title: Regulation of glucocorticoid receptor expression: I. Use of a specific radioimmunoassay and antiserum to a synthetic peptide of the N-terminal domain.
abstract: In order to study glucocorticoid receptor (GR) gene expression at the protein level, we have produced an anti-serum to the GR using a 14 amino acid peptide (14-mer) of amino terminus domain of the human GR, and established a simple and specific RIA to quantitate both the human and rat GR. The antibody was raised in rabbits to the 14-mer coupled to either BSA or keyhole limpet hemocyanin. This antibody immunoblots the Mr = 94,000 bona fide GR in tissue extracts and localizes the GR at the subcellular level by immunocytochemistry. In addition, cytosolic GR, previously labeled by the affinity ligand, [3H]dexamethasone mesylate, was immunoprecipitated by the peptide antibody. The 14-mer was iodinated at its tyrosine residue and used in a standard RIA. The binding of the antibody to the 125I-14-mer was displaced by increasing concentrations of either the 14-mer (standard curve) pure GR or tissue cytosol containing native GR. This RIA reliably detects glucocorticoid receptor level between 20 and 500 fmol/tube in human, rat, and mouse tissues. In two well established cell line systems and their subclones (human CEM and in rat hepatoma tissue culture cells transfected or not with GR cDNA) the GR level, as assessed by this RIA, was compared to GR values using the classical radioreceptor or previously published mRNA assays. The relative amount of GR in wild-type cells and in subclones, as assessed by the novel RIA, was identical to the above-mentioned assays. Using the RIA, we demonstrated the down-regulation of GR level in liver following glucocorticoid administration and its up-regulation following adrenalectomy. This study, which constitutes the first description of an RIA for a steroid receptor using a synthetic peptide, provides a powerful tool for a standardized, sensitive, and simple assay for the GR in human and animal tissues.
authors.affiliation: Department of Anatomy, McGill University, Montreal, Quebec, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Antakly
authors.forename: D
authors.initials: D
authors.lastname: Raquidan
authors.forename: D
authors.initials: D
authors.lastname: O'Donnell
authors.forename: L
authors.initials: L
authors.lastname: Katnick
mesh_terms: D000315:Adrenalectomy; D000345:Affinity Labels; D000818:Animals; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology*; D011863:Radioimmunoassay / Q000379:methods*; D011869:Radioligand Assay; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000276:immunology / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D005938:Glucocorticoids; D007106:Immune Sera; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid