
<ArticleTitle>Molecular identification and characterization of a and b forms of the glucocorticoid receptor.</ArticleTitle>
<AbstractText>The human glucocorticoid receptor (<prot>hGRalpha</prot>) 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 <prot>hGRalpha</prot> 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 <prot>hGRbeta</prot> isoform. We report here that when a single <prot>GR</prot> 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 <prot>hGRalpha</prot> are produced by alternative translation of the same gene and are henceforth termed <prot>GR-A</prot> and <prot>GR-B</prot>. 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, <prot>GR-A</prot>. Moreover, mutagenesis of the initial ATG codon to ACG (Met 1 to Thr) also resulted in production of single, shorter protein species (91 kDa), <prot>GR-B</prot>. Mutagenesis of the Kozak translation initiation sequence strongly indicates that a leaky ribosomal scanning mechanism is responsible for generating the <prot>GR-A</prot> 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 <prot>hGR-A</prot> and <prot>hGR-B</prot> under various glucocorticoid-responsive promoters reveal the shorter <prot>hGR-B</prot> to be nearly twice as effective as the longer <prot>hGR-A</prot> species in gene transactivation, but not in transrepression.</AbstractText>
