0013-7227/90/1273-1087$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Glucocorticoid Receptor Identified on Nuclear Envelopes of Male Rat Livers by Affinity Labeling and Immunochemistry* GILLIAN M. HOWELLf, JAN-AKE GUSTAFSSON, AND YVONNE A. LEFEBVRE Departments of Medicine and Biochemistry, University of Ottawa, Moses and Rose Loeb Research Institute, Ottawa Civic Hospital, Ottawa, Canada Kl Y 4E9; the Bristol Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine (G.M.H.), Houston, Texas 77030; and the Department of Medical Nutrition, Huddinge University Hospital, Karolinska Institute (J.-A.G.), Huddinge, Sweden
ABSTRACT. To exert their action at the genome, steroids must traverse the nuclear envelope, either alone or complexed to their receptor. Our previous studies identified two classes of dexamethasone-binding sites on male rat liver nuclear envelopes: a low capacity, high affinity site and a high capacity, low affinity site. The affinity reagent, [3H]dexamethasone mesylate, labeled peptides at 35-85 kDa, which may be the low affinity glucocorticoid-binding peptides, as these peptides showed the same response to hormonal manipulation as the low affinity [3H]dexamethasone-binding sites previously characterized. With dexamethasone mesylate and a monoclonal antibody against the glucorticoid receptor, we have confirmed that the high affinity binding site on the nuclear envelope is the glucocorticoid receptor. Affinity labeling revealed the presence of a doublet of
I
T IS WELL established that steroids exert their action at the genome of target cells to influence the production of specific mRNAs via interaction of the steroid receptor with specific DNA elements (reviewed in Ref. 1). However, the molecular mechanism by which steroids enter the nucleus to reach the genome is not clear. Early studies based on cell fractionation and autoradiography led to the development of the two-step model of steroid hormone action (2-4). The premise of this model is that free steroid binds to unoccupied receptor, which resides solely in the cytoplasm. Upon binding steroid in the cytoplasm the receptor becomes transformed, i.e. acquires the ability to bind DNA [defined by Grody et al. (5)], and the steroid-receptor complex translocates to the nucleus. However, many investigators have presented Received April 30, 1990. Address all correspondence and requests for reprints to: Dr. Yvonne Lefebvre, Departments of Medicine and Biochemistry, University of Ottawa, Moses and Rose Loeb Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada KlY 4E9. * This work was supported by the Medical Research Council of Canada. t Recipient of a studentship from the Alberta Heritage Foundation for Medical Research.
peptides at 85 and 110 kDa, in the same mol wt range as that reported for the glucocorticoid receptor. Furthermore, these affinity-labeled peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The monoclonal antibody identified a doublet of peptides, a major component of 92-94 kDa and a minor component of 98 kDa. Again, both peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The nuclear envelope-associated glucocorticoid receptor is not extracted by 0.1 M NaCl or 1% Triton X-100. These results show that glucocorticoid hormone interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor, lending support to the two-step model of steroid hormone action. {Endocrinology 127: 1087-1096, 1990)
evidence that tissue fractionation can lead to artefactual redistribution of proteins (6-8). Moreover, steroid receptor transformation is enhanced in vitro in the presence of DNA and so in vivo many represent an intranuclear event (9, 10). More recent studies using cell enucleation and immunohistochemistry provided evidence that the localization of some classes of steroid receptors may be predominantly nuclear (11-15). However, in the case of the glucocorticoid receptor the weight of the evidence continues to support the two-step model (16-22). These studies have involved immunocytochemistry of receptor in target cells (16-21) or transfection of glucocorticoid receptor into nontarget cells, followed by immunocytochemistry (22). We have taken a different approach to the delineation of the mechanism of steroid hormone action. We reasoned that whichever model of steroid hormone action is correct, steroids must traverse the nuclear envelope to reach their site of action within the nucleus. The two models of steroid hormone action predict different modes of nuclear entry for the steroid. A requirement for the two-step model is that steroid enters the nucleus bound to receptor, while the more recently proposed model 1087
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1088
GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
requires that steroid enters the nucleus free of receptor. To resolve the manner in which steroids exert their action, it has been our aim to establish the molecular mechanism by which steroids traverse the nuclear envelope. Based on generally accepted principles of membrane transport, steroids may cross the nuclear envelope in one of three ways: 1) by passive diffusion in an unregulated passage, 2) by carrier-mediated transport, in which the steroid itself would bind to a nuclear envelope carrier, or 3) by carrier-mediated transport, in which the steroid would bind first to the steroid receptor followed by binding of the steroid-receptor complex to a nuclear envelope component carrier. There was no need to consider a fourth possibility, i.e. unregulated passage of the glucocorticoid receptor, as it is known that protein molecules the size of steroid receptors enter the nucleus in a controlled fashion (23). As the first step to determine which of these processes was operable in glucocorticoid hormone action, we characterized the binding of [3H] dexamethasone to male rat liver nuclear envelopes and have identified a class of binding sites demonstrating high affinity and low capacity, which was proteinaceous in nature (24). These studies demonstrated that the machinery exists on the nuclear envelope of the rat liver for regulated carrier-mediated entry of glucocorticoid into the nucleus. In this paper we report the identification, by affinity labeling and immunological techniques, of the dexamethasone-binding sites on the nuclear envelope as the glucocorticoid receptor. The association of the glucocorticoid receptor with the nuclear envelope was found to be glucocorticoid responsive, thus suggesting that glucocorticoid interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor. These data are consistent with the two-step model of steroid hormone action for glucocorticoids.
Materials and Methods Reagents [3H]Dexamethasone (38.0-48.9 Ci/mmol), [3H]dexamethasone mesylate (48.9 Ci/mmol), unlabeled crystalline dexamethasone mesylate, and Enhance spray were purchased from New England Nuclear (Montreal, Quebec, Canada). NCS tissue solubilizer was obtained from Amersham Canada Ltd. (Oakville, Ontario, Canada). Ready Solv was purchased from Beckman (Palo Alto, CA). Af-Tris (hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS), deoxyribonuclease-I (DNase-I; RNase free; shipped on dry ice), dithiothreitol (DTT), and BSA were purchased from Sigma Chemical Co. (St. Louis, MO). Acrylamide, iV,AT-methylene bisacrylamide, and Coomassie brilliant blue R250 were obtained from Serva (Heidelberg, West Germany). Prestained protein standards (high range) for immunoblotting were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD). Hydrogen peroxide (ACS grade
Endo • 1990 Vol 127 • No 3
for immunoblotting) was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Rabbit antirat serum albumin was obtained from U.S. Biochemical Corp. (Cleveland, OH). All other chemicals for electrophoresis, including Tris(hydroxy methyl)aminomethane, the protease substrate gel tablets (for detection of proteolytic activity), and the Immunoblot horseradish peroxidase-conjugated goat antimouse and goat antirabbit immunoglobulin G (IgG) detection kit were purchased from Bio-Rad (Mississauga, Ontario, Canada). All chemicals used in immunoblotting were of at least ACS grade. Animals Male Sprague-Dawley rats, weighing 200-250 g, were obtained from Charles River Canada, Inc. (Montreal, Quebec, Canada), and maintained on a diet of Wayne Lab Blox (Allied Mills, Chicago, IL) and tap water ad libitum. The rats were killed by decapitation. The livers were quickly removed, placed in ice-cold 0.32 M sucrose containing 3 mM MgCl2 and 1 mM DTT that had been adjusted to pH 7.5, rapidly stripped of connective tissue, and weighed. Rats were adrenalectomized under halothane anesthesia and subsequently received 0.9% NaCl in their drinking water. These rats were used 7 days after surgery. Another group of adrenalectomized rats received long term glucocorticoid replacement commencing 7 days after surgery. These rats were injected with dexamethasone phosphate (SABEX, Montreal, Quebec, Canada) in corn oil at a dose of 4 mg/kg, sc, once daily for 14 days. The last injection was given 30-45 min before death. Another group of rats received short term glucocorticoid replacement. They received two injections of dexamethasone phosphate (each 4 mg/kg, sc), one 16-20 h and the second 30-45 min before death. A control group of rats was anesthetized, sham adrenalectomized, and used 7 days after surgery. Preparation of nuclear envelopes All procedures were carried out as rapidly as possible at 0-4 C. Nuclei were isolated by a modified procedure of Widnell and Tata (25). The nuclear envelopes were prepared from these purified nuclei by a modification of the procedure of Kay et al. (26), as described previously (24). However, in some preparations, particularly for affinity labeling and immunoblotting to verify that proteolytic degradation was not occurring, the protease inhibitors phenylmethylsulfonylfluoride (PMSF; 0.1 mM), leupeptin (10 Mg/ml), soybean trypsin inhibitor (100 ng/m\), and benzamidine (10 mM) were included in the homogenization buffer (0.32 M sucrose containing 3 mM MgCl2 and 1 mM DTT, adjusted to pH 7.5). Purified nuclear envelopes were resuspended to a protein concentration of approximately 4 mg/ml in either 25 mM TAPS, pH 8.6, containing 1 mM DTT or 10 mM Tris, pH 7.4, containing 1 mM DTT and immediately frozen and stored overnight in liquid nitrogen. The protein content of the nuclear envelopes was determined by the method of Lowry et al. (27). The yield was 0.16 ± 0.03 mg nuclear envelope protein/g tissue (mean ± SEM; n = 8). Purity was assessed by chemical, enzymatic, and electron micrographic means (24).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:59 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
1089
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- staining of glucocorticoid receptor by the monoclonal antibody PAGE) M7 (29a). M7 is an IgG2a antibody which was raised against the transformed rat liver glucocorticoid receptor. Prestained SDS-PAGE was performed as described by Laemmli (28) in mol wt protein standards were run on gels for blotting. The vertical slab gels which comprised a 4% acrylamide stacking proteins were transferred electrophoretically from the gels onto gel and a 7.5% or 12% acrylamide running gel. Samples were nitrocellulose based on the procedure originally described by run into the gel at 20 mamp/gel and separated at 30 mamp/gel. Towbin et al. (30). The transblot cell was run overnight in a The gels were stained for 1 h in 0.1% Coomassie brilliant blue cold room at 35 V, and then for 1 h at 70 V. The nitrocellulose R-250 and destained sequentially in 40% methanol-10% acetic sheet was removed, briefly air dried, and then blocked with 200 acid solution (vol/vol), followed by 5% methanol-10% acetic ml 1.5% skim milk made up in 20 mM Tris-500 mM NaCl, pH acid solution (vol/vol). 7.5 (Tris-buffered saline), for 2 h at 2 C on a shaking platform. The use of skim milk as a blocking solution has been described Binding assay by Hauri and Bucher (31). The nitrocellulose sheet was then incubated with M7 (1:400 to 1:500) or rabbit antirat serum 3 Binding assays with [ H]dexamethasone were performed at albumin (1:300; the primary antibodies) in Tris-buffered saline 0-4 C in 1.5-ml microfuge tubes in a total volume of 250 ^1 containing 1% BSA for 6-8 h at 20 C on a shaking platform. buffer, as previously described (24). Membrane suspension was The sheets were then washed three times for 15-20 min each added at 25-300 ^g protein/tube. After the addition of 1 ml with buffered saline containing 0.05% Tween-20 and once with excess buffer, the assays were stopped by centrifugation for 1 Tris-buffered saline alone. min in a Microfuge B and aspiration of the supernatant. The The immunodetection procedure was carried out according pellet was then washed twice with vortexing as described above. the instructions in the Bio-Rad immunoblot kit using the to The tip of the tube containing the pellet was cut, and the pellet second antibodies at a 1:1000 dilution and using 4-chloro-lwas incubated overnight at 50 C in 5 ml Ready-Solv containing napthol in the presence of 0.015% hydrogen peroxide to produce 500 (A NCS tissue solubilizier. The radioactivity was deterthe color development. mined by scintillation counting on an LKB 1215 Rackbeta II (Wallach OY, Turku, Finland). The efficiency of counting was Results 30-35%, as determined by use of the external standard. Affinity labeling Purified nuclear envelopes (250 ng protein/tube) were labeled with [3H]dexamethasone mesylate at 0-4 C. Nonspecific binding was determined by addition of the affinity label to samples incubated with unlabeled dexamethasone for 12 h. The assay was terminated as described previously (24). The pellets were dissolved by boiling for 5 min in 150 n\ SDS-sample buffer and run on SDS-PAGE. After staining, the gels were cut into 2mm slices and dissolved in 150 [A 50% hydrogen peroxide-0.8% ammonia solution (19:1, vol/vol) by incubation for several hours at 60-70 C. After the addition of 103 U catalase and 250 lA 10% ascorbic acid (wt/vol) to the cooled tubes, the samples were counted in 10 ml Ready-Solv. Preparation and affinity labeling of cytosol Cytosol was prepared from livers of rats (200-250 g) adrenalectomized 3-7 days before the experiment essentially as described by Eisen et al. (29). The rat liver cytosolic proteins, including glucocorticoid receptor were then ammonium sulfate precipitated at 40% ammonium sulfate and affinity labeled with 50 nM [3H]dexamethasone mesylate in the presence or absence of 1.25 nM unlabeled dexamethasone (250-fold excess) as previously described (29). Immunoblotting of rat liver nuclear envelopes Rat liver nuclear envelopes (300 ng) for immunoblotting were electrophoresed in a 7.5% SDS-gel. Ammonium sulfateprecipitated affinity-labeled glucocorticoid receptor or cytosol (25 to 150 jug protein/lane) was run as a positive control for
Affinity labeling of rat liver nuclear envelope dexamethasone-binding sites Affinity labeling studies were performed at pH 8.6 because it has been reported that the efficiency of labeling of cytosolic glucocorticoid receptor is highest at this pH (32). We first confirmed that dexamethasone mesylate bound to the nuclear envelopes and to the same sites as [3H]dexamethasone by performing a competition study using unlabeled dexamethasone mesylate. The incubation was performed at 0-4 C for 16-18 h, conditions that we had previously shown were optimal for binding of [3H]dexamethasone to the male rat liver nuclear envelope (24). Dexamethasone mesylate competed for the binding of [3H]dexamethasone to rat liver nuclear envelopes, but not as effectively as dexamethasone (Fig. 1). Further, the presence or absence of 1 mM DTT in the incubation buffer did not affect the ability of dexamethasone mesylate to compete for these sites. Thus, the IC50 of dexamethasone mesylate is 3.3 ± 0.3 /xM (n = 2) in the absence of DTT compared to 3.1 ± 0.3 pM (n = 3) in the presence of DTT. The fact that DTT does not interfere with affinity labeling by [3H]dexamethasone mesylate is unexpected, since the a-ketomesylate function of the affinity label would be expected to react with the sulfydryl groups of DTT. Simons et al. (32) observed that in order to terminate the affinity-labeling reaction of [3H]dexamethasone mesylate in HTC cell cytosol, a concentration of 50 mM 0-mercaptoethanol was required. This suggests that the reducing agent is not a preferred
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:59 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
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Endo • 1990 Vol 127 • No 3
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Vol. 127, No. 3 Printed in U.S.A.
Glucocorticoid Receptor Identified on Nuclear Envelopes of Male Rat Livers by Affinity Labeling and Immunochemistry* GILLIAN M. HOWELLf, JAN-AKE GUSTAFSSON, AND YVONNE A. LEFEBVRE Departments of Medicine and Biochemistry, University of Ottawa, Moses and Rose Loeb Research Institute, Ottawa Civic Hospital, Ottawa, Canada Kl Y 4E9; the Bristol Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine (G.M.H.), Houston, Texas 77030; and the Department of Medical Nutrition, Huddinge University Hospital, Karolinska Institute (J.-A.G.), Huddinge, Sweden
ABSTRACT. To exert their action at the genome, steroids must traverse the nuclear envelope, either alone or complexed to their receptor. Our previous studies identified two classes of dexamethasone-binding sites on male rat liver nuclear envelopes: a low capacity, high affinity site and a high capacity, low affinity site. The affinity reagent, [3H]dexamethasone mesylate, labeled peptides at 35-85 kDa, which may be the low affinity glucocorticoid-binding peptides, as these peptides showed the same response to hormonal manipulation as the low affinity [3H]dexamethasone-binding sites previously characterized. With dexamethasone mesylate and a monoclonal antibody against the glucorticoid receptor, we have confirmed that the high affinity binding site on the nuclear envelope is the glucocorticoid receptor. Affinity labeling revealed the presence of a doublet of
I
T IS WELL established that steroids exert their action at the genome of target cells to influence the production of specific mRNAs via interaction of the steroid receptor with specific DNA elements (reviewed in Ref. 1). However, the molecular mechanism by which steroids enter the nucleus to reach the genome is not clear. Early studies based on cell fractionation and autoradiography led to the development of the two-step model of steroid hormone action (2-4). The premise of this model is that free steroid binds to unoccupied receptor, which resides solely in the cytoplasm. Upon binding steroid in the cytoplasm the receptor becomes transformed, i.e. acquires the ability to bind DNA [defined by Grody et al. (5)], and the steroid-receptor complex translocates to the nucleus. However, many investigators have presented Received April 30, 1990. Address all correspondence and requests for reprints to: Dr. Yvonne Lefebvre, Departments of Medicine and Biochemistry, University of Ottawa, Moses and Rose Loeb Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada KlY 4E9. * This work was supported by the Medical Research Council of Canada. t Recipient of a studentship from the Alberta Heritage Foundation for Medical Research.
peptides at 85 and 110 kDa, in the same mol wt range as that reported for the glucocorticoid receptor. Furthermore, these affinity-labeled peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The monoclonal antibody identified a doublet of peptides, a major component of 92-94 kDa and a minor component of 98 kDa. Again, both peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The nuclear envelope-associated glucocorticoid receptor is not extracted by 0.1 M NaCl or 1% Triton X-100. These results show that glucocorticoid hormone interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor, lending support to the two-step model of steroid hormone action. {Endocrinology 127: 1087-1096, 1990)
evidence that tissue fractionation can lead to artefactual redistribution of proteins (6-8). Moreover, steroid receptor transformation is enhanced in vitro in the presence of DNA and so in vivo many represent an intranuclear event (9, 10). More recent studies using cell enucleation and immunohistochemistry provided evidence that the localization of some classes of steroid receptors may be predominantly nuclear (11-15). However, in the case of the glucocorticoid receptor the weight of the evidence continues to support the two-step model (16-22). These studies have involved immunocytochemistry of receptor in target cells (16-21) or transfection of glucocorticoid receptor into nontarget cells, followed by immunocytochemistry (22). We have taken a different approach to the delineation of the mechanism of steroid hormone action. We reasoned that whichever model of steroid hormone action is correct, steroids must traverse the nuclear envelope to reach their site of action within the nucleus. The two models of steroid hormone action predict different modes of nuclear entry for the steroid. A requirement for the two-step model is that steroid enters the nucleus bound to receptor, while the more recently proposed model 1087
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:59 For personal use only. No other uses without permission. . All rights reserved.
1088
GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
requires that steroid enters the nucleus free of receptor. To resolve the manner in which steroids exert their action, it has been our aim to establish the molecular mechanism by which steroids traverse the nuclear envelope. Based on generally accepted principles of membrane transport, steroids may cross the nuclear envelope in one of three ways: 1) by passive diffusion in an unregulated passage, 2) by carrier-mediated transport, in which the steroid itself would bind to a nuclear envelope carrier, or 3) by carrier-mediated transport, in which the steroid would bind first to the steroid receptor followed by binding of the steroid-receptor complex to a nuclear envelope component carrier. There was no need to consider a fourth possibility, i.e. unregulated passage of the glucocorticoid receptor, as it is known that protein molecules the size of steroid receptors enter the nucleus in a controlled fashion (23). As the first step to determine which of these processes was operable in glucocorticoid hormone action, we characterized the binding of [3H] dexamethasone to male rat liver nuclear envelopes and have identified a class of binding sites demonstrating high affinity and low capacity, which was proteinaceous in nature (24). These studies demonstrated that the machinery exists on the nuclear envelope of the rat liver for regulated carrier-mediated entry of glucocorticoid into the nucleus. In this paper we report the identification, by affinity labeling and immunological techniques, of the dexamethasone-binding sites on the nuclear envelope as the glucocorticoid receptor. The association of the glucocorticoid receptor with the nuclear envelope was found to be glucocorticoid responsive, thus suggesting that glucocorticoid interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor. These data are consistent with the two-step model of steroid hormone action for glucocorticoids.
Materials and Methods Reagents [3H]Dexamethasone (38.0-48.9 Ci/mmol), [3H]dexamethasone mesylate (48.9 Ci/mmol), unlabeled crystalline dexamethasone mesylate, and Enhance spray were purchased from New England Nuclear (Montreal, Quebec, Canada). NCS tissue solubilizer was obtained from Amersham Canada Ltd. (Oakville, Ontario, Canada). Ready Solv was purchased from Beckman (Palo Alto, CA). Af-Tris (hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS), deoxyribonuclease-I (DNase-I; RNase free; shipped on dry ice), dithiothreitol (DTT), and BSA were purchased from Sigma Chemical Co. (St. Louis, MO). Acrylamide, iV,AT-methylene bisacrylamide, and Coomassie brilliant blue R250 were obtained from Serva (Heidelberg, West Germany). Prestained protein standards (high range) for immunoblotting were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD). Hydrogen peroxide (ACS grade
Endo • 1990 Vol 127 • No 3
for immunoblotting) was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ). Rabbit antirat serum albumin was obtained from U.S. Biochemical Corp. (Cleveland, OH). All other chemicals for electrophoresis, including Tris(hydroxy methyl)aminomethane, the protease substrate gel tablets (for detection of proteolytic activity), and the Immunoblot horseradish peroxidase-conjugated goat antimouse and goat antirabbit immunoglobulin G (IgG) detection kit were purchased from Bio-Rad (Mississauga, Ontario, Canada). All chemicals used in immunoblotting were of at least ACS grade. Animals Male Sprague-Dawley rats, weighing 200-250 g, were obtained from Charles River Canada, Inc. (Montreal, Quebec, Canada), and maintained on a diet of Wayne Lab Blox (Allied Mills, Chicago, IL) and tap water ad libitum. The rats were killed by decapitation. The livers were quickly removed, placed in ice-cold 0.32 M sucrose containing 3 mM MgCl2 and 1 mM DTT that had been adjusted to pH 7.5, rapidly stripped of connective tissue, and weighed. Rats were adrenalectomized under halothane anesthesia and subsequently received 0.9% NaCl in their drinking water. These rats were used 7 days after surgery. Another group of adrenalectomized rats received long term glucocorticoid replacement commencing 7 days after surgery. These rats were injected with dexamethasone phosphate (SABEX, Montreal, Quebec, Canada) in corn oil at a dose of 4 mg/kg, sc, once daily for 14 days. The last injection was given 30-45 min before death. Another group of rats received short term glucocorticoid replacement. They received two injections of dexamethasone phosphate (each 4 mg/kg, sc), one 16-20 h and the second 30-45 min before death. A control group of rats was anesthetized, sham adrenalectomized, and used 7 days after surgery. Preparation of nuclear envelopes All procedures were carried out as rapidly as possible at 0-4 C. Nuclei were isolated by a modified procedure of Widnell and Tata (25). The nuclear envelopes were prepared from these purified nuclei by a modification of the procedure of Kay et al. (26), as described previously (24). However, in some preparations, particularly for affinity labeling and immunoblotting to verify that proteolytic degradation was not occurring, the protease inhibitors phenylmethylsulfonylfluoride (PMSF; 0.1 mM), leupeptin (10 Mg/ml), soybean trypsin inhibitor (100 ng/m\), and benzamidine (10 mM) were included in the homogenization buffer (0.32 M sucrose containing 3 mM MgCl2 and 1 mM DTT, adjusted to pH 7.5). Purified nuclear envelopes were resuspended to a protein concentration of approximately 4 mg/ml in either 25 mM TAPS, pH 8.6, containing 1 mM DTT or 10 mM Tris, pH 7.4, containing 1 mM DTT and immediately frozen and stored overnight in liquid nitrogen. The protein content of the nuclear envelopes was determined by the method of Lowry et al. (27). The yield was 0.16 ± 0.03 mg nuclear envelope protein/g tissue (mean ± SEM; n = 8). Purity was assessed by chemical, enzymatic, and electron micrographic means (24).
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GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
1089
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- staining of glucocorticoid receptor by the monoclonal antibody PAGE) M7 (29a). M7 is an IgG2a antibody which was raised against the transformed rat liver glucocorticoid receptor. Prestained SDS-PAGE was performed as described by Laemmli (28) in mol wt protein standards were run on gels for blotting. The vertical slab gels which comprised a 4% acrylamide stacking proteins were transferred electrophoretically from the gels onto gel and a 7.5% or 12% acrylamide running gel. Samples were nitrocellulose based on the procedure originally described by run into the gel at 20 mamp/gel and separated at 30 mamp/gel. Towbin et al. (30). The transblot cell was run overnight in a The gels were stained for 1 h in 0.1% Coomassie brilliant blue cold room at 35 V, and then for 1 h at 70 V. The nitrocellulose R-250 and destained sequentially in 40% methanol-10% acetic sheet was removed, briefly air dried, and then blocked with 200 acid solution (vol/vol), followed by 5% methanol-10% acetic ml 1.5% skim milk made up in 20 mM Tris-500 mM NaCl, pH acid solution (vol/vol). 7.5 (Tris-buffered saline), for 2 h at 2 C on a shaking platform. The use of skim milk as a blocking solution has been described Binding assay by Hauri and Bucher (31). The nitrocellulose sheet was then incubated with M7 (1:400 to 1:500) or rabbit antirat serum 3 Binding assays with [ H]dexamethasone were performed at albumin (1:300; the primary antibodies) in Tris-buffered saline 0-4 C in 1.5-ml microfuge tubes in a total volume of 250 ^1 containing 1% BSA for 6-8 h at 20 C on a shaking platform. buffer, as previously described (24). Membrane suspension was The sheets were then washed three times for 15-20 min each added at 25-300 ^g protein/tube. After the addition of 1 ml with buffered saline containing 0.05% Tween-20 and once with excess buffer, the assays were stopped by centrifugation for 1 Tris-buffered saline alone. min in a Microfuge B and aspiration of the supernatant. The The immunodetection procedure was carried out according pellet was then washed twice with vortexing as described above. the instructions in the Bio-Rad immunoblot kit using the to The tip of the tube containing the pellet was cut, and the pellet second antibodies at a 1:1000 dilution and using 4-chloro-lwas incubated overnight at 50 C in 5 ml Ready-Solv containing napthol in the presence of 0.015% hydrogen peroxide to produce 500 (A NCS tissue solubilizier. The radioactivity was deterthe color development. mined by scintillation counting on an LKB 1215 Rackbeta II (Wallach OY, Turku, Finland). The efficiency of counting was Results 30-35%, as determined by use of the external standard. Affinity labeling Purified nuclear envelopes (250 ng protein/tube) were labeled with [3H]dexamethasone mesylate at 0-4 C. Nonspecific binding was determined by addition of the affinity label to samples incubated with unlabeled dexamethasone for 12 h. The assay was terminated as described previously (24). The pellets were dissolved by boiling for 5 min in 150 n\ SDS-sample buffer and run on SDS-PAGE. After staining, the gels were cut into 2mm slices and dissolved in 150 [A 50% hydrogen peroxide-0.8% ammonia solution (19:1, vol/vol) by incubation for several hours at 60-70 C. After the addition of 103 U catalase and 250 lA 10% ascorbic acid (wt/vol) to the cooled tubes, the samples were counted in 10 ml Ready-Solv. Preparation and affinity labeling of cytosol Cytosol was prepared from livers of rats (200-250 g) adrenalectomized 3-7 days before the experiment essentially as described by Eisen et al. (29). The rat liver cytosolic proteins, including glucocorticoid receptor were then ammonium sulfate precipitated at 40% ammonium sulfate and affinity labeled with 50 nM [3H]dexamethasone mesylate in the presence or absence of 1.25 nM unlabeled dexamethasone (250-fold excess) as previously described (29). Immunoblotting of rat liver nuclear envelopes Rat liver nuclear envelopes (300 ng) for immunoblotting were electrophoresed in a 7.5% SDS-gel. Ammonium sulfateprecipitated affinity-labeled glucocorticoid receptor or cytosol (25 to 150 jug protein/lane) was run as a positive control for
Affinity labeling of rat liver nuclear envelope dexamethasone-binding sites Affinity labeling studies were performed at pH 8.6 because it has been reported that the efficiency of labeling of cytosolic glucocorticoid receptor is highest at this pH (32). We first confirmed that dexamethasone mesylate bound to the nuclear envelopes and to the same sites as [3H]dexamethasone by performing a competition study using unlabeled dexamethasone mesylate. The incubation was performed at 0-4 C for 16-18 h, conditions that we had previously shown were optimal for binding of [3H]dexamethasone to the male rat liver nuclear envelope (24). Dexamethasone mesylate competed for the binding of [3H]dexamethasone to rat liver nuclear envelopes, but not as effectively as dexamethasone (Fig. 1). Further, the presence or absence of 1 mM DTT in the incubation buffer did not affect the ability of dexamethasone mesylate to compete for these sites. Thus, the IC50 of dexamethasone mesylate is 3.3 ± 0.3 /xM (n = 2) in the absence of DTT compared to 3.1 ± 0.3 pM (n = 3) in the presence of DTT. The fact that DTT does not interfere with affinity labeling by [3H]dexamethasone mesylate is unexpected, since the a-ketomesylate function of the affinity label would be expected to react with the sulfydryl groups of DTT. Simons et al. (32) observed that in order to terminate the affinity-labeling reaction of [3H]dexamethasone mesylate in HTC cell cytosol, a concentration of 50 mM 0-mercaptoethanol was required. This suggests that the reducing agent is not a preferred
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GLUCOCORTICOID RECEPTOR ON NUCLEAR ENVELOPES
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