Affinity Labeling of Glucocorticoid Receptors: New Methods in Affinity Labeling

Simons, S. Stoney; Thompson, E. Brad

doi:10.1016/b978-0-12-452809-3.50013-0

Cited by 11 publications

(7 citation statements)

References 90 publications

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“…One technique which has the potential to overcome this limitation is electrophilic affinity labeling, whereby an irreversible ligandreceptor complex is formed. This technique makes use of a modified steroid suitably equipped with an electrophilic functional group which still retains both high affinity and specificity for its macromolecular (receptor) binding site (reviewed by Katzenellenbogen, 1977;Simons and Thompson, 1982). The formation of the irreversible receptor-steroid complex requires two steps, i.e., the routine noncovalent binding of the steroid to its receptor followed by the formation of a cova-…”

Section: Validity Of 3h-dm As An Affinity Label For the Localization mentioning

confidence: 99%

“…With the introduction of a covalent affinity label for the GR, dexamethasone 21-mesylate (DM), the biochemical identification of the GR has been facilitated owing to the formation of an irreversible steroid-receptor complex from which the radiolabeled steroid cannot dissociate (Simons and Thompson, 1982). However, the application of affinity labeling for histological localizations of steroid receptors has not been reported.…”

Section: Introductionmentioning

confidence: 98%

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Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

1989

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The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Validity Of 3h-dm As An Affinity Label For the Localization mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

1989

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“…Another technique that potentially can overcome the problem of receptor-steroid dissociation is that of covalent affinity labeling, whereby an irreversible ligandreceptor complex is formed. This technique makes use of a modified ligand (steroid), suitably equipped with an electrophilic functional group, which still retains both high affinity and specificity for its macromolecular binding site (receptor) (reviewed by Katzenellobogen, 1977;Simons and Thompson, 1982). Dexamethasone 21-mesylate (DM) is the most widely accepted GR affinity label in whole-cell and cell-free systems (Simons et al, 1980(Simons et al, ,1983Simons and Thompson, 1981).…”

Section: Introductionmentioning

confidence: 99%

Subcellular distribution of [³H]‐dexamethasone mesylate binding sites in leydig cells using electron microscope radioautography

1991

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The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively. Cytosol (exclusively of all organelles) and nucleus showed comparatively weak labeling, at 9% and 7%, respectively. This study thus clearly establishes with the electron microscope, the localization of glucocorticoid binding sites using DM. It remains to be determined whether or not these DM binding sites represent bona fide glucocorticoid receptors or nonreceptor proteins that bind DM. Whereas the functional significance of the subcellular distribution of DM is not known, the labeling of the cytosol may represent localization of the steroid and GR in their traditional compartment. The steroid antagonistic properties of DM may have prevented the DM-GR complexes from translocating to the nucleus. However, the significant labeling of the sER-rich cytosol and mitochondria was unexpected and raises intriguing questions that are being addressed in current studies.

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“…Dexamethasone 21-mesylate forms covalent linkages with glucocorticoid receptors in both cell extracts and whole cells (6). This steroid derivative also acts as a long-term irreversible anti-glucocorticoid in HTC cells (1)(2)(3). The covalently bound steroid-receptor complexes are activated to DNA binding forms (6)(7)(8) and then bind to the same long-terminal-repeat regions of mouse mammary tumor virus DNA (9) as the noncovalent complexes.…”

mentioning

confidence: 99%

“…Dexamethasone-21-methanesulfonate (dexamethasone 21-mesylate) is an effective affinity label for glucocorticoidbinding proteins (1)(2)(3). The methanesulfonate substituent at C-21 of the steroid derivative is a reactive electrophile that reacts preferentially with thiols, more slowly with amino and carboxylate moieties, but not with hydroxyl and imidazole groups (4).…”

mentioning

confidence: 99%

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

Homma¹,

Listowsky²

1985

Proc. Natl. Acad. Sci. U.S.A.

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(6). This steroid derivative also acts as a long-term irreversible anti-glucocorticoid in HTC cells (1-3). The covalently bound steroid-receptor complexes are activated to DNA binding forms (6-8) and then bind to the same long-terminal-repeat regions of mouse mammary tumor virus DNA (9) as the noncovalent complexes.Glutathione S-transferases (glutathione transferase, EC 2.5.1.18) are a family of enzymes that catalyze reactions of glutathione (GSH) with compounds that contain electrophilic centers. Through this type of reaction, the enzyme can detoxify toxic compounds (10-15). Because these enzymes have the capacity to bind a large number of lipophilic substances including endogenous metabolites (e.g., bilirubin, heme, and bile acids), carcinogens, and various other xenobiotics (16-24) an intracellular transport function has also been ascribed to the glutathione-S-transferases. Multiple forms of the rat enzyme arise from dimeric combinations of subunit types designated as Ya, Yb, and Yc (18,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).Reports that the Yb form of the protein selectively binds certain steroids (36) prompted this study to explore possible reactions of the protein with steroid affinity probes and to determine whether the protein retains steroid-binding capacity in the presence of other cytosolic proteins, notably glucocorticoid receptors. Evidence is presented that the Yb subgroup of rat liver glutathione-S-transferase is the major cytosolic protein covalently labeled with dexamethasone 21-mesylate. MATERIALS AND METHODS[6,7-3H(N)]Dexamethasone 21-mesylate in methanol with a specific activity of 48.9 Ci/mmol (1 Ci = 37 GBq) and dexamethasone 21-mesylate powder (unlabeled) were obtained from New England Nuclear. Sephadex G-100, DEAESephacel, protein-A Sepharose CL-6B, and CNBr-activated Sepharose-4B were obtained from Pharmacia. GSH, Smethyl-GSH and dexamethasone were obtained from Sigma and 1-chloro-2,4-dinitrobenzene from Aldrich. Acrylamide and other reagents used for gel electrophoresis were obtained from Bio-Rad.Protein Preparations. Glutathione-S-transferases were prepared as described (35,36). In brief, cytosol prepared from livers of male Sprague-Dawley rats (175-200 g) (105,000 x g supernatants) in 10 mM Tris HCl/1.0 mM EDTA, pH 8.0 (Tris/EDTA buffer) was fractionated by chromatography on Sephadex G-100. The fractions containing glutathione-Stransferase activity were pooled, applied to a lysyl-GSH affinity column (35), and eluted with 10 mM S-methyl glutathione. The multiple forms of the enzyme were resolved by chromatography using 36). Homogeneous preparations of Yb forms with and without bound GSH were separated as described (37).The IgG fraction of rabbit anti-rat Yb-glutathione-S-transferase antisera (36) was purified by a protein A-Sepharose CL-6B column, and it was coupled to CNBr-activated Sepharose 4B to prepare the immunoadsorbant.Enzymatic activities were measured using 1.0 mM 1-chloro-2,4-dinitrobenzene and 1.0 mM GSH as substrates. Absorbance at 343 nm was measured at 25°C in...

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Affinity Labeling of Glucocorticoid Receptors: New Methods in Affinity Labeling

Cited by 11 publications

References 90 publications

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

Subcellular distribution of [³H]‐dexamethasone mesylate binding sites in leydig cells using electron microscope radioautography

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

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Affinity Labeling of Glucocorticoid Receptors: New Methods in Affinity Labeling

Cited by 11 publications

References 90 publications

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

Subcellular distribution of [3H]‐dexamethasone mesylate binding sites in leydig cells using electron microscope radioautography

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

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Subcellular distribution of [³H]‐dexamethasone mesylate binding sites in leydig cells using electron microscope radioautography