Molecular identification and characterization of a and b forms of the glucocorticoid receptor

The human glucocorticoid receptor (hGRalpha) is a ligand-activated transcription factor that mediates the physiological effects of corticosteroid hormones and is essential for life. Originally cloned in 1986, the transcriptionally active hGRalpha was reported to be a single protein species of 777 amino acids (molecular mass = 94 kDa). Biochemical data, obtained using various mammalian tissues and cell lines, however, have consistently revealed an additional, slightly smaller, second hGR protein (molecular mass = 91 kDa) that is not recognized by antibodies specific for the transcriptionally inactive and dominant negative, non-hormone-binding hGRbeta isoform. We report here that when a single GR cDNA is transfected in COS-1 cells, or transcribed and translated in vitro, two forms of the receptor are observed, similar to those seen in cells that contain endogenous GR. These data suggest that two forms of the hGRalpha are produced by alternative translation of the same gene and are henceforth termed GR-A and GR-B. To test this hypothesis, we have investigated the role of an internal ATG codon corresponding to methionine 27 (M27) as a potential alternative translation initiation site for the GR. Mutagenesis of this ATG codon to ACG in human, rat, and mouse GR cDNA results in generation of a single 94-kDa protein species, GR-A. Moreover, mutagenesis of the initial ATG codon to ACG (Met 1 to Thr) also resulted in production of single, shorter protein species (91 kDa), GR-B. Mutagenesis of the Kozak translation initiation sequence strongly indicates that a leaky ribosomal scanning mechanism is responsible for generating the GR-A and -B isoforms. Western blot analysis using peptide-specific antibodies show both the A and B receptor forms are present in human cell lines. Both receptors exhibit similar subcellular localization and nuclear translocation after ligand activation. Functional analyses of hGR-A and hGR-B under various glucocorticoid-responsive promoters reveal the shorter hGR-B to be nearly twice as effective as the longer hGR-A species in gene transactivation, but not in transrepression.