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Biochemistry 1984, 23, 5376-5384
Limited Proteolysis of Covalently Labeled Glucocorticoid Receptors as a Probe of Receptor Structuret Marsha E. Reichman, Carol M. Foster, Laura P. Eisen, Howard J. Eisen, Barbara F. Torain, and S . Stoney Simons, Jr.*
ABSTRACT: [3H]Dexamethasone 2 1-mesylate affinity-labeled glucocorticoid receptors were subjected to controlled proteolysis by trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on denaturing constant percentage or gradient polyacrylamide gels. The molecular weights ( M I -98 000) and cleavage patterns for rat liver and HTC cell receptors indicated extensive homology between the glucocorticoid receptors from normal rat liver and a transformed rat liver cell line. The major DNA-binding species generated by chymotrypsin treatment was found to be a 42K fragment that was accompanied by several unresolved, slightly lower molecular weight fragments. The meroreceptors obtained after trypsinization were comprised of two species of M, 30 000 and 28 000. Each of the three proteases, despite their differing
specificities, generated fragments with molecular weights close to 42 500, 30 500, and 27 000. Nevertheless, each of the three proteases gave rise to a distinctive "ladder" of labeled fragments. No differences could be detected in the digestion patterns of unactivated and activated HTC cell complexes for all three proteases. Also, native and denatured receptorsteroid complexes yielded surprisingly similar digestion patterns with each enzyme. Digestion of denatured complexes readily generated large amounts of a fragment of MI E 15 000 that was much smaller than the protease-resistant meroreceptors formed from native complexes. The presence of these 15K fragments suggested that the [3H]dexamethasone 21-mesylate labeling of the steroid-binding cavity is restricted to a relatively small segment of the receptor.
S t e r o i d receptors are absolutely required for steroid hormone action and appear to provide excellent model systems for studying the regulation of gene expression in eukaryotes. Nevertheless, details about receptor molecules such as a description of the functional domains and the changes that accompany activation of the receptorsteroid complex to a DNA binding molecule remain largely unknown. In theory, proteases are useful probes of receptor structure and function, and some such studies have been reported. The role of endogenous proteases has been considered in the appearance of certain phenotypes of variant receptors (Stevens & Stevens, 1981; Nordeen et al., 1981; Yamamato et al., 1975). Exogenous proteases have been used to define three different domains of the glucocorticoid receptor that are involved in (1) steroid binding, (2) DNA binding, and (3) the expression of biological activity (Wrange & Gustafsson, 1978). However, the utility of proteolytic analysis has been limited by the two major obstacles of needing purified receptor for further studies and of being unable to identify specific portions of the receptor under conditions that permit higher resolution of the fragments. With the recent development by one of us (Simons, 1981, 1982; Simons & Thompson, 1981) and others (Dure et al., 1980; Nordeen et al., 1981; Westphal et al., 1981; Holmes et al., 1981; Katzenellenbogen et al., 1983) of affinity labels for steroid receptors, it is now possible to overcome both of these problems. The high selectivity of affinity labels enables one to obtain minimally purified receptor preparations in which only the receptor is covalently labeled. The formation of a covalent steroid-receptor bond permits one to follow the steroid binding portion of the receptor even under strongly denaturing conditions.
The types of information that can be obtained from proteolysis of affinity-labeled receptors have just begun to emerge. Birnbaumer et al. (1983) have concluded that the A and B subunits of the chick oviduct progesterone receptor are derived from separate genes. Nordeen et al. (1 98 1) and Gehring & Hotz (1983) have determined that the glucocorticoid receptor of nt', but not nt-, variant S49 mouse lymphoma cells is smaller than the wild-type receptor and similar in properties to a major chymotrypsin fragment of the wild-type receptor. In this study, we have used proteolysis of affinity-labeled receptors to examine the structural homology between glucocorticoid receptors in rat liver and rat hepatoma tissue culture (HTC) cells. In addition, aided by the near quantitative yield of labeled receptors with the electrophilic affinity label dexamethasone 21-mesylate (Simons & Thompson, 198 1; Eisen et al., 1981; Simons et al., 1983), we have identified the denatured, covalently labeled fragments that correspond to the trypsin-generated meroreceptors (Sherman et al., 1976; Wrange & Gustafsson, 1978; Sherman et al., 1983; Vedeckis, 1983a) and to the DNA-binding species observed after chymotrypsin digestion (Wrange & Gustafsson, 1978; Gehring & Hotz, 1983). We have also used exogenous proteases to examine the primary and higher order structures of glucocorticoid receptors in different functional (i.e., activated vs. unactivated) and physical (Le., native vs. denatured) states.
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Materials and Methods Chemicals. [ 3H]Triamcinolone acetonide (33 Ci/mmol), [3H]dexamethasone (46 Ci/mmol), [3H]dexamethasone 21mesylate (46 Ci/mmol), and Aquasol were from New England Nuclear. Hydrofluor was supplied by National Diagnostics. Tricine,' triamcinolone acetonide, dexamethasone, and sodium
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From the Caboratory ofDevelopmenta1 Pharmacology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 (M.E.R., C.M.F., L.P.E., and H.J.E.),and the Laboratory of Chemistry, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 4, Room 132,Bethesda, Maryland 20205 (B.F.T. and S.S.S.).Received December 30, 1983.
Abbreviations: Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyllglycine; TAPS, 3-[[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino]- 1-propanesulfonic acid; NaDodS0,-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPCK, tosylphenylalanine chloromethyl ketone; TLCK, tosyllysine chloromethyl ketone; Tris, tris(hydroxymethyl)aminomethane; DTT, dithiothreitol.
This article not subject to U.S.Copyright. Published 1984 by the American Chemical Society
PROTEOLYSIS OF GLUCOCORTICOID RECEPTORS
molybdate were from Sigma. TAPS was obtained from Sigma or Cal-Biochem. Reagents for NaDodS0,-PAGE including molecular weight standards were from Bio-Rad. Sephacryl S-200, Sephadex G-25, and hydroxylapatite were from Pharmacia. The counting efficiency of all 3H-labeled samples was 40-55%. Trypsin (TPCK treated) was obtained from Worthington or Sigma, chymotrypsin (TLCK treated) was from Sigma, and Staphylococcus aureus V8 protease was from Miles. The molecular weight standards (myosin, MI 200 000; &galactosidase, M , 116 250; phosphorylase b, MI 97 400; albumin, M I 66 300; ovalbumin, MI 45 000; carbonic anhydrase, MI 30 600; soybean trypsin inhibitor, MI 21 500; lysozyme, MI 14 400) were from Bio-Rad. Fluorescent UltEmit autoradiography marker was from New England Nuclear. Buffers and Solutions. TAPS,, buffer was composed of 25 mM TAPS, 50 mM NaCl, 20 mM Na2Mo04,1 mM ethylene diaminetetraacetic acid (EDTA), and 10% glycerol, pH 8.2 at 0 "C. TAPS, buffers used with HTC cell preparations differed from TAPSSobuffer in containing no NaCl or Na2MOO, and in having higher pH values at 0 OC, Le., pH 8.8 and 9.5. The pH 8.8 TAPS, buffer is identical with the pH 8.5 buffer reported in an earlier publication (Simons et al., 1983). The higher pH here was due to the use of an Altex electrode (53 1167 combination electrode with internal silver/silver chloride reference electrode) and the use of temperature-corrected standard buffers at 0 OC (Beckman) instead of room temperature standard buffers with external temperature compensation. Twofold concentrated NaDodSO, sample buffer contained 0.6 M Tris (pH 8.85), 2% NaDodSO,, 0.2 M dithiothreitol, 20% glycerol, and bromphenol blue. Cells and Preparation of Cytosols. Preparation of rat liver cytosol and (NH,),SO, precipitation of rat liver glucocorticoid receptors have been reported previously (Eisen et al., 1981). Growth of HTC cells in spinner and monolayer cultures has been described (Thompson, 1979). The preparation of HTC cell cytosol has been modified from our previous procedure (Simons et al., 1979) as follows. After the cells were washed in phosphate-buffered saline (without calcium), the cell pellet was quick frozen in dry ice for 45 min and then stored at -20 OC. After the pellet was thawed, an equal volume of pH 9.5 TAPSo buffer was added, and the solution was gently refluxed 20 times in a pipet. At this point >95% of the cells were ruptured, as determined by staining with Azur A. Subsequent centrifugation steps were as previously described (Simons et al., 1979), Le., 15 rnin a t 17000g followed by 90 rnin at 200000g. The 200000g supernatant, or cytosol, was used immediately or quick frozen in liquid nitrogen. Cytosol frozen in this manner retained its original receptor binding capacity for at least 61/2 months (data not shown) when the frozen cytosol was rapidly thawed and used immediately. Covalent Labeling of Cytosols. The labeling of (NH4)2S04-precipitatedrat liver receptors has been previously described (Eisen et al., 1981). The published procedures for HTC cell receptors were modified only by using TAPS, buffers instead of the Tricine-based buffer A. Thus, the receptors in the thawed, HTC cell cytosol were treated with [3H]dexamethasone 21mesylate & 80-fold excess unlabeled dexamethasone in 0.67 volume of pH 9.5 TAPS, buffer. At this point the pH of the cytosol solution was usually 8.8 or slightly less. All further dilutions were with pH 8.8 TAPS, buffer unless otherwise indicated. Procedures for Partial Purification of Labeled Receptors. Sephacryl S-200 chromatography was carried out at 4 OC in TAPS5o buffer plus 1 mM dithiothreitol on columns with dimensions of 80 cm length and 2 cm diameter. One column
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was used for initial size fractionation of crude cytosol and a second for trypsin-treated material. DNA-cellulose chromatography was conducted as previously described (Simons et al., 1983). DEAE-cellulose (Whatman DE52) chromatography was performed on 2.5 (diameter) X 3 (length) cm columns using cytosol solutions that first had been chromatographed on Sephadex G-25 PD-10 columns (Pharmacia) equilibrated with 5 mM potassium phosphate buffer containing 20 mM Na,MOO, (pH 7.8 at 0 OC). This material (2.1-2.8 mL) was then loaded onto the DEAE-cellulosecolumns equilibrated with the same buffer. The column was developed with two sample volumes of the above phosphate buffer followed by 10 mL each of 50 mM potassium phosphate/lO mM Na2Mo0,, 75 mM potassium phosphate/lO mM Na2Mo04,and 300 mM potassium phosphate/20 mM Na2Mo0, all at pH 7.8-7.9 at 0 "C. Noncovalent [3H]dexamethasone and covalent [3H]dexamethasone 21-mesylate labeled complexes exhibited identical step-elution and gradient-elution profiles (data not shown). Under these conditions, [3H]dexamethasone-bound receptors contained only 6% activated complexes (data not shown). It should be pointed out, however, that increasing concentrationsof N a 2 M d 4in the potassium phosphate buffers were found to decrease the potassium phosphate concentrations required to elute activated and unactivated complexes (data not shown). (NH4)2S04precipitation of affinity-labeled HTC cell receptors was performed as previously described (Eisen et al., 1981) except that all of the (",),SO, was added at once to samples in TAPSo buffer (final pH 8.8) containing 20 mM Na2Mo04in 1.5-mL screw-capped conical centrifuge tubes (Sarstedt no. 72.692). After incubation at 0 "C for 30 rnin, the precipitate was centrifuged (0 OC/1 min in a microfuge), resuspended in pH 8.2 TAPS,, buffer containing 20 mM Na2Mo04,quick frozen at -80 OC, and stored at -20 OC. In view of the fact that all of the activated complexes and only some of the unactivated complexes are precipitated by 40% (NH4)2S04(Dahmer et al., 1981), it could be calculated from the yield of receptor-steroid complex precipitation (-40%) and from the amount of activated complexes initially present (-6%; see above) that the unactivated complexes constituted 280% of the total complexes isolated by (NH,)2S0, precipitation of unactivated, labeled cytosols. By the criterion of binding to DNA-cellulose columns in pH 8.2 TAPSo buffer containing 20 mM Na2Mo04, (NH4)2S04 precipitated, unactivated HTC cell complexes contained
