Proc. Nat. Acad. Sci. USA
Vol. 72, No. 8, pp. 3060-3064, August 1975 Biochemistry
Effect of chemical inactivating agents on glucocorticoid receptor proteins in mouse and hamster cells (dexamethasone/N-ethylmaleimide/iodoacetamide/hormone binding site/DNA binding site)
HOWARD A. YOUNG, WADE P. PARKS, AND EDWARD M. SCOLNICK National Cancer Institute, Bethesda, Maryland 20014
Communicated by C. B. Anfinsen, June 5,1975
hormones to the cytoplasmic receptor protein, we can protect the receptor from inactivation with N-ethylmaleimide. In addition, we have investigated the second step in the action of the hormone by treating the hormone-receptor complex with iodoacetamide. Although the hormone-receptor complex remains intact after iodoacetamide treatment, the IAA-treated complex is no longer able to bind to DNA. The results suggest that the binding of [3H]glucocorticoids to the cytoplasmic receptor protein may involve a sulfhydryl group, and that the subsequent binding to DNA either involves a second site on the receptor molecule separate from the hormone binding site, or that alternatively another protein(s) may be involved in the DNA binding step.
ABSTRACT The effect of N-ethylmaleimide and iodoacetamide on the glucocorticoid receptor activity extracted from the cytosol of either mouse or hamster cells has been investigated. Treatment of mouse or hamster cytosol with Nethylmaleimide or iodoacetamide rapidly inactivates the [3Hlglucocorticoid hormone binding activity of either cytosol. Prebinding the glucocorticoid hormone, dexamethasone, to the cytosol receptor blocks the rapid inactivation of the receptor by N-ethylmaleimide. Treatment of the prebound hormone-receptor complex with iodoacetamide prevents the subsequent binding of the hormone-receptor complex to DNA without causing a dissociation of the complex. Although the conclusions may be limited by the lack of purity of the receptor, the results suggest that a sulfhydryl group is involved in the binding of glucocorticoid hormones to the receptor protein. In addition, the results suggest that iodoacetamide is inactivating a separate chemical site which is necessary for the binding of the hormone-receptor complex to DNA.
MATERIALS AND METHODS Materials. [3H]Dexamethasone (28 Ci/mmol) was obtained from Amersham-Searle Corp. Other [3H]hormones, [3H]hydrocortisone (83 Ci/mmol), [3H]aldosterone (109 Ci/ mmol), [3H]corticosterone (83 Ci/mmol), [3H]progesterone (46 Ci/mmol), [3H]deoxycorticosterone (46 Ci/mmol), were obtained from New England Nuclear. With the exception of [3H]aldosterone, hormones, which were sold in benzene-ethanol mixtures (9:1), were dried under N2 and dissolved in absolute ethanol before use. N-ethylmaleimide (NEM) was obtained from Calbiochem, and iodoacetamide (IAA) was purchased from the Aldrich Chemical Co. CF-1l cellulose was purchased from Calbiochem, and DNA-cellulose was prepared as previously described (11) with DNA from NIH 3T3 mouse cells. The DNA content was 2.6 ,tg/mg of cellulose. Activated charcoal was from Merck (Rahway, N.J.). Buffers. Tris-glycerol buffer (TG) contained 10% (v/v) glycerol, 0.01 M Tris-HCl, pH 7.9, 0.05 M NaCl, 100 Mg/ml of bovine serum albumin, 1 mM EDTA, and 1 mM dithiothreitol. NEM was used as a 0.25 M stock solution in 0.001 M Tris-HCl, pH 7.4, and iodoacetamide was 0.50 M in 0.001 M Tris-HCl, pH 7.4. Methods. Preparation of cytosol and binding of [3H]hormone to receptor was measured by the charcoal binding assay as previously described (6), with the exception that Tris-glycerol buffer was now utilized during the extraction of receptor from cells. All binding studies were performed at 40 and represented high-affinity, saturable specific steroid binding as documented in an earlier publication (6). Cytosol preparations had protein concentrations ranging from 20 to 30 mg/ml and were stored in aliquots at -170°, and used after only one freeze-thaw cycle. Binding to DNA-cellulose was done in the manner described by Andre and Rochefort for estrogen receptor (12). Details are described in the appropriate legends.
The steroid hormones, which include glucocorticoids, estrogens, androgens, and progestins, have many similarities in their mode of action (1). Each hormone first binds with high affinity to a specific cytoplasmic receptor protein. Subsequently, the hormone-receptor complex becomes "activated" and transported to nuclei, and stimulates the transcription of RNA from DNA (1-4). Recently, the effect of glucocorticoid hormones has been studied in cell cultures derived from mouse mammary tumors. The glucocorticoid hormones have been observed, both in primary cell cultures (5) and in cell lines derived from murine mammary tumors, to stimulate an increase in the production of murine mammary tumor virus (MMTV) particles (2). This increase in production of MMTV particles is accompanied by a specific increase in MMTV RNA sequences (2, 3). Importantly, the glucocorticoid hormone effect on MMTV RNA synthesis is mediated by the binding of the hormone to a cytoplasmic receptor protein which has many properties in common with receptor proteins studied in other glucocorticoid induction systems (6). In earlier experiments on the mechanism of action of glucocorticoid hormones, the cytoplasmic receptor protein has been shown to be sensitive to inactivation with chemical reagents which can interact with sulfhydryl groups on proteins (7-10). In an effort to understand further the mode of action of the cytoplasmic receptor protein, we have investigated the effects of various chemical reagents which can inactivate the ability of the cytoplasmic receptor protein to bind [3H]glucocorticoid hormones. By prebinding glucocorticoid Abbreviations: MMTV, mouse mammary tumor virus; BHK, baby hamster kidney; NEM, N-ethylmaleimide; IAA, iodoacetamide; TG, Tris-glycerol; Dxm, dexamethasone. 3060
Biochemistry: Young et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Protection of receptor by dexamethasone (Dxm)
E z
w < 80
Charcoal-resistant radioactive dexamethasone Addition to cytosol
[3H]Dxm [3H] Dxm + 500-fold unlabeled Dxm [3H]Dxm+6mMNEM [3H]Dxm + [6 mM NEM + 50 mM dithiothreitol] [3H]Dxm + 12 mM IAA [3H]Dxm + [12 mM IAA + 50 mM dithiothreitol] [3H]Dxm prebound 90 min at
cpm
'
B. 'I ' --40--
15%); therefore all studies were performed with 50-100 pmol of [3H]dexamethasone per ml. Addition of N-ethylmaleimide or iodoacetamide prior to the [3H]dexamethasone leads to complete inactivation of the cytoplasmic receptor activity. If a 10-fold excess of dithiothreitol is first added to the N-ethylmaleimide or iodoacetamide, no inactivation of the receptor binding is observed. If the [3H]dexamethasone is prebound to the cytoplasmic receptor and N-ethylmaleimide or iodoacetamide is subsequently added for an additional period of 30 min, little destruction
0* a:
o0
40 20 0 15 30 4560 75900 15 30 4560 7590 TIME (MINUTES)
FIG. 1. Kinetics of inactivation of preformed complexes: Aliquots (2 ml) of either mouse cell cytosol (20-30 mg/ml) or hamster cytosol (19-25 mg/ml) were preincubated at 4° for 90 min with either [3H]dexamethasone, [3H]hydrocortisone, [3H]corticosterone, or [3H]aldosterone (50-60 pmol/ml). An 0.05 ml aliquot of 0.25 M NEM was then added (final concentration 6 mM) and at various periods of time 0.2 ml aliquots were removed, mixed with 0.025 ml of charcoal (100 mg/ml) to a final concentration of 10 mg/ml, vortexed, and immediately centrifuged at 5000 X g for 3 min at 4°. Aliquots (0.15 ml) of the supernatant were removed and radioactivity was determined. Nonspecific binding (in the presence of a 1000fold excess of unlabeled dexamethasone) represented 10-15% of the charcoal-resistant radioactivity and was subtracted from all values. 100% values represent, for mouse cytosol, 30,000 cpm for dexamethasone, 82,000 cpm for hydrocortisone, 78,000 for corticosterone, and 29,000 cpm for aldosterone; 100% values for hamster cytosol are: 20,000 cpm for dexamethasone; 34,000 cpm for hydrocortisone; 23,000 cpm for corticosterone; and 10,000 cpm for aldosterone. (A) * - *, mouse cytosol + dexamethasone; & - &, hamster cytosol + dexamethasone; * - *, mouse cytosol + corticosterone; o - o, hamster cytosol + corticosterone. (B 0 --- *, mouse cytosol + hydrocortisone; 0 --- 0, hamster cytosol + hydrocortisone; 0 - *, mouse cytosol + aldosterone; 0 - 0, hamster cytosol + aldosterone.
of the preformed complex occurs. The results indicate that prebinding of the [3H]dexamethasone can protect the cytoplasmic binding activity from inactivation by N-ethylmaleimide or iodoacetamide. In order to investigate the kinetics of inactivation of the cytoplasmic receptor with various glucocorticoid hormones bound to it, a series of hormone-receptor complexes were formed, and the inactivation of these complexes by N-ethylmaleimide was followed. The results are shown with receptor from mouse cells and receptor from hamster cells in Fig. 1A and B. In each case, N-ethylmaleimide inactivated the receptor activity at 4° within 2 min of NEM addition if added prior to any of the hormones. In the case of the mouse cell receptor activity, if the hormone dexamethasone (1A) or hydrocortisone (IB) was first bound to the receptor, slight loss of the complex, approximately 10-15% of the radioactivity with dexamethasone (1A) and 15-20% with hydrocortisone (1B) was observed. When a similar experiment was performed with hamster cell receptor, virtually no destruction of the complex was observed with the subsequent addition of NEM over the next 90 min of reaction at 4°. When similar experiments were performed by prebinding corticosterone, as shown in Fig. 1A, a somewhat different result was obtained. Over the course of the 90 min reaction with NEM, the prebound mouse cell corticosterone receptor complex was inactivated by reaction with the NEM. Presumably, this occurred by virtue of a reversible equilibrium between the corticosterone and the receptor protein, allowing the NEM to act on the sulfhydryl-sensitive group(s) in the protein. A similar result occurred with the hamster cell receptor, although the rate of inactivation by NEM of th4;
3062
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Proc. Nat. Acad. Sci. USA 72 (1975)
Table 2. Rate of inactivation of hormone-receptor complex by N-ethylmaleimide
Table 3. Conditions for binding to DNA-cellulose
Conditions
Time of 50% inactivation (min)
Hamster Hormone complex
Mouse receptor
receptor
Dexamethasone Hydrocortisone Corticosterone Aldosterone Deoxycorticosterone Progesterone
> 180 > 180
> 180
20-35 15-20 10-15 180 > 180 60 15-20 N.T.
Mouse or hamster cell cytosol, 2.0 ml (20-30 mg/ml), was incubated with 50-60 pmol/ml of each [3H]steroid for 90 min at 40. An 0.05 ml aliquot of a 0.25 M NEM solution was added per 2.0 ml reaction to a final concentration of 6 mM NEM. Aliquots of 0.2 ml were removed after various periods of time at 40 and charcoal-resistant radioactivity was determined. Time of 50% inactivation represents that point where charcoal-resistant radioactivity had decreased to one-half of that present at zero time. Progesterone binding to hamster receptor was not tested due to high nonspecific binding. In parallel reaction mixtures incubated minus NEM, over 95% of the radioactivity remained charcoal-resistant for up to 180 min.
preformed corticosterone complex was much slower in this cytosol. In Fig. 1B, a similar experiment was performed utilizing the hormone aldosterone bound to the glucocorticoid receptor protein. Again, the mouse hormone-receptor complex was relatively rapidly inactivated by the NEM addition, whereas the hamster cell aldosterone-receptor complex was inactivated much less rapidly. The results indicate that several glucocorticoid hormones can be used to protect the receptor protein from inactivation with N-ethylmaleimide and that the rates of inactivation differ with different hormones prebound to the receptor. In addition, the rates of inactivation with prebound corticosterone and aldosterone are different for the receptor found in BHK cells and the receptor found in mouse cells. The results of a series of such inactivations with NEM of the preformed hormone-receptor complex are summarized in Table 2. The time of 50% inactivation (in minutes) for each preformed hormone complex is indicated for the mouse cytosol receptor and the hamster cytosol receptor. In addition to the hormones indicated in Fig. 1A and B, deoxycorticosterone and progesterone were also tested. The deoxycorticosterone and progesterone binding complexes were rapidly inactivated by the NEM treatment, with the progesterone complex being the most rapidly inactivated. The hamster receptor could not be tested with progesterone due to a high background of progesterone binding in the hamster cytosol which was nonspecific as defined by the inability of unlabeled'dexamethasone to compete with it. In general, the hamster hormone-receptor complex is more slowly inactivated than the mouse hormone-receptor complex. Chemical inactivation of nuclear binding The ability of the hormone-receptor complex to stimulate transcription is dependent upon activation of this complex, which somehow increases the affinity of the hormone-receptor complex for DNA. Although the specificity involved in DNA binding with the glucocorticoid-receptor complex is not understood, the rate of this activation can be increased by exposing the hormone-receptor complex either to elevated temperature or to salt. In
order to
investigate the chemi-
cal nature of the DNA binding sites, we first explored condi-
Dexamethasone bound
Temperature, °C 4 4 16 16
16 16
Cation
-
Ca++ -
Ca++ (10 mM) Mg++ (10 mM) Mn++ (5 mM)
(cpm)
% Binding
3,600
6.5 6.5 14.9 47.2 21.8 25.4
3,600 8,200 26,000 12,000 14,000
Mouse cell cytosol, 5.0 ml (20-30 mg/ml), was preincubated for 90 min at 40 with [3H]dexamethasone (about 200 pmol/ml). Duplicate portions, 0.5 ml, of the cytosol were adjusted as shown above to the cation concentrations by the addition of 0.005 ml of 1.0 M CaCl2, 0.005 ml of 1.0 M MgCl2, or 0.005 ml of 0.50 M MnCl2, and were immediately charcoal treated to remove free hormone. Aliquots of 0.20 ml (approximately 55,000 cpm of [3H]dexamethasone) were then mixed with 20 mg of dry DNA-cellulose per aliquot (2.6 gg of DNA per mg of cellulose) and incubated for 30 min at 160. At that time, the tubes were chilled to 40, and cellulose was sedimented out of the suspension by centrifugation at 5000 x g for 5 min at 40 and washed twice with 1 ml of TG buffer, and cpm were determined by mixing the DNA-cellulose pellet with 10 ml of Beckman Redissolve VI. One hundred percent cpm represents the sum of those cpm bound to DNA-cellulose and the charcoal-resistant radioactivity remaining in the supernatant.
tions for maximal binding of the complex to DNA-cellulose. The results are shown in Table 3. To obtain maximal binding, activation in the presence of calcium was required. Magnesium or manganese over a range of concentrations could not substitute for calcium. With [3H]dexamethasone prebound to the receptor, the complex in the presence of calcium could be bound to DNA-cellulose after raising the temperature to 16° for 30 min. No binding (less than 2%) was observed with cellulose prepared in parallel but without DNA. As shown in Table 4, iodoacetamide, a reagent capable of reacting with both sulfhydryl groups and other groups on amino acids in proteins (12), had a marked effect on the ability of the hormone-receptor complex to bind to DNAcellulose. If during the 30 min treatment at 16° iodoacetamide was included, the binding to DNA cellulose was prevented. Similar results were obtained if nuclei were used instead as the source of DNA. Importantly, the hormone-receptor complex remained intact after IAA treatment, as assayed by the inability of charcoal to adsorb the [3H]dexamethasone, indicating that the receptor had retained its ability to bind the [3H]dexamethasone more strongly than the charcoal. In addition, the [3H]dexamethasone was still excluded from a Sephadex G-25 column, whereas free [3H]dexamethasone was not. Treatment with low levels of NEM in similar experiments gave equivocal results, but some loss at 160 of the receptor-hormone complex as assayed by charcoal resistance was observed and this reagent was not pursued. In control experiments, IAA-treated DNAcellulose still was able to bind as well the [3H]glucocorticoidreceptor complex. In order to ascertain if prebinding the hormone-receptor complex to DNA-cellulose would protect it from iodoacetamide treatment, a chase experiment was performed as shown in Fig. 2. The [3H]dexamethasone-receptor complex was preformed and binding to DNA-cellulose was carried -out as indicated earlier. Any receptor not bound to DNAcellulose was removed by centrifugation and washing the
Biochemistry: Young et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
3063
Table 4. Iodoacetamide inactivates- DNA binding of hormone-receptor complex
Condition 30 minutes,
40
Percent dexamethDexamethasone asone radioactivity bound remaining char% Binding coal-resistant (cpm)
4,200
12
100
18,000
51
96
30 minutes,
160 30 minutes, 160+ IAA (0.025 M)
TIME (MINUTES)
3,900
11
90
Five milliliters of mouse cell cytosol (20-30 mg/ml) was incubated with [3H]dexamethasone (180-200 pmol/ml cytosol) for 90 min at 40. Calcium chloride, 0.025 ml of a 2.0 M solution, was then added at 40 to give a final concentration of 10 mM CaCl2; 0.50 ml of a 100 mg/ml charcoal suspension was added (final concentration 10 mg/ml), the suspension was mixed on a vortex device, and the mixture was clarified by centrifugation for 10 min at 5000 X g at 40. Duplicate 0.2 ml aliquots were mixed with 20 mg of dry DNAcellulose; 0.01 ml of 0.50 M iodoacetamide was added as indicated and the suspensions were incubated. After 30 min, the mixtures were clarified by centrifugation at 40, and the supernates were carefully removed and charcoal treated. The DNA-cellulose pellets were washed twice with TG buffer at 40, and radioactive content was determined as described in the legend to Table 3. Total charcoal-resistant radioactivity represents that bound to DNA-cellulose plus that in the supernatant remaining charcoal-resistant.
DNA-cellulose. In order to assess the ability of the hormonereceptor complex to subsequently bind to DNA after it had been bound to DNA-cellulose, a chase experiment was performed (11). The DNA-cellulose-receptor-hormone complex was allowed to dissociate at 160 in the presence or absence of added free DNA in the reaction mixture (Fig. 2A). The added DNA in the reaction mixture led to an increased rate in the dissociation of [3H]dexamethasone in a charcoalresistant form, implying that the hormone-receptor was dissociating from DNA-cellulose and binding to the free DNA in solution. Parallel aliquots of hormone-receptor complex, and hormone-receptor complex bound to DNA-cellulose were then treated with iodoacetamide. The iodoacetamide, as shown in Table 4, inactivated by over 95% the ability of the receptor-hormone complex to subsequently bind to DNA-cellulose. But, the hormone-receptor complex bound to DNA-cellulose, which had been treated with iodoacetamide, subsequently dissociated to bind to the free DNA in the medium at the same rate as the untreated complex. These results indicate that, if prebound to DNA-cellulose, the hormone-receptor complex is protected from inactivation by iodoacetamide.
DISCUSSION The chemical modification of the active sites in several enzymes has been important to the understanding of their mechanism of action (13). Such an understanding into the nature of the active sites of hormone receptors would be a useful contribution to the basic understanding of DNA binding proteins in mammalian cells. While definitive information on active site structure can only be gained by working with pure proteins, we have undertaken some experiments of the inactivation of crude fractions of glucocorticoid hormone binding proteins in order to begin to ascertain the active sites involved in binding of glucocorticoid hormones
FIG. 2. Effect of iodoacetamide on complex bound to DNAcellulose: Aliquots (5.0 ml) of cytosol (26 mg/ml) were incubated for 90 min at 40 with [3H]dexamethasone (200 pmol/ml). An 0.025 ml aliquot of 2.0 M CaCl2 was then added to a final concentration of 10 mM, and the cytosol was then charcoal treated as described in earlier legends to remove free hormone. Aliquots of 0.2 ml were then mixed with 20 mg of DNA-cellulose and incubated for 30 min at 160. In (A), the cellulose was then removed by centrifugation and washed once with TG buffer. An 0.2 ml portion of buffer (0.01 M Tris-HCl, pH 7.4, 10% glycerol; 1 mM EDTA; 1 mM dithiothreitol) with or without calf thymus DNA (200 gg) was then mixed with the sedimented cellulose and incubated at 16° for various periods of time. The cellulose was then removed by centrifugation, washed once with TG buffer, and resuspended in 12 ml of Redissolve VI, and radioactivity was determined. In (B), after the initial incubation at 16° for 30 min, 0.01 ml of 0.50 M iodoacetamide was added per 0.2 ml DNA-cellulose mixture for 30 min at 16° to final concentration of 25 mM. The cellulose was then removed by centrifugation, washed once with TG buffer (minus dithiothreitol), and then incubated with buffer or calf thymus DNA as described above. 0-0 minus DNA; *-* plus DNA.
and the binding of the hormone-receptor complex to DNA. Earlier experiments have indicated that the glucocorticoid hormone receptor protein can be inactivated by treatment with reagents which are known to interact with sulfhydryl groups on proteins (7-10). The results presented here have confirmed these experiments and further indicated that by prebinding a variety of glucocorticoid hormones to the glucocorticoid receptor proteins, the complex can be protected to varying degrees from inactivation with the chemical inactivating reagent N-ethylmaleimide. The most stable complexes were formed with either dexamethasone or hydrocortisone and the least stable with corticosterone, aldosterone, deoxycorticosterone, and progesterone. However, in each case the hormone-receptor complex was more protected than if no prebinding of hormone had been performed. Similar results were seen with the receptor isolated from either mouse cytosol or baby hamster kidney cytosol. However, importantly, the rate of destruction of the preformed complex differed in the mouse cell and in the hamster cell when either corticosterone or aldosterone was used to form the prebound complex. These results suggest that chemical modification of cytosol receptor proteins might be a useful mode of distinguishing in somatic cell hybrids which receptor is present. Since somatic cell hybrids have been used to study the inducibility of various enzymes by glucocorticoids (14, 15), this approach could be useful in distinguishing which species of receptor is responsible for the induction. Such studies could help elucidate questions of the molecular specificity of DNA binding of the receptor protein. In addition, we have investigated the mechanism of action of the second step of the glucocorticoid hormone-receptor complex binding to DNA. By preforming the hormone-receptor complex and then treating it during "activation" with iodoacetamide, we could prevent the binding of the receptor
3064
Biochemistry: Young et al.
complex to DNA without destroying the hormone-receptor complex. By prebinding the hormone-receptor complex to DNA cellulose, the hormone-receptor complex could be protected from inactivation by iodoacetamide. Clearly the mode of inactivation of receptor activity and DNA binding by these reagents is difficult to interpret in crude extracts. However, because of the high degree of specificity in the protection of stereospecific glucocorticoids, the results suggest that the binding of glucocorticoid hormones to steroid receptors involves sulfhydryl groups. The simplest interpretation of the protection by dexamethasone or hydrocortisone from the NEM inactivation of the receptor protein would suggest that a sulfhydryl group is directly involved in the active site of binding of glucocorticoid hormones. It is also possible that the protection of the glucocorticoid hormone is occurring by an indirect mechanism such as induction of a conformational change in the molecule which protects subsequently a sulfhydryl group from inactivation with NEM. Further studies will be necessary with more purified protein preparations to distinguish between these two possibilities. Similarly, the treatment of the preformed hormone-receptor complex with iodoacetamide clearly distinguishes a second chemical site necessary for the binding of the hormone-receptor complex to DNA. The iodoacetamide could be acting at a second site on a single receptor protein, thus preventing its binding to DNA. Alternatively, iodoacetamide could be inactivating a second protein in the cytosol preparation which is necessary for the activation or binding of the complex to DNA. Again, further purification of the cytosol binding activity will be necessary to distinguish between these alternatives. However, these biochemical results are consistent with recent genetic experiments of Tomkins and collaborators (16-18). They selected mouse lymphoma cells resistant to killing by high doses of dexamethasone and found among the survivors classes of mutants with differing defects in the steroid hormone receptor binding activity (16-18). They have suggested that there is a class of receptor mutants in the steroid-resistant lymphoma cells which are capable of binding the glucocorticoids but which are unable to subsequently bind the hormone to DNA. However, the magnitude of the effects observed with the various NT-
Proc. Nat. Acad. sci. usA 72 0975)
lymphoma cell mutants is much less than the chemical effect of iodoacetamide in these experiments. The current results suggest that the steps in the interaction of glucocorticoid hormones with their cytoplasmic receptor proteins and the subsequent binding of this protein to DNA can be studied with the use of chemical reagents used to probe the active sites of various enzymes. It is hoped that further studies with more purified preparations of cytoplasmic receptors and the use of such variously modified proteins in in vitro transcription systems where the receptor stimulates the synthesis of the murine mammary tumor virus RNA will be useful in elucidating mechanisms of transcription of RNA in mammalian cells. 1. Jensen, C. V. & DeSombre, E. R. (1972) Annu. Rev. Biochem. 41,203-230. 2. Parks, W. P., Scolnick, E. M. & Kozikowski, E. H. (1974) Science 184,158-160. 3. Parks, W. P., Ransom, J. C., Young, H. A. & Scolnick, E. M. (1975) J. Blol. Chem. 250,3330-336. 4. Schutz, G., Killewich, L., Chen, G. & Feigelson, P. (1975) Proc. Nat. Acad. Sci. USA 72,1017-1020. 5. McGrath, C. M. (1971) J. Nat. Cancer Inst. 47,455-459. 6. Young, H. A., Scolnick, E. M. & Parks, W. P. (1975) J. Biol. Chem. 250,3337-343. 7. Schaumberg, B. P. (1972) Biochim. Biophys. Acta 261, 219235. 8. Gardner, D. G. & Wittliff, J. I. (1973) Biochim. Blophys. Acta 320,617-627. 9. Watanabe, H., Orth, D. N. & Toft, D. 0. (1973) J. Biol. Chem. 248,7625-7630. 10. Mayer, M., Kaiser, N., Milholland, R. J. & Rosen, F. (1974) J. Biol. Chem. 250,1207-1211. 11. Litman, R. M. (1968) J. Biol. Chem. 243,6222-6233. 12. Andre, J. & Rochefort, H. (1975) FEBS Lett. 50, 319-322. 13. Vallee, B. L. & Riordan, J. F. (1969) Annu. Rev. Biochem. 39, 733-794. 14. Thompson, E. B. & Gelehrter, T. D. (1971) Proc. Nat. Acad. Sd. USA 68,2589-2593. 15. Croce, C. M., Koprowski, H. & Litwack, G. (1974) Nature
249,839-841. 16. Sibley, C. H. & Tomkins, G. M. (1974) Cell 2,221-227. 17. Gehring, I. & Tomkins, G. M. (1974) Cell 3,301-306. 18. Yamamoto, K. R., Stampfer, M. R. & Tomkins, G. M. (1974) Proc. Nat. Acad. Sci. USA 71, 3901-3905.
Vol. 72, No. 8, pp. 3060-3064, August 1975 Biochemistry
Effect of chemical inactivating agents on glucocorticoid receptor proteins in mouse and hamster cells (dexamethasone/N-ethylmaleimide/iodoacetamide/hormone binding site/DNA binding site)
HOWARD A. YOUNG, WADE P. PARKS, AND EDWARD M. SCOLNICK National Cancer Institute, Bethesda, Maryland 20014
Communicated by C. B. Anfinsen, June 5,1975
hormones to the cytoplasmic receptor protein, we can protect the receptor from inactivation with N-ethylmaleimide. In addition, we have investigated the second step in the action of the hormone by treating the hormone-receptor complex with iodoacetamide. Although the hormone-receptor complex remains intact after iodoacetamide treatment, the IAA-treated complex is no longer able to bind to DNA. The results suggest that the binding of [3H]glucocorticoids to the cytoplasmic receptor protein may involve a sulfhydryl group, and that the subsequent binding to DNA either involves a second site on the receptor molecule separate from the hormone binding site, or that alternatively another protein(s) may be involved in the DNA binding step.
ABSTRACT The effect of N-ethylmaleimide and iodoacetamide on the glucocorticoid receptor activity extracted from the cytosol of either mouse or hamster cells has been investigated. Treatment of mouse or hamster cytosol with Nethylmaleimide or iodoacetamide rapidly inactivates the [3Hlglucocorticoid hormone binding activity of either cytosol. Prebinding the glucocorticoid hormone, dexamethasone, to the cytosol receptor blocks the rapid inactivation of the receptor by N-ethylmaleimide. Treatment of the prebound hormone-receptor complex with iodoacetamide prevents the subsequent binding of the hormone-receptor complex to DNA without causing a dissociation of the complex. Although the conclusions may be limited by the lack of purity of the receptor, the results suggest that a sulfhydryl group is involved in the binding of glucocorticoid hormones to the receptor protein. In addition, the results suggest that iodoacetamide is inactivating a separate chemical site which is necessary for the binding of the hormone-receptor complex to DNA.
MATERIALS AND METHODS Materials. [3H]Dexamethasone (28 Ci/mmol) was obtained from Amersham-Searle Corp. Other [3H]hormones, [3H]hydrocortisone (83 Ci/mmol), [3H]aldosterone (109 Ci/ mmol), [3H]corticosterone (83 Ci/mmol), [3H]progesterone (46 Ci/mmol), [3H]deoxycorticosterone (46 Ci/mmol), were obtained from New England Nuclear. With the exception of [3H]aldosterone, hormones, which were sold in benzene-ethanol mixtures (9:1), were dried under N2 and dissolved in absolute ethanol before use. N-ethylmaleimide (NEM) was obtained from Calbiochem, and iodoacetamide (IAA) was purchased from the Aldrich Chemical Co. CF-1l cellulose was purchased from Calbiochem, and DNA-cellulose was prepared as previously described (11) with DNA from NIH 3T3 mouse cells. The DNA content was 2.6 ,tg/mg of cellulose. Activated charcoal was from Merck (Rahway, N.J.). Buffers. Tris-glycerol buffer (TG) contained 10% (v/v) glycerol, 0.01 M Tris-HCl, pH 7.9, 0.05 M NaCl, 100 Mg/ml of bovine serum albumin, 1 mM EDTA, and 1 mM dithiothreitol. NEM was used as a 0.25 M stock solution in 0.001 M Tris-HCl, pH 7.4, and iodoacetamide was 0.50 M in 0.001 M Tris-HCl, pH 7.4. Methods. Preparation of cytosol and binding of [3H]hormone to receptor was measured by the charcoal binding assay as previously described (6), with the exception that Tris-glycerol buffer was now utilized during the extraction of receptor from cells. All binding studies were performed at 40 and represented high-affinity, saturable specific steroid binding as documented in an earlier publication (6). Cytosol preparations had protein concentrations ranging from 20 to 30 mg/ml and were stored in aliquots at -170°, and used after only one freeze-thaw cycle. Binding to DNA-cellulose was done in the manner described by Andre and Rochefort for estrogen receptor (12). Details are described in the appropriate legends.
The steroid hormones, which include glucocorticoids, estrogens, androgens, and progestins, have many similarities in their mode of action (1). Each hormone first binds with high affinity to a specific cytoplasmic receptor protein. Subsequently, the hormone-receptor complex becomes "activated" and transported to nuclei, and stimulates the transcription of RNA from DNA (1-4). Recently, the effect of glucocorticoid hormones has been studied in cell cultures derived from mouse mammary tumors. The glucocorticoid hormones have been observed, both in primary cell cultures (5) and in cell lines derived from murine mammary tumors, to stimulate an increase in the production of murine mammary tumor virus (MMTV) particles (2). This increase in production of MMTV particles is accompanied by a specific increase in MMTV RNA sequences (2, 3). Importantly, the glucocorticoid hormone effect on MMTV RNA synthesis is mediated by the binding of the hormone to a cytoplasmic receptor protein which has many properties in common with receptor proteins studied in other glucocorticoid induction systems (6). In earlier experiments on the mechanism of action of glucocorticoid hormones, the cytoplasmic receptor protein has been shown to be sensitive to inactivation with chemical reagents which can interact with sulfhydryl groups on proteins (7-10). In an effort to understand further the mode of action of the cytoplasmic receptor protein, we have investigated the effects of various chemical reagents which can inactivate the ability of the cytoplasmic receptor protein to bind [3H]glucocorticoid hormones. By prebinding glucocorticoid Abbreviations: MMTV, mouse mammary tumor virus; BHK, baby hamster kidney; NEM, N-ethylmaleimide; IAA, iodoacetamide; TG, Tris-glycerol; Dxm, dexamethasone. 3060
Biochemistry: Young et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Protection of receptor by dexamethasone (Dxm)
E z
w < 80
Charcoal-resistant radioactive dexamethasone Addition to cytosol
[3H]Dxm [3H] Dxm + 500-fold unlabeled Dxm [3H]Dxm+6mMNEM [3H]Dxm + [6 mM NEM + 50 mM dithiothreitol] [3H]Dxm + 12 mM IAA [3H]Dxm + [12 mM IAA + 50 mM dithiothreitol] [3H]Dxm prebound 90 min at
cpm
'
B. 'I ' --40--
15%); therefore all studies were performed with 50-100 pmol of [3H]dexamethasone per ml. Addition of N-ethylmaleimide or iodoacetamide prior to the [3H]dexamethasone leads to complete inactivation of the cytoplasmic receptor activity. If a 10-fold excess of dithiothreitol is first added to the N-ethylmaleimide or iodoacetamide, no inactivation of the receptor binding is observed. If the [3H]dexamethasone is prebound to the cytoplasmic receptor and N-ethylmaleimide or iodoacetamide is subsequently added for an additional period of 30 min, little destruction
0* a:
o0
40 20 0 15 30 4560 75900 15 30 4560 7590 TIME (MINUTES)
FIG. 1. Kinetics of inactivation of preformed complexes: Aliquots (2 ml) of either mouse cell cytosol (20-30 mg/ml) or hamster cytosol (19-25 mg/ml) were preincubated at 4° for 90 min with either [3H]dexamethasone, [3H]hydrocortisone, [3H]corticosterone, or [3H]aldosterone (50-60 pmol/ml). An 0.05 ml aliquot of 0.25 M NEM was then added (final concentration 6 mM) and at various periods of time 0.2 ml aliquots were removed, mixed with 0.025 ml of charcoal (100 mg/ml) to a final concentration of 10 mg/ml, vortexed, and immediately centrifuged at 5000 X g for 3 min at 4°. Aliquots (0.15 ml) of the supernatant were removed and radioactivity was determined. Nonspecific binding (in the presence of a 1000fold excess of unlabeled dexamethasone) represented 10-15% of the charcoal-resistant radioactivity and was subtracted from all values. 100% values represent, for mouse cytosol, 30,000 cpm for dexamethasone, 82,000 cpm for hydrocortisone, 78,000 for corticosterone, and 29,000 cpm for aldosterone; 100% values for hamster cytosol are: 20,000 cpm for dexamethasone; 34,000 cpm for hydrocortisone; 23,000 cpm for corticosterone; and 10,000 cpm for aldosterone. (A) * - *, mouse cytosol + dexamethasone; & - &, hamster cytosol + dexamethasone; * - *, mouse cytosol + corticosterone; o - o, hamster cytosol + corticosterone. (B 0 --- *, mouse cytosol + hydrocortisone; 0 --- 0, hamster cytosol + hydrocortisone; 0 - *, mouse cytosol + aldosterone; 0 - 0, hamster cytosol + aldosterone.
of the preformed complex occurs. The results indicate that prebinding of the [3H]dexamethasone can protect the cytoplasmic binding activity from inactivation by N-ethylmaleimide or iodoacetamide. In order to investigate the kinetics of inactivation of the cytoplasmic receptor with various glucocorticoid hormones bound to it, a series of hormone-receptor complexes were formed, and the inactivation of these complexes by N-ethylmaleimide was followed. The results are shown with receptor from mouse cells and receptor from hamster cells in Fig. 1A and B. In each case, N-ethylmaleimide inactivated the receptor activity at 4° within 2 min of NEM addition if added prior to any of the hormones. In the case of the mouse cell receptor activity, if the hormone dexamethasone (1A) or hydrocortisone (IB) was first bound to the receptor, slight loss of the complex, approximately 10-15% of the radioactivity with dexamethasone (1A) and 15-20% with hydrocortisone (1B) was observed. When a similar experiment was performed with hamster cell receptor, virtually no destruction of the complex was observed with the subsequent addition of NEM over the next 90 min of reaction at 4°. When similar experiments were performed by prebinding corticosterone, as shown in Fig. 1A, a somewhat different result was obtained. Over the course of the 90 min reaction with NEM, the prebound mouse cell corticosterone receptor complex was inactivated by reaction with the NEM. Presumably, this occurred by virtue of a reversible equilibrium between the corticosterone and the receptor protein, allowing the NEM to act on the sulfhydryl-sensitive group(s) in the protein. A similar result occurred with the hamster cell receptor, although the rate of inactivation by NEM of th4;
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Proc. Nat. Acad. Sci. USA 72 (1975)
Table 2. Rate of inactivation of hormone-receptor complex by N-ethylmaleimide
Table 3. Conditions for binding to DNA-cellulose
Conditions
Time of 50% inactivation (min)
Hamster Hormone complex
Mouse receptor
receptor
Dexamethasone Hydrocortisone Corticosterone Aldosterone Deoxycorticosterone Progesterone
> 180 > 180
> 180
20-35 15-20 10-15 180 > 180 60 15-20 N.T.
Mouse or hamster cell cytosol, 2.0 ml (20-30 mg/ml), was incubated with 50-60 pmol/ml of each [3H]steroid for 90 min at 40. An 0.05 ml aliquot of a 0.25 M NEM solution was added per 2.0 ml reaction to a final concentration of 6 mM NEM. Aliquots of 0.2 ml were removed after various periods of time at 40 and charcoal-resistant radioactivity was determined. Time of 50% inactivation represents that point where charcoal-resistant radioactivity had decreased to one-half of that present at zero time. Progesterone binding to hamster receptor was not tested due to high nonspecific binding. In parallel reaction mixtures incubated minus NEM, over 95% of the radioactivity remained charcoal-resistant for up to 180 min.
preformed corticosterone complex was much slower in this cytosol. In Fig. 1B, a similar experiment was performed utilizing the hormone aldosterone bound to the glucocorticoid receptor protein. Again, the mouse hormone-receptor complex was relatively rapidly inactivated by the NEM addition, whereas the hamster cell aldosterone-receptor complex was inactivated much less rapidly. The results indicate that several glucocorticoid hormones can be used to protect the receptor protein from inactivation with N-ethylmaleimide and that the rates of inactivation differ with different hormones prebound to the receptor. In addition, the rates of inactivation with prebound corticosterone and aldosterone are different for the receptor found in BHK cells and the receptor found in mouse cells. The results of a series of such inactivations with NEM of the preformed hormone-receptor complex are summarized in Table 2. The time of 50% inactivation (in minutes) for each preformed hormone complex is indicated for the mouse cytosol receptor and the hamster cytosol receptor. In addition to the hormones indicated in Fig. 1A and B, deoxycorticosterone and progesterone were also tested. The deoxycorticosterone and progesterone binding complexes were rapidly inactivated by the NEM treatment, with the progesterone complex being the most rapidly inactivated. The hamster receptor could not be tested with progesterone due to a high background of progesterone binding in the hamster cytosol which was nonspecific as defined by the inability of unlabeled'dexamethasone to compete with it. In general, the hamster hormone-receptor complex is more slowly inactivated than the mouse hormone-receptor complex. Chemical inactivation of nuclear binding The ability of the hormone-receptor complex to stimulate transcription is dependent upon activation of this complex, which somehow increases the affinity of the hormone-receptor complex for DNA. Although the specificity involved in DNA binding with the glucocorticoid-receptor complex is not understood, the rate of this activation can be increased by exposing the hormone-receptor complex either to elevated temperature or to salt. In
order to
investigate the chemi-
cal nature of the DNA binding sites, we first explored condi-
Dexamethasone bound
Temperature, °C 4 4 16 16
16 16
Cation
-
Ca++ -
Ca++ (10 mM) Mg++ (10 mM) Mn++ (5 mM)
(cpm)
% Binding
3,600
6.5 6.5 14.9 47.2 21.8 25.4
3,600 8,200 26,000 12,000 14,000
Mouse cell cytosol, 5.0 ml (20-30 mg/ml), was preincubated for 90 min at 40 with [3H]dexamethasone (about 200 pmol/ml). Duplicate portions, 0.5 ml, of the cytosol were adjusted as shown above to the cation concentrations by the addition of 0.005 ml of 1.0 M CaCl2, 0.005 ml of 1.0 M MgCl2, or 0.005 ml of 0.50 M MnCl2, and were immediately charcoal treated to remove free hormone. Aliquots of 0.20 ml (approximately 55,000 cpm of [3H]dexamethasone) were then mixed with 20 mg of dry DNA-cellulose per aliquot (2.6 gg of DNA per mg of cellulose) and incubated for 30 min at 160. At that time, the tubes were chilled to 40, and cellulose was sedimented out of the suspension by centrifugation at 5000 x g for 5 min at 40 and washed twice with 1 ml of TG buffer, and cpm were determined by mixing the DNA-cellulose pellet with 10 ml of Beckman Redissolve VI. One hundred percent cpm represents the sum of those cpm bound to DNA-cellulose and the charcoal-resistant radioactivity remaining in the supernatant.
tions for maximal binding of the complex to DNA-cellulose. The results are shown in Table 3. To obtain maximal binding, activation in the presence of calcium was required. Magnesium or manganese over a range of concentrations could not substitute for calcium. With [3H]dexamethasone prebound to the receptor, the complex in the presence of calcium could be bound to DNA-cellulose after raising the temperature to 16° for 30 min. No binding (less than 2%) was observed with cellulose prepared in parallel but without DNA. As shown in Table 4, iodoacetamide, a reagent capable of reacting with both sulfhydryl groups and other groups on amino acids in proteins (12), had a marked effect on the ability of the hormone-receptor complex to bind to DNAcellulose. If during the 30 min treatment at 16° iodoacetamide was included, the binding to DNA cellulose was prevented. Similar results were obtained if nuclei were used instead as the source of DNA. Importantly, the hormone-receptor complex remained intact after IAA treatment, as assayed by the inability of charcoal to adsorb the [3H]dexamethasone, indicating that the receptor had retained its ability to bind the [3H]dexamethasone more strongly than the charcoal. In addition, the [3H]dexamethasone was still excluded from a Sephadex G-25 column, whereas free [3H]dexamethasone was not. Treatment with low levels of NEM in similar experiments gave equivocal results, but some loss at 160 of the receptor-hormone complex as assayed by charcoal resistance was observed and this reagent was not pursued. In control experiments, IAA-treated DNAcellulose still was able to bind as well the [3H]glucocorticoidreceptor complex. In order to ascertain if prebinding the hormone-receptor complex to DNA-cellulose would protect it from iodoacetamide treatment, a chase experiment was performed as shown in Fig. 2. The [3H]dexamethasone-receptor complex was preformed and binding to DNA-cellulose was carried -out as indicated earlier. Any receptor not bound to DNAcellulose was removed by centrifugation and washing the
Biochemistry: Young et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
3063
Table 4. Iodoacetamide inactivates- DNA binding of hormone-receptor complex
Condition 30 minutes,
40
Percent dexamethDexamethasone asone radioactivity bound remaining char% Binding coal-resistant (cpm)
4,200
12
100
18,000
51
96
30 minutes,
160 30 minutes, 160+ IAA (0.025 M)
TIME (MINUTES)
3,900
11
90
Five milliliters of mouse cell cytosol (20-30 mg/ml) was incubated with [3H]dexamethasone (180-200 pmol/ml cytosol) for 90 min at 40. Calcium chloride, 0.025 ml of a 2.0 M solution, was then added at 40 to give a final concentration of 10 mM CaCl2; 0.50 ml of a 100 mg/ml charcoal suspension was added (final concentration 10 mg/ml), the suspension was mixed on a vortex device, and the mixture was clarified by centrifugation for 10 min at 5000 X g at 40. Duplicate 0.2 ml aliquots were mixed with 20 mg of dry DNAcellulose; 0.01 ml of 0.50 M iodoacetamide was added as indicated and the suspensions were incubated. After 30 min, the mixtures were clarified by centrifugation at 40, and the supernates were carefully removed and charcoal treated. The DNA-cellulose pellets were washed twice with TG buffer at 40, and radioactive content was determined as described in the legend to Table 3. Total charcoal-resistant radioactivity represents that bound to DNA-cellulose plus that in the supernatant remaining charcoal-resistant.
DNA-cellulose. In order to assess the ability of the hormonereceptor complex to subsequently bind to DNA after it had been bound to DNA-cellulose, a chase experiment was performed (11). The DNA-cellulose-receptor-hormone complex was allowed to dissociate at 160 in the presence or absence of added free DNA in the reaction mixture (Fig. 2A). The added DNA in the reaction mixture led to an increased rate in the dissociation of [3H]dexamethasone in a charcoalresistant form, implying that the hormone-receptor was dissociating from DNA-cellulose and binding to the free DNA in solution. Parallel aliquots of hormone-receptor complex, and hormone-receptor complex bound to DNA-cellulose were then treated with iodoacetamide. The iodoacetamide, as shown in Table 4, inactivated by over 95% the ability of the receptor-hormone complex to subsequently bind to DNA-cellulose. But, the hormone-receptor complex bound to DNA-cellulose, which had been treated with iodoacetamide, subsequently dissociated to bind to the free DNA in the medium at the same rate as the untreated complex. These results indicate that, if prebound to DNA-cellulose, the hormone-receptor complex is protected from inactivation by iodoacetamide.
DISCUSSION The chemical modification of the active sites in several enzymes has been important to the understanding of their mechanism of action (13). Such an understanding into the nature of the active sites of hormone receptors would be a useful contribution to the basic understanding of DNA binding proteins in mammalian cells. While definitive information on active site structure can only be gained by working with pure proteins, we have undertaken some experiments of the inactivation of crude fractions of glucocorticoid hormone binding proteins in order to begin to ascertain the active sites involved in binding of glucocorticoid hormones
FIG. 2. Effect of iodoacetamide on complex bound to DNAcellulose: Aliquots (5.0 ml) of cytosol (26 mg/ml) were incubated for 90 min at 40 with [3H]dexamethasone (200 pmol/ml). An 0.025 ml aliquot of 2.0 M CaCl2 was then added to a final concentration of 10 mM, and the cytosol was then charcoal treated as described in earlier legends to remove free hormone. Aliquots of 0.2 ml were then mixed with 20 mg of DNA-cellulose and incubated for 30 min at 160. In (A), the cellulose was then removed by centrifugation and washed once with TG buffer. An 0.2 ml portion of buffer (0.01 M Tris-HCl, pH 7.4, 10% glycerol; 1 mM EDTA; 1 mM dithiothreitol) with or without calf thymus DNA (200 gg) was then mixed with the sedimented cellulose and incubated at 16° for various periods of time. The cellulose was then removed by centrifugation, washed once with TG buffer, and resuspended in 12 ml of Redissolve VI, and radioactivity was determined. In (B), after the initial incubation at 16° for 30 min, 0.01 ml of 0.50 M iodoacetamide was added per 0.2 ml DNA-cellulose mixture for 30 min at 16° to final concentration of 25 mM. The cellulose was then removed by centrifugation, washed once with TG buffer (minus dithiothreitol), and then incubated with buffer or calf thymus DNA as described above. 0-0 minus DNA; *-* plus DNA.
and the binding of the hormone-receptor complex to DNA. Earlier experiments have indicated that the glucocorticoid hormone receptor protein can be inactivated by treatment with reagents which are known to interact with sulfhydryl groups on proteins (7-10). The results presented here have confirmed these experiments and further indicated that by prebinding a variety of glucocorticoid hormones to the glucocorticoid receptor proteins, the complex can be protected to varying degrees from inactivation with the chemical inactivating reagent N-ethylmaleimide. The most stable complexes were formed with either dexamethasone or hydrocortisone and the least stable with corticosterone, aldosterone, deoxycorticosterone, and progesterone. However, in each case the hormone-receptor complex was more protected than if no prebinding of hormone had been performed. Similar results were seen with the receptor isolated from either mouse cytosol or baby hamster kidney cytosol. However, importantly, the rate of destruction of the preformed complex differed in the mouse cell and in the hamster cell when either corticosterone or aldosterone was used to form the prebound complex. These results suggest that chemical modification of cytosol receptor proteins might be a useful mode of distinguishing in somatic cell hybrids which receptor is present. Since somatic cell hybrids have been used to study the inducibility of various enzymes by glucocorticoids (14, 15), this approach could be useful in distinguishing which species of receptor is responsible for the induction. Such studies could help elucidate questions of the molecular specificity of DNA binding of the receptor protein. In addition, we have investigated the mechanism of action of the second step of the glucocorticoid hormone-receptor complex binding to DNA. By preforming the hormone-receptor complex and then treating it during "activation" with iodoacetamide, we could prevent the binding of the receptor
3064
Biochemistry: Young et al.
complex to DNA without destroying the hormone-receptor complex. By prebinding the hormone-receptor complex to DNA cellulose, the hormone-receptor complex could be protected from inactivation by iodoacetamide. Clearly the mode of inactivation of receptor activity and DNA binding by these reagents is difficult to interpret in crude extracts. However, because of the high degree of specificity in the protection of stereospecific glucocorticoids, the results suggest that the binding of glucocorticoid hormones to steroid receptors involves sulfhydryl groups. The simplest interpretation of the protection by dexamethasone or hydrocortisone from the NEM inactivation of the receptor protein would suggest that a sulfhydryl group is directly involved in the active site of binding of glucocorticoid hormones. It is also possible that the protection of the glucocorticoid hormone is occurring by an indirect mechanism such as induction of a conformational change in the molecule which protects subsequently a sulfhydryl group from inactivation with NEM. Further studies will be necessary with more purified protein preparations to distinguish between these two possibilities. Similarly, the treatment of the preformed hormone-receptor complex with iodoacetamide clearly distinguishes a second chemical site necessary for the binding of the hormone-receptor complex to DNA. The iodoacetamide could be acting at a second site on a single receptor protein, thus preventing its binding to DNA. Alternatively, iodoacetamide could be inactivating a second protein in the cytosol preparation which is necessary for the activation or binding of the complex to DNA. Again, further purification of the cytosol binding activity will be necessary to distinguish between these alternatives. However, these biochemical results are consistent with recent genetic experiments of Tomkins and collaborators (16-18). They selected mouse lymphoma cells resistant to killing by high doses of dexamethasone and found among the survivors classes of mutants with differing defects in the steroid hormone receptor binding activity (16-18). They have suggested that there is a class of receptor mutants in the steroid-resistant lymphoma cells which are capable of binding the glucocorticoids but which are unable to subsequently bind the hormone to DNA. However, the magnitude of the effects observed with the various NT-
Proc. Nat. Acad. sci. usA 72 0975)
lymphoma cell mutants is much less than the chemical effect of iodoacetamide in these experiments. The current results suggest that the steps in the interaction of glucocorticoid hormones with their cytoplasmic receptor proteins and the subsequent binding of this protein to DNA can be studied with the use of chemical reagents used to probe the active sites of various enzymes. It is hoped that further studies with more purified preparations of cytoplasmic receptors and the use of such variously modified proteins in in vitro transcription systems where the receptor stimulates the synthesis of the murine mammary tumor virus RNA will be useful in elucidating mechanisms of transcription of RNA in mammalian cells. 1. Jensen, C. V. & DeSombre, E. R. (1972) Annu. Rev. Biochem. 41,203-230. 2. Parks, W. P., Scolnick, E. M. & Kozikowski, E. H. (1974) Science 184,158-160. 3. Parks, W. P., Ransom, J. C., Young, H. A. & Scolnick, E. M. (1975) J. Blol. Chem. 250,3330-336. 4. Schutz, G., Killewich, L., Chen, G. & Feigelson, P. (1975) Proc. Nat. Acad. Sci. USA 72,1017-1020. 5. McGrath, C. M. (1971) J. Nat. Cancer Inst. 47,455-459. 6. Young, H. A., Scolnick, E. M. & Parks, W. P. (1975) J. Biol. Chem. 250,3337-343. 7. Schaumberg, B. P. (1972) Biochim. Biophys. Acta 261, 219235. 8. Gardner, D. G. & Wittliff, J. I. (1973) Biochim. Blophys. Acta 320,617-627. 9. Watanabe, H., Orth, D. N. & Toft, D. 0. (1973) J. Biol. Chem. 248,7625-7630. 10. Mayer, M., Kaiser, N., Milholland, R. J. & Rosen, F. (1974) J. Biol. Chem. 250,1207-1211. 11. Litman, R. M. (1968) J. Biol. Chem. 243,6222-6233. 12. Andre, J. & Rochefort, H. (1975) FEBS Lett. 50, 319-322. 13. Vallee, B. L. & Riordan, J. F. (1969) Annu. Rev. Biochem. 39, 733-794. 14. Thompson, E. B. & Gelehrter, T. D. (1971) Proc. Nat. Acad. Sd. USA 68,2589-2593. 15. Croce, C. M., Koprowski, H. & Litwack, G. (1974) Nature
249,839-841. 16. Sibley, C. H. & Tomkins, G. M. (1974) Cell 2,221-227. 17. Gehring, I. & Tomkins, G. M. (1974) Cell 3,301-306. 18. Yamamoto, K. R., Stampfer, M. R. & Tomkins, G. M. (1974) Proc. Nat. Acad. Sci. USA 71, 3901-3905.
