. 19. Kalimi M. and Banerji A.: Role of sulfhydryl modifying reagents in the binding and activation of chick oviduct progesterone-receptor complex. J. steroid Biochem. 14, (1981) 593-597. 20. Baker M. E. and Fanestil D. D.: Diethylpyrocarbonate inhibition of estrogen binding to rat alpha-fetoprotein. Evidence than one or more histidine residues regulate estrogen binding. Biochem. biophys. Res. Comm& 98 (1981) 976-982. 21. Ikeda M.: Identification of thiol group at the estrogenbinding site on the cytoplasmic receptor from rabbit uterus by affinity labeling. Biochim. biophys. Acta 718 (1982) 66-73. 22. Kuhn R. W. and Raymoure W. J.: The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig. J. sreroid Biochem. 17 (1982) 459460. 23. Le Gaillard F., Racadot A. and Aubert J. P.: Modification of human transcortin by tetranitromethane. Evidence for the implication of tyrosine residue in cortisol binding. Biochimie 64 (1982) 153158. 24. Shyamala G. and Daveluy A.: Inhibition of the binding of dexamethasone to mammary cytoplasmic glucocorticoid receptor by phenylglyoxal. J. biol. Chem. 20 (1982) 11976-11981. 25. Baker M. E. and Terry L. S.: Diethylpyrocarbonate, a histidine selective reagent inhibits progestin binding to chick oviduct cytosol. Steroids 42 (1983) 593602. 26. Holmes S. D. and Smith R. G.: Identification of histidine and methionine residues in the active site of the human uterine progesterone receptor with the afhnity labels 1la- and 16x-(bromoacetoxy)progesterone. Biochemistry 22 (1983) 1729-l 734. 27. Chang C. H., Lob1 T. J., Rowley D. R. and Tindall D. J.: Affinity labeling of the androgen receptor in rat prostate cytosol with 17B-[(bromoacetyl)oxy]-5aandrostan-3-one. Biochemistry 23 (1984) 2527-2533. 28. Baker M. E., Sklar D. H., Terry L. S. and Hedges M. R.: Diethylpyrocarbonate, a histidine reagent, inhibits estrogen binding to receptor protein in rat uterus cytosoL Biochem. jnr. 11 (1385) i33-238. 29. Khan M. S. and Rosner W.: 17B-bromoacetoxvdihydrotestosterone is an affinity Isabel for human testosterone-estradiol binding globulin (TeBG). Endocrine Society Meeting (1986) Abst. n”70. 30. Miles E. W.: Modification of histidyl residues in proteins by diethylpyrocarbonate. Meth. Enzym. 47 (1977) 43143. 31. Melchior W. B. and Fahmey D.: Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with a-chymotrypsin, papain and pancreatic ribbnuclease at pH 4. Biochemisrrv 9 (1970) 251-258. 32. Gross S. C., Kumar ‘S. A. and Dickerman H. W.: Diethylpyrocarbonate inhibition of estradiol receptor binding to oligonucleotides. Molec. cell. Endocr. 22 (1981) 371-384. and turbidimetric 33. Layne E.: Spectrophotometric methods for measuring proteins. Meth. Enzym. 3 (1957) 444454. 34. Blondeau J. P. and Robe1 P.: Determination

of proteinligand binding constants at equilibrium in biological samples. Eur. J. Biochem. 55 (1975) 375-384.

436

JEAN Louts BORGNAand

35. Korenman S. G.: Relation between estrogen inhibitory activity and binding to cytosol of rabbit and human uterus. Endocrinology 87 (1970) 1119-I 123. 36. Borgna J. L. and Rochefort H.: High-affinity binding to the estrogen receptor of [‘H]4_hydroxytamoxifen, an active antiestrogen metabolite. Molec. ceil. Endocr. 20 (1980) 71-85.

JACQUELINE SCALI

37. Riordan J. F. and Vallee B. L.: Nitration with tetranitromethane. Merh. Enzym. U (1972) 515-521. 38. Inano H. and Tamaoki B. I.: Functional site of human placental estradiol l’ljl-dehydrogenase involves tyrosine residues, as evidenced by reaction to p-nitrobenzenesulfonylfluoride. J. steroid Biochem. 19 (1983) 1677-1679.