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A rabbit immunized with a highly purified preparation of rat liver [3H]triamcinolone-receptor complex developed antibodies to the receptor. Although precipitating reactions were not detected, complexes formed between IgG and the receptor could be detected by Staphylococcus aureus protein A-Sepharose and gel permeation chromatography. IgG was purified and covalently immobilized on Sepharose CL-4B; this affinity matrix adsorbed the ligand-free receptor and both activated and nonactivated forms of the [3H]triamcinolone-receptor complex. Rat liver cytosol proteins adsorbed by control and immune immunoglobulin-Sepharoses were eluted with 0.1 M acetic acid and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with molecular weight 78,000 was the major species eluted from immune immunglobulin-Sepharose, and it was not present in eluates from control columns. Rat transcortin, glucocorticoid binder IB, and an estrogen-binding protein from rat liver were not adsorbed by immune IgG-Sepharose. Mouse and hamster liver glucocorticoid receptors showed only limited adsorption. Thus, the antiserum does not crossreact with other major glucocorticoid-binding proteins and demonstrates species specificity.

Antiserum prepared against highly purified glucocorticoid receptor was used for immunocytochemical studies. Rat liver and pituitary were chemically fixed, and histological sections were examined for immunoreactivity by an indirect immunoperoxidase procedure. In liver, immunoperoxidase staining was observed in the nuclei and cytoplasm of most hepatocytes. Kupffer, endothelial, and bile duct cells were not significantly stained. Control sections treated with preimmune serum remained unstained; preadsorption of antisera with purified liver glucocorticoid receptor resulted in a significant decrease in immunocytochemical staining. In adrenalectomized rats, nuclear staining was markedly reduced, whereas in adrenalectomized rats treated with cortisol acetate, the density of nuclear staining was comparable to and often slightly higher than that in intact animals. In the anterior pituitary, numerous cells were immunoreactive; their nuclei, cytoplasm, or both were stained. Alternate histological sections to those stained with antireceptor antibodies were processed for the localization of ACTH. It was found that the number of anterior pituitary cells that stained with the antireceptor antibodies exceeded the number of corticotrophs. Cells of the intermediate lobe pituitary were devoid of staining, whereas cells in the posterior lobe were stained. The presence of immunoreactive glucocorticoid receptors in pituitary cytosolic fractions was biochemically confirmed by immunoadsorption studies with [3H]triamcinolone acetonide-receptor complexes. This morphological localization of glucocorticoid receptor in liver and pituitary tissues demonstrated that immunocytochemistry can be successfully used to study localization of the glucocorticoid receptor. The known lack of response of intermediate pituitary cells to glucocorticoid may be secondary to a very small number or even an absence of glucocorticoid receptors.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.

Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [3H]dexamethasone 21-mesylate [( 3H]DM) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7. Antisera were obtained following immunization of New Zealand White rabbits with [3H]triamcinolone acetonide [( 3H]TA)-glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography. The presence of anti-human glucocorticoid receptor antibodies was verified by: (a) adsorption of [3H]TA-receptor-antibody complexes to Protein A; (b) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [3H]TA-receptor complexes incubated with immune serum; and (c) the ability of immune serum to displace [3H]TA-receptor complexes on sucrose gradients. These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of approximately 90,000, three minor competable components with molecular weights of approximately 78,000, approximately 51,000, and approximately 38,500, and at least 21 other noncompetable components. Following immunoprecipitation of [3H]DM-labeled cytosol with immune serum, only the Mr 90,000 and 78,000 components were seen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]DM-labeled CEM-C7 cytosol revealed a larger number of [3H]DM-labeled components. However, after immunoprecipitation of [3H]DM-labeled CEM-C7 cytosol, a predominant competable component with a molecular weight of 90,000 was easily identified. This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used. Thus, the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors.

Lymphoid cells contain specific receptors for glucocorticoids. We have used [3H]dexamethasone-21-mesylate to label covalently glucocorticoid receptors in rat thymic lymphocytes and in neoplastic cells obtained from patients with acute lymphoblastic leukemia and malignant lymphoma. The covalently labeled glucocorticoid receptors were identified by polyacrylamide gel electrophoresis (in the presence of 0.1% sodium dodecyl sulfate). In cytosolic fractions prepared from rat thymic lymphocytes, [3H]-dexamethasone-21-mesylate labels a protein (Mr approximately equal to 95,000) which was identified as the glucocorticoid receptor by the following criteria: (a) labeling of this moiety is inhibited by treatment with a 100-fold molar excess of glucocorticoids, such as dexamethasone and triamcinolone acetonide; and (b) the covalently labeled Mr approximately equal to 95,000 protein is activated (by heating at 20 degrees for 30 min) to a form that binds to DNA-cellulose. When intact thymocytes are treated with [3H]dexamethasone-21-mesylate, an Mr approximately equal to 95,000 moiety is also labeled covalently. Approximately 35% of the glucocorticoid receptors can be labeled covalently when intact thymocytes are treated with 100 nM [3H]dexamethasone-21-mesylate for 30 min at 4 degrees. Neoplastic cells from acute lymphoblastic leukemia and malignant lymphoma were treated with [3H]dexamethasone-21-mesylate. In all samples, an Mr approximately equal to 95,000 moiety was labeled covalently; labeling was inhibited by excess glucocorticoid. Smaller moieties were also identified by competition experiments; these may represent proteolytic fragments of the Mr approximately equal to 95,000 receptor. Thus, in rat and human lymphoid cells, [3H]dexamethasone-21-mesylate can be used to label covalently the glucocorticoid receptor.

[3H]Dexamethasone 21-mesylate affinity-labeled glucocorticoid receptors were subjected to controlled proteolysis by trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on denaturing constant percentage or gradient polyacrylamide gels. The molecular weights (Mr congruent to 98 000) and cleavage patterns for rat liver and HTC cell receptors indicated extensive homology between the glucocorticoid receptors from normal rat liver and a transformed rat liver cell line. The major DNA-binding species generated by chymotrypsin treatment was found to be a 42K fragment that was accompanied by several unresolved, slightly lower molecular weight fragments. The meroreceptors obtained after trypsinization were comprised of two species of Mr 30 000 and 28 000. Each of the three proteases, despite their differing specificities, generated fragments with molecular weights close to 42 500, 30 500, and 27 000. Nevertheless, each of the three proteases gave rise to a distinctive "ladder" of labeled fragments. No differences could be detected in the digestion patterns of unactivated and activated HTC cell complexes for all three proteases. Also, native and denatured receptor-steroid complexes yielded surprisingly similar digestion patterns with each enzyme. Digestion of denatured complexes readily generated large amounts of a fragment of Mr congruent to 15 000 that was much smaller than the protease-resistant meroreceptors formed from native complexes. The presence of these approximately 15K fragments suggested that the [3H]dexamethasone 21-mesylate labeling of the steroid-binding cavity is restricted to a relatively small segment of the receptor.

We have explored the possibility of using antibodies against purified rat liver glucocorticoid receptors to study the immunochemical properties of glucocorticoid receptors from murine and human malignant lymphocytes. For this purpose, purified immune immunoglobulin G was covalently linked to Sepharose CL-4B. We then examined the ability of the affinity gel to recognize cytosolic [3H]triamcinolone acetonide-receptor complexes from the corticoid-sensitive (CS) and -resistant strains of mouse lymphoma P1798, from CS lymphocytes of patients with chronic lymphatic leukemia, and from a CS clone of human leukemic lymphoblasts in tissue culture (CH6). Mouse thymus was used as a source of glucocorticoid receptor from normal CS lymphocytes. Whereas the immunoaffinity column retained 70 to 84% of the 58- to 62-A (Stokes radius) [3H]triamcinolone acetonide-receptor complexes characteristic of the CS mouse and human lymphocytes, it failed to recognize the 27- to 28-A (Stokes radius) glucocorticoid receptor present in corticoid-resistant mouse lymphoma P1798 cells. Therefore, under appropriate experimental conditions, it was possible to demonstrate cross-reactivity between the antiserum against rat liver glucocorticoid receptor and the 58- to 62-A (Stokes radius) glucocorticoid receptor from species as diverse as mouse and humans.