Anti-glucocorticoid Receptor Antibody Research Articles

The Anti-Glucocorticoid Receptor Antibody Clone 5E4: Raising Awareness of Unspecific Antibody Binding.

Unspecific antibody binding takes a significant toll on researchers in the form of both the economic burden and the disappointed hopes of promising new therapeutic targets. Despite recent initiatives promoting antibody validation, a uniform approach addressing this issue has not yet been developed. Here, we demonstrate that the anti-glucocorticoid receptor (GR) antibody clone 5E4 predominantly targets two different proteins of approximately the same size, namely AMP deaminase 2 (AMPD2) and transcription intermediary factor 1-beta (TRIM28). This paper is intended to generate awareness of unspecific binding of well-established reagents and advocate the use of more rigorous verification methods to improve antibody quality in the future.

Expression of the glucocorticoid receptor in early and late passage C-6 glioma cells and in normal astrocytes derived from aged mouse cerebral hemispheres

The presence of the glucocorticoid receptor in early and late passage C-6 glioma cells 2B clone and in astrocytes derived from aged mouse cerebral hemispheres has been documented by immunoblotting and/or immunofluorescence labelling. All cell types studied express the glucocorticoid receptor of molecular weight 97 KDa. In addition, in astrocytes derived from aged mouse cerebral hemispheres a smaller molecular weight polypeptide reacting with anti-glucocorticoid receptor antibody was also demonstrated. No difference in the amount of the 97 KDa glucocorticoid receptor between early and late C-6 2B cells was observed, whereas the astrocytes from aged cerebral hemispheres contained considerably reduced amounts of the glucocorticoid receptor compared to C-6 2B cells. Late passage C-6 2B cells were immunofluorescence labelled with the anti-glucocorticoid antibody, the receptor being almost exclusively present in the cytoplasm, with particular concentration in the perinuclear region. The presence of glucocorticoid receptor of molecular weight 97 KDa in glial cells corroborates and expands the existing data based on radioligand binding and immunocytochemical studies. These cell populations can be exploited as a model system for the study of the effects of glucocorticoids on senescence and brain aging.

Production and flow cytometric application of a monoclonal anti-glucocorticoid receptor antibody

Detection and monitoring the expression and level of intracellular glucocorticoid receptor (GCR) is necessary in many clinical and experimental situations. Binding of radioactive steroids ( 3 H dexamethasone) to the cytosolic fractions of cells has been recently used. However, it is an expensive, time-consuming technique difficult to use in routine diagnostics. In this article we describe a novel, simple method for GCR detection, using a FITC-conjugated anti-GCR monoclonal antibody (mAb) for flow cytometric measurements in permeabilized cells. The monoclonal antibody was raised against a conserved sequence (150–176 amino acids) of the regulatory part of the receptor. Synthetic peptide (called APTEK-26) fragment of the receptor conjugated to different carriers (TG, BSA) was used for immunization and screening of the hybridomas. The a-GCR 8E9, 3C8 and 5E4 clones (IgG1) were further characterized by immunoserological methods for their reactivity against overlapping synthetic peptide fragments of the receptor and by Western blot technique on cytosolic fraction of HEP G2 cells (containing the GCR). Furthermore the mAbs could be used for the FACS based detection of GCR, despite its low number of antigen structure within the cells. Solving the problem of nonspecific binding of the secondary antibodies we used our high affinity IgG1 a-GCR mAbs directly labeled with the fluorescent dye FITC. The fluorescent labeling of the GCRs in HEP G2 cell line and human peripheral blood mononuclear cells (PBMC) were demonstrated by flow cytometric analysis after fixation with 4% paraformaldehyde and permeabilization with saponin. Competition with molar excess of unlabelled antibodies and with the GCR peptide fragment confirmed the specific binding of the 8E9 and 5E4 mAbs to the GCRs. Monitoring the GCR level by flow cytometry would be useful in clinical diagnostics, e.g., in steroid-treated patients and in steroid-resistant states.

Interference between DNA binding activities of AP-1 and GR transcription factors in rat thymocytes undergoing dexamethasone-induced apoptosis.

The early molecular events of glucocorticoid-induced apoptosis have been investigated by studying glucocorticoid receptor levels, as well as binding activities to GRE and AP-1 sequences, using nuclear extracts from dexamethasone (Dex)-treated rat thymocytes. When the time-course of glucocorticoid-receptor complexes in nuclei of thymocytes was evaluated by binding studies using the tritiated ligand, we found that nuclear accumulation of radioactive complexes occurred in the first hour of incubation, and was followed by a progressive decline. This trend was confirmed by immunoblotting of nuclear proteins using a monoclonal anti-glucocorticoid receptor antibody. When the kinetics of binding activity to AP-1 and GRE sequences were studied, using nuclear extracts prepared from Dex-treated thymocytes in gel shift assays, we found peaks at 1 and 2 h after Dex treatment, and a return to basal levels in the following hours. Binding specificity was proved by competition studies using non-radioactive sequences, including mutated AP-1. Unexpectedly, however, protein binding to GRE was better competed for by AP-1 sequence than by GRE itself. Data obtained using the super gel shift assay suggested that AP-1/Jun can be responsible for the high affinity for the GRE sequence. Thus, we report here for the first time that an interference between AP-1 and GR in the binding to DNA consensus sequences-previously described in other biological systems-also occurs during apoptosis induced by glucocorticoids in lymphoid cells.

Immunochemical detection of glucocorticoid receptors within rat cochlear and vestibular tissues

A monoclonal antibody, BuGR2, to liver glucocorticoid receptor sites was tested for its reactivity and specificity in inner ear tissue supernatants by an Enzyme Linked Immuno Sorbant Assay (ELISA) and a Western blotting technique. Results demonstrated that this antibody specifically recognized a protein of 93 kDa in inner ear supernatant fractions, which conformed to the reported molecular weights of the glucocorticoid receptor in other tissues. Antigenic sites were determined to be higher within cochlear supernatant fractions compared to vestibular supernatant fractions by ELISA. This anti-glucocorticoid receptor antibody combined with the quantitative ELISA provides a sensitive means to further investigate the inner ear glucocorticoid receptor system.

Microsomal dexamethasone binding sites identified by affinity labelling

Binding studies with [3H]dexamethasone† identified a class of binding sites on male rat liver microsomes. The binding sites were glucocorticoid-dependent and specific for glucocorticoids and progestins. Scatchard binding parameters, competition studies with triamcinolone acetonide, a synthetic glucocorticoid which competes well for the glucocorticoid receptor, and immunoblotting with an antiglucocorticoid receptor antibody indicated that these sites are distinct from the cytosolic glucocorticoid receptor. Affinity labelling experiments with [3H]dexamethasone 21-mesylate revealed two specifically labelled peptides, one at approx. 66 kDa and a doublet at 45 kDa. The 66 kDa peptide had been previously identified in serum and may be present as a result of serum contamination of the microsomal preparation. The 45 kDa doublet, on the other hand, had been shown to be absent from rat serum. The characteristics of the 45 kDa peptide(s) were identical to those of the dexamethasone binding site identified in the binding studies. [3H]Dexamethasone binding characteristics and affinity labelling of microsomal subfractions, separated by isopycnic centrifugation, showed that the binding sites are located in the endoplasmic reticulum. The identification and role of the 45 kDa peptide doublet remain to be determined.

Glucocorticoid-receptor complexes are associated with small RNA in vitro

Identification of RNA associated with soluble glucocorticoid-receptor complexes of HeLa cells was performed by immunoprecipitation of receptor complexes with a monoclonal antibody raised against rat liver glucocorticoid receptor. Polyacrylamide gel electrophoresis of RNA extracted from immunoprecipitates of cytosolic complexes revealed the presence of eight RNA bands, consisting of 28S, 18S, and small RNAs, including 5.8S, 5S and tRNA. A comparison of RNA species immunoprecipitated by monoclonal anti-glucorcorticoid receptor antibody and IgG purified from normal mouse serum showed that four small RNAs were preferentially recovered after immunoprecipitation with anti-glucocorticoid receptor antibody. When these species were analyzed on sequencing gels, their nucleotide lengths coincided with those of 7-3, 7S, U2, and U1 RNA. Immunoprecipitation of nuclear extracts containing glucocorticoid-receptor-RNA complexes showed that the same set of small RNAs was preferentially immunoprecipitated by anti-glucocorticoid receptor antibody. The four small RNAs we detected represented minor species in whole extracts, and their preferential immunoprecipitation by anti-glucorcorticoid receptor antibody was prevented by removal of glucocorticoid-receptor complexes from HeLa cell extracts. We conclude that 7-3, 7S, U2, and U1 RNA are associated with glucocorticoid-receptor complexes in vitro, and hypothesize that post-transcriptional effects of glucocorticoids may in part be mediated through interaction of receptor complexes with these small RNAs.

Interactions between glucocorticoid receptor-bearing chromatin and antireceptor antibody preparations

Recent evidence indicates that the control of gene expression by steroid hormones is mediated by hormone-receptor complexes bound at specific chromosomal locations. The isolation of these in vivo sites of binding would be useful in an analysis of the mechanism of this control. We have therefore examined the interaction between two different antiglucocorticoid receptor antibody preparations and a defined chromatin fraction containing bound glucoeorticoid receptors in order to test the feasibility of this approach. Both antibody preparations, when attached to sepharose, removed nucleosome-bound and nucleosome free receptor from solution, indicating that chromatin-bound receptor was exposed and available for reaction with antibody. The bulk of the chromatin, not containing receptor, was mainly unaffected during these reactions, showing that the antibodies exhibited significant specificity. To determine whether the nucleosome-bound receptor remained attached to the nucleosome during reaction with antibody, studies using soluble antibody were performed. One of the antibodies caused a shift in the sedimentation rate of the nucleosome-bound receptor from 11 S to 11.5S, suggesting that an intact ternary complex of antibody-receptor-nucleosome had formed. The other antibody produced various-sized aggregates of the free and bound receptors. Surprisingly, we found that one of the antibodies reacted strongly with free and nucleosome-bound estrogen receptors as well as glucocorticoid receptors. These studies suggest that an antibody preparation with appropriate characteristics should permit isolation of chromosomal receptor binding sites.

Activation of the rat kidney mineralocorticoid receptor.

Activation of the rat kidney mineralocorticoid receptor was investigated using diethylaminoethyl (DEAE)-cellulose, DNA-cellulose, and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with [3H]aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of [3H]aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BURG-1. The unactivated aldosterone-mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (195 mM KCl) concentration, and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation, the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose and decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. Our results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form.

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRGl could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.

Mineralcorticoid receptor in rat kidney and hippocampus: characterization and quantitation by isoelectric focusing.

The mineralcorticoid receptor in cytosol from the kidneys and hippocampi of adrenalectomized rats was characterized and quantified by use of isoelectric focusing in slabs of polyacrylamide gel. The mineralcorticoid receptor was labeled with [3H]aldosterone after selective blockade of the glucocorticoid receptor sites with a synthetic pure glucocorticoid RU 26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propionyl)androsta-1,4,6-trien-3-one]. The apparent dissociation constant for aldosterone was 5.1 X 10(-9) M, and the ligand specificity studies revealed a mineralcorticoid pattern. The glucocorticoid and mineralocorticoid receptors were compared by limited trypsin digestion, followed by isoelectric focusing. Completely different fragmentation patterns were obtained for the two receptors in the same cytosol from either kidney or hippocampus. However, comparison of the mineralcorticoid receptor from the kidney with that from the hippocampus gave rise to identical fragmentation patterns. An antiglucocorticoid receptor antibody showed no crossreactivity with the mineralcorticoid receptor. Cytosol from kidney and hippocampus contained 28 +/- 24 and 155 +/- 115 fmol mineralcorticoid receptors/mg cytosol protein, respectively. Significant amounts of RU 26988-resistant [3H]aldosterone-binding sites were also present in cerebrum, cerebellum, spleen, and colon.