0013-7227/90/1275-2530$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 5 Printed in U.S.A.

Localization of the Vicinal Dithiols Involved in Steroid Binding to the Rat Glucocorticoid Receptor PRADIP K. CHAKRABORTI, WOLFGANG HOECK, BERND GRONER, AND S. STONEY SIMONS, JR. The Steroid Hormones Section (P.K.C., S.S.S.), NIDDK/LAC, National Institutes of Health, Bethesda, Maryland 20892; and the Friedrich Miescher-Institut (W.H., B.G.), Basel, Switzerland

ABSTRACT. Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to be Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with

A

LL steroid receptors contain multiple cysteine residues (Ref. 1 and references therein), but very little is known about their oxidation state or function in the native protein. The importance of some free thiols in steroid binding to cognate receptors has been recognized for many years (2, 3) and, for the glucocorticoid receptor, has led to the tenet that reduced thiols are required for steroid binding (4, 5). Recently, however, we have shown that this concept needs refinement. Experiments with the sterically small, thiol-specific reagent methyl methanethiolsulfonate (MMTS) revealed that steroids can still bind to oxidized glucocorticoid receptors if the steric bulk of the oxidized thiols is sufficiently small (6). A similar situation may exist for the estrogen receptor (7). Six lines of evidence led us to propose a model (see Fig. 1) in which 2 closely spaced thiols (or vicinal dithiols) are involved in glucocorticoid binding (6, 8): 1) [3H]dexamethasone (Dex) binding, but not affinity labeling by dexamethasone 21-mesylate (Dex-Mes) (9), is

Received May 14, 1990. Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, NIDDK/LMCB National Institutes of Health, Bethesda, Maryland 20892.

MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aPl) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr837Arg673. {Endocrinology 127: 2530-2539, 1990)

retained after pretreatment of the receptors with >1 mM MMTS; 2) lower concentrations of MMTS (-0.3 mM) block steroid binding via a slow, intramolecular reaction; 3) additional MMTS could not convert the 0.3 mM MMTS pretreated, steroid nonbinding form of receptor (IV) to the steroid binding form (II); 4) added dithiothreitol (DTT) readily restored steroid-binding activity to the 0.3 mM MMTS pretreated, nonbinding receptors; 5) increasing amounts of DTT converted those receptors pretreated with >1 mM MMTS first to the steroid nonbinding form (IV) and then to the original binding form of the receptor; and 6) arsenite, which specifically reacts with vicinal dithiols, is extremely potent in blocking steroid binding. In each of these studies, it appeared that Cys-656 of the rat receptor, which is affinity labeled by Dex-Mes (10), was 1 of the 2 thiols that participate in steroid binding (6, 8). The carboxyl terminal cysteine (Cys-754 of the rat receptor) is a candidate for the other part of the vicinal dithiol grouping, which reacts with MMTS and arsenite to block steroid binding, because Cys-754 is photoaffinity labeled by triamcinolone acetonide (11). However, this conclusion is complicated by fact that, at least for glu-

2530

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

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Chemicals

FIG. 1. Model of chemical modifications of the vicinal dithiols in the steroid-binding cavity of glucocorticoid receptors. A hypothetical steroid-binding cavity in part of the receptor protein and Cys-656 are shown. Steroid can bind to the fully reduced, unmodified receptor (I) and to the mixed disulfide form (II), but not to the intramolecular disulfide form (IV) or to the arsenite-complexed form (V). Affinity labeling of only the unmodified receptor (I) is possible.

cocorticoid receptors, high affinity steroid binding occurs only to steroid-free and unactivated1 complexes, which are thought to be associated with several nonreceptor molecules (12, 13). These molecules appear to include hsp90 (13-15), which is actually a mixture of the 2 proteins hsp84 and hsp86 (16, 17), a 59 K protein (18), and possibly a 72 K (19) and 40 K protein (20). Thus, in addition to the 20 thiols of the glucocorticoid receptor (21), there are the 6 and 7 thiols of hsp84 and hsp86, respectively (17), and probably other thiols of the other proteins, that could be involved in forming the kinetically defined intramolecular disulfide resulting from treatment of the receptor with MMTS (6). The objective of the present study was to locate the two thiols that form the MMTS-induced disulfide. Two different techniques were used. First, binding studies with two receptor fragments that had been preincubated with various concentrations of MMTS or arsenite were conducted to localize the vicinal dithiols. Second, Western blot analysis was used to determine the size of variously treated forms of receptor. Our results indicate that the vicinal dithiols, which are intimately involved in steroid binding and which react with MMTS to form an intramolecular disulfide, are restricted to a small region of the steroid-binding domain.

Nonradioactive Dex (Sigma, St. Louis, MO), [3H]Dex (40 or 47 Ci/mmol, Amersham Corp., Arlington Heights, IL), and [3H]Dex-Mes (37.3 or 49.9 Ci/mmol, DuPont-New England Nuclear, Boston, MA) were commercially available. TAPS (Ultrol grade) was purchased from Calbiochem (La Jolla, CA); sodium arsenite was from Baker (Sanford, ME). MMTS and iodoacetamide (IA, both stored at 0 C), were used as received from Aldrich (Milwaukee, WI). Trypsin (tosylphenylalanine chloromethylketone treated) and chymotrypsin (tosyllysyl chloromethylketone treated) were obtained from Worthington (Freehold, NJ) or Sigma (St. Louis, MO). 4-Chloro-l-napthol was purchased from Sigma. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), including Coomassie blue R-250 and Tween 20 (EIA grade), were from Bio-Rad (Richmond, CA). Fluorescent Ult-Emit auto radiography marker was from DuPont-New England Nuclear. ABC reagent for immunoperoxidase staining of Western blots was acquired from Vector Labs (Burlingame, CA). All nHlabeled samples were counted in Hydrofluor (National Diagnostics, Somerville, NJ) at 40 to 55% counting efficiency in a Beckman 5801 liquid scintillation counter with automatic cpmto-dpm conversion. Antibodies The monoclonal antireceptor antibody BUGR-2 (22) and the polyclonal anti-hsp90 antibody (23) were gifts from Dr. R. Harrison (University of Arkansas for Medical Science) and Drs. E. Appella and S. Ulrich (NCI, NIH), respectively. A polyclonal antibody (aPl) against the carboxyl terminal region of the rat glucocorticoid receptor has been described (24). Biotinylated antimouse and antirabbit second antibodies for Western blotting were purchased from Vector Labs. Buffers and solutions TAPS buffer is composed of 25 mM TAPS, 1 mM EDTA, and 10% glycerol. The pH of the TAPS buffer is adjusted to 8.2, 8.8, or 9.5 at 0 C with sodium hydroxide. Two-fold concentrated SDS sample buffer (2 X SDS) contains 0.6 M Tris (pH 8.85), 2% SDS, 0.2 M DTT, 20% glycerol, and bromphenol blue. Transfer buffer for Western blotting contains 25 mM Tris, 192 mM glycine, 20% methanol in water (pH ~8.3 at room temperature). Tris-buffered saline (TBS) was 20 mM Tris and 0.28 M NaCl in water (pH 7.5 at room temperature). Cells and preparation and binding/labeling of receptors

Materials and Methods Unless otherwise indicated, all operations were performed at OC. 1 The terms "activate" and "activation" are used in this article to describe the currently unknown mechanism by which initially formed receptor-steroid complexes, with little or no affinity for DNA or nuclei, are converted by manipulations such as heat, dilution, increased salt, or increased pH to complexes with relatively high affinity for DNA or nuclei.

The growth of HTC cells in spinner and monolayer cultures of Swim's S77 medium supplemented with 5% fetal and 5% newborn bovine serum (Biofluids, Rockville, MD) and 0.03% glutamine has been described (25). HTC cell cytosol containing the steroid-free receptors was prepared, stored in liquid N2, and labeled as described (26, 27). Briefly, 30% cytosol solutions containing 21 mM Na2MoO4 were prepared with three parts HTC cell cytosol, 2 parts pH 9.5 TAPS buffer, and 5 parts pH 8.8 TAPS buffer. The additional EtOH in those experiments

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

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with MMTS was kept constant at 1%. Steroid binding (or affinity labeling) was achieved by the addition of 20x stocks of [3H]Dex ± 550x nonradioactive Dex (final [3H]Dex concentration = 3 - 5 x 10""8 M) or of [3H]Dex-Mes ± 100X nonradioactive Dex (final [3H]Dex-Mes concentration -1.5 X 10~7M). After incubation for 2.5 h, the [3H]Dex-Mes-labeled solutions were quick-frozen at -78 C for subsequent SDS-PAGE analysis. The specifically bound [3H]Dex was determined by first adding a 10% dextran-coated charcoal solution (added volume, 20% of reaction solution volume) to remove free steroid and then subtracting the nonspecific binding seen in the presence of excess nonradioactive Dex. Protease digestion of receptors

Solutions of crude receptors (30%) were treated for 1 h with 14 Mg/ml of chymotrypsin (for 42 K fragments) or with 14 fig/ ml of trypsin (for 16 K fragments). A 10-fold excess (wt/wt) of protease inhibitor (aprotinin with chymotrypsin and soybean trypsin inhibitor with trypsin) was added to block further digestion. The integrity of the proteolyzed receptors was always ascertained by affinity labeling with [3H]Dex-Mes followed by SDS-polyacrylamide gel analysis and fluorography. The receptor fragments were used immediately.

Endo • 1990 Vol 127 • No 5

1:1000 (aPl), 1:50 (anti-hsp90 antibody), or 1:20 (BUGR-2 tissue culture medium) in TTBS] was added for > 2 h and then removed with 3 x 5 min washes of TTBS. The incubation with biotinylated secondary antibody and the subsequent immunoperoxidase staining with ABC reagent were conducted as recommended by Vector Labs. Immunoadsorption of receptors Immunopurified receptors were obtained by incubating 50 /A 30% cytosol solutions that had been labeled with 1.5 x 10~7 M [3H]Dex-Mes ± 100-fold excess nonradioactive Dex with 1:30 diluted aPl for 2 h. Slurries (100 nl) of Pansorbin (Calbiochem) prewashed in HEG100 [10 mM HEPES, 1 mM EDTA, 10% glycerol, 100 mM NaCl (pH 7.6 at room temperature)] were added, and the solutions mixed gently for 1 h. Workup consisted of cadding 1 ml of HEGioo followed by centrifugation at 15,000 x g for 5 min. The resultant Pansorbin pellets were extracted with 170 nl of 2 x SDS for 5 min at 100 C. After centrifugation (2,320 X g for 2 min), the supernatants containing the extracted immunopurified receptors were analyzed directly by SDSPAGE on 15% gels, which were fluorographed as described above.

Results

PAGE The preparation of samples for reducing gels and the procedures for electrophoresis are as described (28). For nonreducing gels, the samples were treated with 2 x SDS without DTT. Constant percentage acrylamide gels (between 9 and 15% with a 1:40 ratio of bisacrylamide to acrylamide) were run in a watercooled (15 C) Protean II slab gel apparatus (Bio-Rad) at 30 mA/gel (25 mA/gel for 15% gels; 20 mA/gel while in the stacking gel for all gels). Gels were fixed and stained in 50% methanol, 7.5% acetic acid containing 0.01% Coomassie blue R-250, destained in 10% methanol, 7.5% acetic acid, incubated for 1 h in Enhance (DuPont-New England Nuclear) and 30 to 60 min in 10% Carbowax PEG 8000 (formerly PEG 6000; Fisher, Pittsburgh PA) with constant shaking at room temperature, dried on a Bio-Rad model 443 gel drier at 60 C with a sheet of dialysis membrane backing (Bio-Rad) directly over the gel to prevent cracking, marked with Ult-Emit at the positions of the mol wt markers (from Pharmacia P-L Biochemicals), and fluorographed for 7 to 12 days at -80 C with Kodak XOMAT XAR-5 film. Western blotting The procedure of Towbin et al. (29) was used. Briefly, the SDS-polyacrylamide gel was equilibrated in transfer buffer for at least 30 min at room temperature. Electrophoretic transfer to nitrocellulose was conducted in a well ventilated area (or at 4 C) in a Transblot (Bio-Rad) apparatus (~15 h at 100 mA, then ~250 mA for 90 min). The nitrocellulose was stained with Ponceau S (0.02% Ponceau S and 0.04% glacial acetic acid in water) to visualize the transferred protein, incubated with blocking solution (2% Carnation nonfat dried milk in TBS) for 45 min, and washed with 0.1% Tween in TBS (TTBS) for 15 min. Primary antibody [diluted 1:2000 (BUGR-2 acites fluid),

Both vicinal dithiols are present in the 42 K chymotrypsin fragment of steroid-free receptors Digestion of both unactivated and activated [3H]DexMes-labeled glucocorticoid receptors (26) and of steroidfree receptors (data not shown) with moderate concentrations of chymotrypsin at 0 C yields exclusively a 42 K fragment. The dissociation constant (Kd) and binding capacity of [3H]Dex to this truncated receptor were, within experimental error, identical to that of the intact receptor (data not shown). The amino terminus of this chymotrypsin fragment is known to be heterogeneous but tightly clustered around positions 409 and 413 (30). The carboxyl terminus is probably the same as that of the native receptor (31). Thus, the 42 K fragment lacks 5 of the 20 cysteines of the rat receptor. Nevertheless, the effects of MMTS and of arsenite on the steroidbinding properties of the 42 K fragment were found to be identical to those seen for the intact receptor (6, 8). Preincubation of the 42 K chymotrypsin fragment with MMTS gave a bimodal dose-response curve for [3H]Dex binding (Fig. 2A). The decrease in [3H]Dex-Mes labeling of the truncated receptor paralleled the decrease in [3H] Dex binding, but did not return after incubation with higher concentrations of MMTS (Fig. 2A inset). DexMes labeling of the 42 K receptor was inhibited by lower MMTS concentrations than was required for many other proteins (Fig. 2A insert and data not shown). The effects of 0.3, but not 3.0, mM MMTS on steroid binding and labeling were very time dependent (data not shown). All effects of MMTS were readily reversed by DTT. In

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2533

Cont rol

VICINAL DITHIOLS INVOLVED IN STEROID BINDING -

fa

100

MMTS(mM): 0 ['H]Dex: - +

3

0.3

J_l

ent

"o

80

o CD CL C/)

CO O) c

c

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X CD Q X



60

— — -

\

40

\

20 _ n •

«

/ •

i V^i

io-4

i , ., 10 3

1

i

i

i i 11 n

10 2

10"

MMTS (M)

•_

DTT(mM): ['H]Dex:

1 -

2 +

-

+

-

120 _

10 +

100 S 80 60 40 =6 CD

20

10

10" 1 0

10'

10' 3

10 2

S

10'

DTT (M) 3

FIG. 2. Effect of MMTS on [ H]Dex binding to 42 K chymotrypsin fragment of HTC cell receptors. A, Effect of MMTS preincubation on subsequent [3H]Dex binding. Duplicate samples of 42 K receptor fragments were pretreated with various concentrations of MMTS in absolute

EtOH for 2.5 h before the addition of [3H]Dex ± excess nonradioactive Dex (['HJDex). After a further 2.5-h incubation, the specific binding to receptors was determined after adding dextran-coated charcoal to remove free steroid (see Materials and Methods) and plotted against the concentration of added MMTS. The inset shows effect of MMTS on covalent labeling by [3H]Dex-Mes ± excess nonradioactive Dex in a separate experiment. B, DTT reversal of 3 x 10~4M MMTS inhibition of [3H]Dex binding to 42 K receptor fragments. Samples of 42 K receptor fragments that had been preincubated with 1% EtOH ± 3 x 1(T4 M MMTS for 2.5 h were treated with solutions of pH 8.8 TAPS buffer ± DTT. After 2.5 h of incubation, [3H]Dex ± nonradioactive Dex was added to duplicate tubes for 2.5 h. The average specific binding to receptors was determined as in A, expressed as percentage of EtOH control binding, and plotted vs. the final DTT concentration. The inset shows DTT reversal of MMTS inhibition of covalent labeling by [3H]Dex-Mes ± excess nonradioactive Dex in the same experiment. C, DTT reversal of 3.0 x 1(T;) M MMTS preincubation. Samples of 42 K receptor fragments were preincubated ± 3.0 x 10"3 M MMTS for 2.5 h followed by various concentrations of DTT for 2.5 h and then [3H]Dex ± nonradioactive Dex as in B. The average specifically bound [3H]Dex was determined and plotted as in B. The inset shows DTT reversal of MMTS inhibition of covalent labeling by [3H]Dex-Mes ± excess nonradioactive Dex in the same experiment. For each insert, the arrow (—>) indicates the 42 K fragment.

addition, 42 K receptors pretreated with 0.3-mM MMTS gave a simple sigmoidal dose-response curve for the regeneration of steroid-binding activity (Fig. 2B). A more complex, bimodal curve was seen for fragments pretreated with 3 mM MMTS (Fig. 2C), as expected from our model (Fig. 1). Very low concentrations of sodium arsenite (8) eliminated steroid binding and affinity labeling (Fig. 3). A 30min incubation at 0 C with a 2.5-fold excess of DTT reversed > 93% of the arsenite inhibition (data not shown). DTT also reversed the arsenite-induced blockage of affinity labeling by [3H]Dex-Mes (data not shown). Collectively, these data show that the 42 K fragment

retains the vicinal dithiols that are involved in steroid binding. Therefore, the five cysteines in the region of amino acids 1 to 400 can be eliminated as candidates for the vicinal dithiols. Both vicinal dithiols are present in the 16 K steroidbinding core fragment of the receptor We have recently shown that trypsin digestion of the native steroid-free rat receptor yields a 16 K fragment, which can be viewed as the core binding unit of the glucocorticoid receptor. The sequence of the 16 K core is Thr537-Arg673 and contains only 3 cysteines (31). MMTS

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Endo•1990 Vol 127* No 5

VICINAL DITHIOLS INVOLVED IN STEROID BINDING

2534

120 ;MMTS(|JM): _p__25_ 50 70 100 300 1000 3000

100

10

105

10"

['H]Dex: - + -- + Z T - * + - + - + - + - + ' S-

10 4

10_ 120

g

0

100

activity of these receptors was then assayed by incubating duplicate

preincubation of the 16 K fragment for 30 min caused a bimodal dose-response curve for [3H]Dex binding (Fig. 4A). This is in marked contrast to the 2.5-h preincubation that is required for such a response with the 98 K receptor (6). The low amount of binding at >1 mM MMTS appears to be due to a reduced affinity of the MMTS-modified 16 K fragment (see Discussion). As expected from our model (Fig. 1), MMTS inhibition of [3H]Dex-Mes labeling showed a simpler dose-response curve, with complete inhibition occurring at the same low MMTS concentration that blocked [3H]Dex binding (Fig. 4A inset). The inhibition of [3H]Dex binding to the 16 K core by all concentrations of MMTS could be reversed by DTT if the incubations were conducted in the presence of 20 mM Na2MoO4. In incubations without Na2MoO4, the effects of 0.3 mM MMTS were totally reversible by DTT, whereas those of 3 mM MMTS could be reversed to an extent of only about 10% (data not shown). Arsenite caused the same blockage of [3H]Dex binding and [3H]Dex-Mes labeling of the 16 K core binding fragment (Fig. 4B) as seen with the 98 K (8) and 42 K (Fig. 3) receptor species. However, the inhibition of steroid binding to 16 K fragments occurred at somewhat lower concentrations of arsenite than for the larger receptor species (cf. Fig. 3 and ref. 6). DTT rapidly (30 min at 0 C) reversed the effects of arsenite. A 3-fold excess of DTT regenerated about 40% of the initial binding activity, whereas a 30-fold excess restored > 85% of the activity (data not shown).

Arsenite ((jM):

4

6

8

10 4 0 100

['H]Dex: - + - + - + - + - +

3

samples with [ H]Dex ± excess nonradioactive Dex for 2.5 h and removing free steroid with added dextran-coated charcoal as in Materials and Methods. The amount of specific binding of [3H]Dex was expressed as a percent of the untreated control. The inset shows arsenite inhibition of covalent labeling by [3H]Dex-Mes ± excess nonradioactive Dex in the same experiment. The arrow (—») indicates the 42 K fragment.

10-2

MMTS (M)

Arsenite (M)

FlG. 3. Effect of arsenite on [3H]Dex binding to 42 K chymotrypsin fragment. Steroid-free 42 K fragments were treated with various concentrations of arsenite in pH 8.8 TAPS buffer for 30 min. The binding

10 3

%

80

«

60

mm

- +

to

| m

40

1 20 o

10'

10" 6

Arsenite (M)

FIG. 4. Effect of MMTS (A) and arsenite (B) on [3H]Dex binding to 16 K trypsin core fragment of HTC cell receptors. Steroid-free 16 K fragments were treated with various concentrations of MMTS, or arsenite, in pH 8.8 TAPS buffer for 30 min. The binding activity of duplicate samples was determined and plotted as a percent of the untreated control as described in Fig. 2. The inserts show MMTS and arsenite inhibition of covalent labeling by [3H]Dex-Mes ± excess nonradioactive Dex in the same experiment. S and L identify the mol wt markers soybean trypsin inhibitor and lysozyme respectively. The arrow (—>) indicates the 16 K core fragment.

Antibody aPl recognizes the 16 K steroid-binding core fragment Western blotting was used to further examine those receptors that contain MMTS-induced intramolecular disulfides and no longer bind steroid. Detection of the 16 K core fragment required an antibody that recognizes the N-terminal half of the steroid-binding domain (3). Unfortunately, such antibodies are extremely rare (3235). However, we now find that aPl, a polyclonal antibody raised against the bacterially expressed segment of 440-795 of the rat glucocorticoid receptor (24), does recognize the 16 K fragment of 537-673. aPl immunoadsorbed 15 ± 4% of the [3H]Dex-bound, 16 K complexes. This low efficiency of adsorption appears to be a property of the 16 K complexes because both aPl and the monoclonal antireceptor antibody BUGR-2 (22) immunoadsorbed an identical higher amount of intact 98 K [3H] Dex-bound receptors [46 ± 11% (SD, n = 5) and 52 ±

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

7% (SD, n = 5) respectively]. Furthermore, aPl selectively recognized [3H]Dex-Mes-labeled 16 K core fragments (Fig. 5). Finally, Western blotting of trypsin digest preparations of 16 K steroid-free fragments with aPl detected one major band at Mr = 15,470 ± 730 (SD, n = 10). This band was not seen in the Western blots of receptor digested with lysylendopeptidase C (data not shown), which is incapable of forming the 16 K fragment (31). Thus, we conclude that aPl recognizes at least one epitope in the region of Thr537-Arg673 of the rat receptor. The MMTS-induced intramolecular disulfide does not involve cross-linking the receptor with a nonreceptor protein The 42 K chymotrypsin fragment appears to contain all of the nonreceptor proteins that are associated with the intact 98 K holoreceptor (12). Similarly, hsp90 has been found to be associated with the 16 K steroid-binding core fragment (Chakraborti, P. K., and S. S. Simons Jr., manuscript in preparation). Thus, the inhibition of steroid binding seen after preincubation with 0.3 mM MMTS could arise from the cross-linking of a nonreceptor protein to the receptor. In an effort to detect such Antibody: Buffer [1H]Dex:

-

aP1

+ -

+

FIG. 5. Fluorograph of [3H]Dex-Mes labeled 16 K core fragments immunoprecipitated by aPl antibody. [3H]Dex-Mes-labeled, 16 K complexes were incubated first with aPl antibody and then with Pansorbin. Those receptors that were immobilized on Pansorbin were extracted, analyzed on SDS-polyacrylamide gels, and fluorographed as in Materials and Methods. The observed Mr of the specifically labeled species in the third lane is 15,600 (calculated Mr = 15,521 without steroid). The arrow (—>) indicates the 16 K core fragment. The positions of the mol wt markers (P, phosphorylase b, Mr 97,400; B, BSA, Mr 66,300; O, ovalbumin, Mr 45,000; C, carbonic anhydrase, Mr 30,600; S, soybean trypsin inhibitor, Mr 21,500; L, a-lactalbumin, Mr 14,400) are as indicated.

2535

high mol wt complexes, we have Western blotted 98, 42, and 16 K receptors that had been pretreated with MMTS and then separated on nonreducing SDS-polyacrylamide gels. Control experiments with mol wt standards and bovine immunoglobulin G (IgG) confirmed that simply omitting DTT from the SDS sample buffer was enough to preserve most intramolecular disulfides, even if the samples were heated at 100 C for 5 min. In the absence of added DTT, those proteins with intramolecular disulfides displayed a lower apparent mol wt (presumably because of a more condensed tertiary structure), whereas proteins crosslinked by intermolecular disulfides, such as IgG, had a higher apparent mol wt (data not shown). However, in crude receptor solutions, we expected that multiple thioldisulfide rearrangements could present problems. In fact, simply omitting DTT from cytosol solutions gave a more diffuse band at a slightly lower Mr for the 98 K holoreceptor (lane 9 vs. 10 of Fig. 6A; see also below), whereas no band was seen for the 42 K fragment (lane 1 of Fig. 6A). Therefore, all samples were treated with high concentrations of either IA (5 mM) or MMTS (10 mM) before gel analysis to block the remaining free thiol groups while preserving the existing disulfides. Under these conditions, the 42 K receptor fragment could now be visualized (lane 2 of Fig. 6A) and a sharper band was obtained for the 98 K receptor (lane 10 vs. 9 of Fig. 6A). Western blot analysis of 98, 42, and 16 K receptor species pretreated with various concentrations of MMTS (followed by 10 mM MMTS to block free -SH groups) was conducted with antireceptor and with anti-hsp90 antibodies. In all cases, no new higher Mr species (for receptor or hsp90) were observed after preincubation with those concentrations of MMTS (i.e. 0.1-0.4 mM) that eliminate steroid binding and cause the formation of an intramolecular disulfide (Fig. 6, lanes 3-5 and 1113; Fig. 7, lanes 9 and 10). The species at 122 K in Fig. 6 appears to be an artifact (see Discussion). Therefore, MMTS inhibition of [3H]Dex binding does not cause the cross-linking of proteins to the receptor. In fact, we did not observe the cross-linking of any proteins either to the receptor (Figs. 6A and 7) or to hsp90 (Fig. 6B) at MMTS concentrations < 3 mM. It should be noted that hsp90 (Fig. 6B) and hsp70 (data not shown) were relatively resistant to chymotrypsin digestion. This is consistent with our earlier observations that the native receptor protein is more sensitive to proteolysis than are most other proteins (26, 31). We consistently observed a decrease in the intensity of the Western blot signal of either 98 or 42 K receptors after pretreatment with 0.07-1 mM MMTS (see Fig. 6A, lanes 3-6 and 11-14). However, this decrease was unrelated to the intramolecular disulfide that caused a loss of steroid binding. The signal was equally weak for

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

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42 kDa Chymotrypsin Fragment Lane:

1

[3H]Dex Binding as % of Control:

3

5

100 20 2

Pre-MMTS(4x10-"M): 102MMMTS: Post-DTT: +

98 kDa Holoreceptor

7

9

1 61 52 16

11

13

100 25 5

Endo • 1990 Vol 127* No 5

15

5 58 46 23

—s

Pre-MMTS (mM):

-

-

Post-MMTS(IOmM):

-

+

0.1 0.2 0.4 1 +

+

+

+

3 30 +

+

-

- 0.1 0.2 0.4 1 +

+

+

+

+

3 30 +

+ Lane

B

Lane:

1

13

15

FIG. 6. Western blot analysis of intact and chymotrypsin-treated receptors and of hsp90 on nonreducing SDS-polyacrylamide gels after MMTS treatment. Steroid-free 98 or 42 K receptors in pH 8.8 TAPS buffer were preincubated with various concentrations of MMTS for 2.5 h, split, and treated with either [3H]Dex ± excess ^HJDex (to determine steroid binding) or an excess of MMTS (10 mM) for 30 min (to consume all of the remaining free -SH groups). The 10-mM MMTS-treated samples were mixed with an equal volume of SDS sample buffer without DTT and subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose. The samples in lanes 1 and 2 and 9 and 10 were identical except that only the material of lanes 2 and 10 were treated with 10 mM MMTS. Western blotting with (A) an antireceptor antibody (BUGR-2) or (B) an anti-hsp90 antibody was visualized by immunoperoxidase staining of peroxidase-coupled, biotinylated second antibodies as described in Materials and Methods. The solid arrows (—») indicate the positions of the undigested 98 K receptor and hsp90; the open arrow (=>) shows the position of the 42 K receptor fragment. Note that the indicated MMTS concentrations apply to both panels A and B.

receptors pretreated with 0.4 and 1 mM MMTS even though the [3H]Dex binding was 1-5% and 61-58%, respectively. The reason for this decreased Western blot signal intensity is not known, but it was independent of whether an excess of IA or MMTS was used to block the remaining free thiols, was not due to saturation of the nitrocellulose membrane, and was not observed with hsp90 (Fig. 6B). The intensity differences with the MMTS-pretreated receptors persisted when DTT was added either during the transfer of protein to the nitro-

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FIG. 7. Effect of thiol reagents on the apparent mol wt of 16 K core fragments on nonreducing SDS-polyacrylamide gels. Steroid-free 16 K core fragments were treated with buffer, 10 mM DTT for 30 min, 10 mM MMTS for 30 min, or 4 x 10"4 M MMTS for 2.5 h and then 10 mM MMTS for 30 min before being analyzed on nonreducing gels followed by electrophoretic transfer to nitrocellulose. Western blotting with an antireceptor antibody (aPl) was visualized by immunoperoxidase staining of peroxidase-coupled biotinylated second antibodies as described in Materials and Methods. The arrow (—*) indicates the 16 K fragment. The material at ~50 K is nonreceptor, cross-reacting material (24). The positions of the mol wt markers (P, phosphorylase b, Mr 97,400; B, BSA, Mr 66,300; O, ovalbumin, Mr 45,000; C, carbonic anhydrase, Mr 30,600; S, soybean trypsin inhibitor, Mr 21,500; L, alactalbumin, Mr 14,400) are as indicated.

cellulose membrane, during the blocking of the nitrocellulose before the addition of antireceptor antibody, or during the incubation with antireceptor antibody. The absence of a signal in the stacking gel argues that the missing material did not simply fail to enter the gel (data not shown).

Discussion Various characteristics of the thiol group (e.g. hydrogen bonding, high nucleophilicity, and ease of reversible oxidation) make it an important component in determining the molecular properties of proteins. This report describes our use of steroid binding and Western blotting to examine the role of vicinal dithiols in native and chemically modified rat glucocorticoid receptors and receptor fragments. Our previous studies on the effects of MMTS (6) and arsenite (8) established that a vicinal dithiol group is involved in the binding of steroids to the rat glucocorticoid receptor. The current studies with the 42 K chymotrypsin fragment and the 16 K trypsin fragment show that the same dose-response curves for the inhibition of steroid binding and of affinity labeling are also obtained for these smaller forms of the receptor. The low binding of 16 K core fragments pretreated with

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

> 1 mM MMTS appears to be due to the fact that the affinity of Dex for the unmodified 16 K fragment is ~23 fold lower than for the 98 K holoreceptor (31) and reaction with > 1 mM MMTS is expected to lower the affinity further by a factor of 5 (6). Thus, the concentration of [3H]Dex used in Fig. 4A would be below the Kd of 16 K fragments modified by >1 mM MMTS, and only part of the binding capacity would be detected. Because the affinity of Dex for 42 and 98 K receptors was the same, the predicted 5-fold reduced affinity of > 1 mM MMTS pretreated 42 K fragments will have a minimal effect on the quantity of [3H]Dex binding. Collectively these data demonstrate that all of the thiols in the receptor except for Cys-640, -656, and -661 of the 16 K core fragment can be eliminated as potential components of the vicinal dithiol. Cys-754 was a prime candidate for 1 of the 2 thiols because it is photoaffinity labeled by triamcinolone acetonide (11), but it is now eliminated. The vicinal dithiols that react with 0.3 mM MMTS to form a disulfide, thus blocking steroid binding, were defined to be intramolecular on the basis of kinetic data (6). However, both thiols do not have to be on the receptor; the kinetic data are also consistent with one thiol being supplied by any nonreceptor protein that is tightly associated with the steroid-free receptor. One such protein, hsp90, has been covalently attached to the 98 K receptor using the zero length cross-linker Cu ++ /ophenanthroline (36). High concentrations of other zerolength cross-linkers {i.e. sodium tetrathionate, disulfiram, and iodosobenzoate) also caused cross-linking and an increase in the Mr of receptor by ~40 K (20). Unfortunately, the precise mol wt of this cross-linked protein cannot be determined because the SDS-polyacrylamide gels were run under nonreducing conditions. In contrast, we see no cross-linking of either the glucocorticoid receptor or hsp90 to any other proteins with < 10 mM MMTS. In this respect, Tienrungroj et al. (4) also found that 3 to 5 mM MMTS did not cross-link hsp90 to receptor-steroid complexes. We do see a higher mol wt species of about 122 K after 10 mM MMTS treatment when using both the antireceptor and the anti-hsp90 antibody (lanes 1 vs. 2 and 9 vs. 10 in Fig. 6). However, this band appears to be an artifact of the second antibody used in the Western blot because it is seen both when the primary antibody is omitted (data not shown) and when preparations of either 98 or 42 K receptors are used. In conclusion, the vicinal dithiols that are crosslinked by low concentrations of MMTS to block steroid binding are not on two different proteins. Another mechanism that theoretically could be compatible with our results is that the vicinal dithiols are both on a nonreceptor protein that is tightly associated with the receptor. In this scenario, formation of a disulfide in a nonreceptor molecule would block the binding

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to and the labeling of the receptor. This explanation appears unlikely for two reasons. First, the 5-fold reduced affinity of most glucocorticoids for receptors in which both of the vicinal dithiols have been modified by MMTS (6) suggests that these thiols somehow increase the binding affinity. However, when the receptor-steroid complex is activated and the nonreceptor molecules have dissociated (37, 38), the affinity of the bound steroid, as reflected by the reciprocal of the rate of steroid dissociation (39), actually increases (40, 41; Lamontagne, N., and S. S. Simons Jr., unpublished observations). Second, it is known that steroid-free glucocorticoid and estrogen receptors are associated with several identical nonreceptor proteins (13-15, 18). Because arsenite does not block steroid binding to estrogen receptors (Lopez, S., Y. Miyashita, and S. S. Simons Jr., manuscript submitted for publication), it can be argued that the specificity of inhibition by arsenite (and the vicinal dithiols) resides with the glucocorticoid receptor. It may be possible to use point mutagenesis to examine this question, although most mutations in the steroid-binding domain result in the loss of steroid-binding activity (42-44). Thiol-disulfide exchange reactions occur easily. Because both thiols and disulfides are present in many proteins, there is always the possibility that such exchange reactions will interfere with the analysis of a protein. Although our experiments with mol wt standards and IgG indicated that there were no major rearrangements in the absence of added DTT, the diffuse bands for the 98, and especially the 42, K receptors argue that rearrangements do occur. In fact, it has been shown that thiols in the receptor can participate in thiol-disulfide exchange reactions (45, 46). Therefore, we have added 5 mM IA or 10 mM MMTS to our solutions [which contain ~0.8 mM thiol (6)] to block all of the accessible thiols and prevent any rearrangements. Under these conditions, the 98 and 42 K receptor forms consistently displayed a slightly lower Mr than the fully reduced species and sharper bands than the untreated receptors (Fig. 6A and data not shown). These results are most easily explained by the presence of a disulfide in the native receptor, which undergoes thiol-disulfide rearrangements under nonreducing conditions to give new species. Treatment with excess MMTS (or IA) blocks these rearrangements. The absence of higher Mr forms argues that any disulfide of the native receptor would reside entirely in the receptor protein. Because the Mr of both the MMTS-treated and the fully reduced receptors are very similar, we propose that a disulfide exists in the native receptor and involves 2 cysteine residues that are closely spaced in the primary structure. Dex-Mes-labeled receptors have also been reported (20) to give diffuse bands on nonreducing gels, whereas added DTT or IA afforded an identical sharper, higher

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING

Mr reduced receptor band (i.e. ar in Fig. 2 of Ref. 20). Low concentrations of oxidizing agent gave lower Mr forms of the affinity-labeled receptors, which were proposed to result from the formation of new intramolecular disulfides (20). Because IA cannot reduce disulfides, our data indicate that the diffuse band seen in Ref. 20 is due to the same thiol-disulfide rearrangements that we have observed, and that the sharper, higher Mr band (ar) can be formed either by reduction (with DTT) or by blocking the disulfide exchange reactions of free -SH groups (with IA). These results demonstrate the difficulty in making conclusions regarding the existence of disulfides, at least in the glucocorticoid receptor, when the free thiols are not prevented from undergoing thiol/disulfide exchange. The extensive disulfide bond rearrangements that appear to occur in the untreated 42 K receptor may reflect an increased conformational mobility of the 42 K fragment. Other data indicate that the 16 K core fragment is even more conformationally mobile. If the reaction with arsenite or MMTS involves some spatial reorganization of the protein, an increased conformational mobility of the 16 K fragment would readily explain the greater potency of arsenite and the more rapid formation of the intramolecular disulfide with MMTS. Increased conformational mobility could also account for the greater loss of 16 K steroid-binding activity after treatment with high us. low concentrations of MMTS in the absence of Na2MoO4.2 Treatment of molybdate-free solutions of the 16 K core with 0.3 mM MMTS, to cause the formation of an intramolecular disulfide, appeared to lock the receptor into a conformation from which steroid-binding activity could be recovered at a later time simply by adding DTT. Such a protective effect on the 16 or 98 (6) K receptors was not seen with higher concentrations of MMTS, which also do not yield the same intramolecular disulfide. Thus, several lines of evidence indicate that the steroid-binding domain of the receptor protein may be conformationally mobile. This mobility offers a mechanism by which the structure of the bound steroid can be translated into receptor-steroid complexes with different tertiary structures. Such altered tertiary structures could, in turn, be responsible for the different steroid-dependent gel shift mobilities of receptor-steroid complexes bound to specific DNA sequences (47, 48) and may, in combination with tissue specific factors, be important for the expression of agonist us. antagonist activity. Acknowledgments We thank Drs. Robert Harrison, Ettore Appella, and Steven Ulrich 2 It should be noted that the presence of molybdate, which also interacts with thiols, does not block any of the effects of either MMTS or arsenite (see Refs. 6, and 8 and Figs. 2-4).

Endo • 1990 Voll27-No5

for their generous gift of antibodies, Dr. Frank Sistare for help on the experiments concerning MMTS reaction with the 16 K core fragment, Mr. Hung Luu for general technical assistance, and Drs. Ettore Appella, Stephen Ullrich, and Alice Cavanaugh for their critical reading of the manuscript.

References 1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889-895 2. O'Malley BW, Birnbaumer L (eds) 1978 Receptors and Hormone Action. Academic Press, New York, vol II 3. Ratajczak T, Samec AM, Hahnel R 1982 Requirement for a reduced sulfhydryl entity in the protection of molybdate-stabilized estrogen receptor. FEBS Lett 149:80-84 4. Tienrungroj W, Meshinchi S, Sanchez ER, Pratt SE, Grippo JF, Holmgren A, Pratt WB 1987 The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor. J Biol Chem 262:6992-7000 5. Mendel DB, Bodwell JE, Smith LI, Munck A 1987 Structure and function of cytosolic glucocorticoid receptors in WEHI-7 mouse thymoma cells: receptor composition and phosphorylation. In: TC Spelsberg, R Kumar (eds) Steroid and Sterol Hormone Action. Martinus Nijhoff, Boston, pp 175-193 6. Miller NR, Simons Jr SS 1988 Steroid binding to hepatoma tissue culture cell glucocorticoid receptors involves at least two sulfhydryl groups. J Biol Chem 263:15217-15225 7. Harlow KW, Smith DN, Katzenellenbogen JA, Greene GL, Katzenellenbogen BS 1989 Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor. J Biol Chem 264:17476-17485 8. Simons Jr SS, Chakraborti PK, Cavanaugh AH 1990 Arsenite and cadium(II) as probes of glucocorticoid receptor structure and function. J Biol Chem 265:1938-1945 9. Simons Jr SS, Thompson EB 1981 Dexamethasone 21-mesylate: an affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells. Proc Natl Acad Sci USA 78:3541-3545 10. Simons Jr SS, Pumphrey JG, Rudikoff S, Eisen HJ 1987 Identification of cysteine-656 as the amino acid of HTC cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate. J Biol Chem 262:9676-9680 11. Carlstedt-Duke J, Stromstedt P-E, Persson B, Cederlund E, Gustafsson J-A, Jornvall H 1988 Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors. J Biol Chem 263:6842-6846 12. Gehring U, Arndt H 1985 Heteromeric nature of glucocorticoid receptors. FEBS Lett 179:138-142 13. Bresnick EH, Dalman FC, Sanchez ER, Pratt WB 1989 Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J Biol Chem 264:4992-4997 14. Catelli MG, Binart N, Jung-Testas I, Renoir JM, Baulieu EE, Feramisco JR, 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 15. Sanchez ER, Meshinchi S, Tienrungroj W, Schlesinger MJ, Toft DO, Pratt WB 1987 Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor. J Biol Chem 262:6986-6991 16. Mendel BD, Orti E 1988 Isoform composition and stoichiometry of the ~90-kDa heat shock protein associated with glucocorticoid receptors. J Biol Chem 263:6695-6702 17. Moore SK, Kozak C, Robinson EA, Ullrich SJ, Appella E 1989 Murine 86- and 84kDa heat shock proteins, cDNA sequences, chromosome assignments, and evolutionary origins. J Biol Chem 264:5343-5351 18. Tai P-KK, Maeda Y, Nakao K, Wakim NG, Duhring JL, Faber LE 1986 A 59-kilodalton protein associated with progestin, estrogen, androgen, and glucocorticoid receptors. Biochemistry 25:5269-

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VICINAL DITHIOLS INVOLVED IN STEROID BINDING 5275 19. Wrange O, Okret S, Radojcic M, Carlstedt-Duke J, Gustafsson JA 1984 Characterization of the purified activated glucocorticoid receptor from rat liver cytosol. J Biol Chem 259:4534-4541 20. Silva CM, Cidlowski JA 1989 Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. J Biol Chem 264:6638-6647 21. Miesfeld R, Rusconi S, Godowski PJ, Maler BA, Okret S, Wikstrom A-C, Gustafsson J-A, Yamamoto KR 1986 Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell 46:389-399 22. Gametchu B, Harrison RW 1984 Characterization of a monoclonal antibody to the rat liver glucocorticoid receptor. Endocrinology 114:274-279 23. Ullrich SJ, Robinson EA, Law LW, Willingham M, Appella E 1986 A mouse tumor-specific transplantation antigen is a heat shockrelated protein. Proc Natl Acad Sci USA 83:3121-3125 24. Hoeck W, Rusconi S, Groner B 1989 Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment. J Biol Chem 264:14396-14402 25. Thompson EB 1979 Liver cells (HTC). Methods Enzymol 58:544551 26. Reichman ME, Foster CM, Eisen LP, Eisen HJ, Torain BF, Simons Jr SS 1984 Limited proteolysis of covalently labeled glucocorticoid receptors as a probe of receptor structure. Biochemistry 23:53765384 27. Simons Jr SS, Miller PA 1984 Comparison of DNA binding properties of activated, covalent and noncovalent glucocorticoid receptor-steroid complexes from HTC cells. Biochemistry 23:6876-6882 28. Simons Jr SS 1987 Selective covalent labeling of cysteines in bovine serum albumin and in HTC cell glucocorticoid receptors by dexamethasone 21-mesylate. J Biol Chem 262:9669-9675 29. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:43504354 30. Carlstedt-Duke J, Stromstedt P-E, Wrange 0, Bergman T, Gustafsson J-A, Jornvall H 1987 Domain structure of the glucocorticoid receptor protein. Proc Natl Acad Sci USA 84:4437-4440 31. Simons Jr SS, Sistare FD, Chakraborti PK 1989 Steroid binding activity is retained in a 16 kDa fragment of the steroid binding domain of rat glucocorticoid receptors. J Biol Chem 264:1449314497 32. Carlstedt-Duke J, Okret S, Wrange O, Gustafsson J-A 1982 Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNAbinding domains. Proc Natl Acad Sci USA 79:4260-4264 33. Greene GL, Sobel NB, King WJ, Jensen EV 1984 Immunochemical studies of estrogen receptors. J Steroid Biochem 20:51-56

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34. Urda LA, Yen PM, Simons Jr SS, Harmon JM 1989 Regionspecific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid. Mol Endocrinol 3:251-260 35. Vu Hai MT, Jolivet A, Ravet V, Lorenzo F, Perrot-Applanat M, Citerne M, Milgrom E 1989 Novel monoclonal antibodies against human uterine progesterone receptor. Mapping of receptor iraraunogenic domains. Biochem J 260:371-376 36. Rexin M, Busch W, Gehring U 1988 Chemical cross-linking of heteromeric glucocorticoid receptors. Biochemistry 27:5593-5601 37. Gehring U, Mugele K, Arndt H, Busch W 1987 Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors. Mol Cell Endocrinol 53:33-44 38. Pratt WB 1987 Transformation of glucocorticoid and progesterone receptors to the DNA-binding state. J Cell Biochem 35:51-68 39. Pratt WB, Kaine JL, Pratt DV 1975 The kinetics of glucocorticoid binding to the soluble specific binding protein of mouse fibroblasts. J Biol Chem 250:4584-4591 40. Cardo PP, Gambetti M, Vignale B, Divano MC 1983 Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus. Eur J Biochem 137:173-178 41. Danze PM, Richard C, Formstecher P, Dautrevaux M 1990 Steroid receptor exchange assay in the presence of acetonitrile: application to the study of glucocorticoid- and anti-glucocorticoid-receptor complexes. Steroids 55:10-16 42. Danielsen M, Northrop JP, Ringold GM 1986 The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2522 43. Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645652 44. Rusconi S, Yamamoto KR 1987 Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. EMBO J 6:1309-1315 45. Idziorek T, Formstecher P, Danze P-M, Sablonniere B, Lustenberger P, Richard C, Dumur V, Dautrevaux M 1985 Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex. Eur J Biochem 153:6574 46. Bodwell JE, Holbrook NJ, Munck A 1984 Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors. Biochemistry 23:4237-4242 47. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145156 48. Lees JA, Fawell SE, Parker MG 1989 Identification of two transactivation domains in the mouse oestrogen receptor. Nucleic Acids Res 17:5477-5488

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Localization of the vicinal dithiols involved in steroid binding to the rat glucocorticoid receptor.

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have...
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