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

The alpha isoform of the glucocorticoid receptor (GR alpha) binds glucocort icoids and functions as a ligand-dependent transcription factor. Although G R alpha is expressed in almost all tissues and cells, its subcellular distr ibution is controversial. Many studies have reported that GR alpha transloc ates from the cytoplasm to the nucleus in a hormone-dependent manner wherea s others have concluded that GR alpha is constitutively located in the nucl eus. These conflicting data may result from the use of antibodies that do n ot discriminate GR alpha from a splice variant of the GR gene termed GR bet a. Using a CR beta-specific antibody, we have recently demonstrated that CR beta resides in the nucleus of cells independent of glucocorticoid treatme nt. In the following study we have generated a novel GR alpha-specific anti body (AShGR) in order to assess, unambiguously, the subcellular distributio n of GR alpha. AShGR recognizes recombinant GR alpha on Western blots and i n immunoprecipitation experiments but does not cross-react with recombinant GR beta. Endogenous GR alpha is detected by AShGR in a variety of human ce ll lines including HeLa S-3, CEM-C7, HEK-293,MCF-7, Hep G2, and secondary l ung epithelial cells. In addition, AShGR detects endogenous rat and mouse G R alpha. Immunocytochemistry was performed with AShGR on COS-1 cells transf ected with human GR alpha and on HTC rat hepatoma cells expressing endogeno us 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 al pha. Published by Elsevier Science Inc.