Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.     

Relationship between glucocorticoid receptor steroid-binding capacity and association of the Mr 90,000 heat shock protein with the unliganded receptor

Treatment of rat liver cytosol with hydrogen peroxide (H2O2) or sodium molybdate (MoO4(2-)) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25 degrees C. Dithiothreitol (DTT) prevents the stabilization of receptors by H2O2. Heating (25 degrees C) of immune pellets formed by immunoadsorption of L-cell murine glucocorticoid receptor complexes to protein-A-Sepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heat shock protein (hsp90) from the steroid binding protein. Such thermal-induced dissociation of hsp90 is inhibited by H2O2. Pretreatment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H2O2 to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.     

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.     

Analysis of glucocorticoid receptor activation by high resolution two-dimensional electrophoresis of affinity-labeled receptor

To determine if activation of the glucocorticoid receptor involves covalent charge modification of the steroid-binding protein, unactivated and activated IM-9 cell glucocorticoid receptors were examined by high resolution two-dimensional gel electrophoresis. As previously reported (Smith, A. C., and Harmon, J. M. (1985) Biochemistry 24, 4946-4951), two-dimensional electrophoresis of immunopurified, [3H]dexamethasone mesylate-labeled, steroid-binding protein from unactivated receptors resolves two 92-kDa isoforms (pI congruent to 5.7 and 6.0-6.5). After activation, the apparent pI of neither isoform was altered, indicating that there had been no covalent charge modification of the steroid-binding protein. Thus, the physicochemical changes observed after activation of the steroid receptor cannot be explained by dephosphorylation or other models which involve covalent charge modification of the steroid-binding protein. This conclusion was consistent with the observation that treatment of immunopurified, affinity-labeled receptors with calf intestine alkaline phosphatase did not alter the apparent pI values or distribution of the steroid-binding protein isoforms. However, chromatography of activated steroid-receptor complexes on DNA-cellulose revealed that only the more basic of the two steroid-binding protein isoforms bound to DNA. Therefore, the charge heterogeneity of the steroid-binding protein may be important in regulating the ability of the steroid-binding protein to interact with DNA.     

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

Ketoconazole binds to the human androgen receptor.

Ketoconazole, an imidazole anti-fungal agent, has often produced features of androgen deficiency including decreased libido, gynecomastia, impotence, oligospermia, and decreased testosterone levels, in men being treated for chronic mycotic infections. Based on these potent effects on gonadal function in vivo as well as previous work in vitro demonstrating affinity of ketoconazole for receptor proteins for glucocorticoids and 1,25(OH)2 vitamin D3 and for sex steroid binding globulin (SSBG), the binding of ketoconazole to human androgen receptors (AR) in vitro was also examined. Ketoconazole competition with [3H]methyltrienolone (R1881) for androgen binding sites in dispersed, intact cultured human skin fibroblasts was determined at 22 degrees C. Fifty percent displacement of [3H]R1881 binding to AR was achieved by 6.4 +/- 1.8 (SE) x 10(-5) M ketoconazole. Additional binding studies performed with ketoconazole in the presence of increasing amounts of [3H]R1881 showed that the interaction of ketoconazole with AR was competitive when the data were analyzed by the Scatchard method. It should be noted, however, that the dose of ketoconazole required for 50% occupancy of the androgen receptor is not likely to be achieved in vivo, at least in plasma. Finally, androgen binding studies performed with other imidazoles, such as clotrimazole, miconazole, and fluconozole, revealed that in this class of compounds only ketoconazole appears to interact with the androgen receptor. Ketoconazole appears to be the first example of a non-steroidal compound which binds competitively to both SSBG and multiple steroid hormone receptors, suggesting that the ligand binding sites of these proteins share some features in common.     

Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor

Using L cell glucocorticoid receptors that have been immunopurified by adsorption to protein A Sepharose with a monoclonal antireceptor antibody, we have developed an assay to study the requirements for maintenance of steroid-binding capacity. After rapid purification by immunoadsorption, heteromeric receptor complexes retain the ability to bind glucocorticoid hormone. When the receptor complexes are warmed at 20 degrees C, steroid-binding capacity is lost, and the 90-kDa heat shock protein (hsp90) dissociates from the receptor. The rates of both temperature- and salt-dependent dissociation of hsp90 parallel the rates of loss of hormone-binding activity. Molybdate and hydrogen peroxide stabilize the hsp90-receptor complex against temperature-dependent dissociation. Molybdate, however, is much more effective in stabilizing steroid-binding capacity than peroxide. Receptors that have been inactivated in the absence of molybdate or peroxide cannot be reactivated. Inactivation of steroid-binding capacity occurs in the presence or absence of reducing agent, and inactivation is not accompanied by receptor cleavage or dephosphorylation. Under no conditions does an hsp90-free receptor bind steroid. Receptor bound to hsp90 can be cleaved to the 27-kDa meroreceptor in the presence of molybdate with retention of both hsp90 and steroid-binding activity. These observations lead us to propose that hsp90 is necessary but not sufficient for maintaining a competent high affinity glucocorticoid-binding site. Although the 27-kDa meroreceptor fragment is not itself sufficient for a competent binding site, it is sufficient when it is associated with hsp90.     

Mechanism of androgen-receptor augmentation. Analysis of receptor synthesis and degradation by the density-shift technique

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) contains receptors for, and is stimulated by, androgens. Cells cultured in the absence of androgens maintain a basal level of androgen receptors. Following incubation with various concentrations of the synthetic androgen methyltrienolone (R1881) for 1-6 h, the concentration of these receptors increased from 6.0 to 12.2 fmol/micrograms of DNA, while the equilibrium dissociation constant (Kd) of 0.5 nM for this steroid remained unchanged. The steroid-induced increase in androgen receptor levels was specific for androgens and dependent upon protein synthesis. The mechanism of receptor augmentation was examined by utilization of isotopically dense amino acids to determine rates of receptor appearance and degradation in the presence or absence of [3H]R1881. In the absence of androgens, the half-life of the androgen receptor was 3.1 h, with a rate constant (kD) of 0.22/h. In the presence of 1 nM [3H]R1881, however, the half-life was 6.6 h, with kD = 0.11/h. The rate constant for receptor synthesis (ks) in the absence or presence of [3H]R1881 was calculated to be 1.35 and 2.23 fmol/micrograms of DNA/h, respectively. Thus, androgen-induced androgen-receptor augmentation is explained by an increase both in receptor half-life and in rates of receptor synthesis.     

Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25 degrees C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37 degrees C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.     

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molybdate-stabilized L-cell glucocorticoid receptor isolated by affinity chromatography or with a monoclonal antibody is associated with a 90-92-kDa nonsteroid-binding phosphoprotein

We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein A-Sepharose-bound material resulting from incubating the monoclonal antibody with a mixture of 32P-labeled cytosol and [3H]dexamethasone mesylate-labeled cytosol demonstrates identity of the 98-100-kDa [3H]dexamethasone mesylate-labeled band with the 98-100-kDa 32P-labeled band and clear separation from the nonsteroid-binding 90-92-kDa phosphoprotein. The results of immunoblot experiments demonstrate that the 90-92-kDa protein is structurally distinct from the 98-100-kDa steroid-binding protein. As the 90-92-kDa nonsteroid-binding phosphoprotein co-purified with the 98-100-kDa uncleaved form of the glucocorticoid receptor by two independent methods, one of which is based on recognizing a steroid-binding site and the other of which is based on recognizing an antibody binding site, we propose that the 90-92-kDa phosphoprotein is a component of the molybdate-stabilized, untransformed glucocorticoid-receptor complex in L-cell cytosol.     

Androgen receptor in rat liver: characterization and separation from a male-specific estrogen-binding protein

Many liver processes are sexually dimorphic. In particular, the microsomal content of specific enzymes and the synthesis of specific proteins are under sex steroid hormone control. Because the liver of male rats is strikingly androgen responsive, we sought evidence for an androgen receptor in this tissue. We detected and characterized both cytosolic and nuclear androgen-binding proteins. Both forms bind [3H]R1881 (methyltrienolone, 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one) with the high affinity, low capacity, and specificity for androgens and antiandrogens characteristic of androgen receptors. No high-affinity binding of [3H]DHT could be detected in unfractionated cytosol because of the rapid metabolism of this ligand; however, binding of a DHT metabolite to the high-capacity male-specific estrogen binder (MEB) of cytosol was observed. Both gel filtration and heparin-Sepharose affinity chromatography separate the cytosolic androgen receptor from MEB. Incubation of cytosol in the absence of sodium molybdate resulted in androgen-binding activity which was retained by DNA-cellulose. Castration of male rats results in a time-dependent loss of both cytosolic and nuclear androgen binding, as well as a loss in MEB activity. Androgen-binding activity is low in livers from female rats, but can be induced by testosterone treatment. An intact pituitary is necessary for maintenance of androgen-binding activity, as hypophysectomy results in complete loss of activity.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.     

Selective molybdate-directed covalent modification of sulfhydryl groups in the steroid-binding versus the DNA-binding domain of the glucocorticoid receptor

Hydrogen peroxide produces all of the effects on glucocorticoid receptors that are produced by molybdate, including stabilization of the receptor 90-kDa heat shock protein (hsp90) complex (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). When the glucocorticoid receptor is exposed simultaneously to molybdate and peroxide at concentrations that are optimal for receptor stabilization if each agent is present alone, there is an irreversible loss of steroid binding activity. The effect is accompanied by a covalent modification of the receptor, which is demonstrated by an increase in its apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of the receptor with the sulfhydryl-modifying reagents methyl methanethiosulfonate or N-ethylmaleimide prevents covalent modification, suggesting that cysteine moieties are the site of attack. The covalently modified receptor can still bind to DNA. Molybdate-peroxide treatment does not covalently modify the 15-kDa tryptic fragment containing the DNA-binding domain and 11 of the 20 cysteine moieties in the receptor. However, the 27-kDa tryptic fragment, which contains the steroid-binding domain and 5 cysteines, is covalently modified. The 27-kDa tryptic fragment is covalently modified by the molybdate-peroxide combination when [3H]dexamethasone 21-mesylate is covalently bound to Cys-644. This leaves some combination of 4 cysteines in the steroid-binding domain (628, 649, 671, and 742) as the modified groups. These modifications occur in a region of the receptor that is known to contain its sites of interaction with both hsp90 and molybdate, with the latter having a well-established avidity for sulfur. These observations raise the possibility that the covalent modification caused by the molybdate-peroxide combination represents a modification of sulfur ligands involved in molybdate stabilization of the receptor.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

A new method for labeling and autoradiographic localization of androgen receptors

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.