Immunocytochemical analysis of the glucocorticoid receptor alpha isoform (GRα) using a GRα-specific antibody

The alpha isoform of the glucocorticoid receptor (GR alpha) binds glucocorticoids and functions as a ligand-dependent transcription factor. Although GR alpha is expressed in almost all tissues and cells, its subcellular distribution is controversial. Many studies have reported that GR alpha translocates from the cytoplasm to the nucleus in a hormone-dependent manner whereas others have concluded that GR alpha is constitutively located in the nucleus. These conflicting data may result from the use of antibodies that do not discriminate GR alpha from a splice variant of the GR gene termed GR beta. Using a CR beta-specific antibody, we have recently demonstrated that CR beta resides in the nucleus of cells independent of glucocorticoid treatment. In the following study we have generated a novel GR alpha-specific antibody (AShGR) in order to assess, unambiguously, the subcellular distribution of GR alpha. AShGR recognizes recombinant GR alpha on Western blots and in immunoprecipitation experiments but does not cross-react with recombinant GR beta. Endogenous GR alpha is detected by AShGR in a variety of human cell lines including HeLa S-3, CEM-C7, HEK-293,MCF-7, Hep G2, and secondary lung epithelial cells. In addition, AShGR detects endogenous rat and mouse GR alpha. Immunocytochemistry was performed with AShGR on COS-1 cells transfected with human GR alpha and on HTC rat hepatoma cells expressing endogenous GR alpha. In both systems, GR alpha was found in the cytoplasm of cells in the absence of hormone and in the nucleus after hormone treatment. These studies mark the first time a GR alpha-specific antibody has been employed to examine the expression and subcellular distribution of endogenous GR alpha. Published by Elsevier Science Inc.