Molecular identification and characterization of A and B forms of the glucocorticoid receptor

The human glucocorticoid receptor (hGR alpha) is a ligand-activated transcr iption factor that mediates the physiological effects of corticosteroid hor mones and is essential for life. Originally cloned in 1986, the transcripti onally active hGR alpha was reported to be a single protein species of 777 amino acids (molecular mass = 94 kDa). Biochemical data, obtained using var ious mammalian tissues and cell lines, however, have consistently revealed an additional, slightly smaller, second hGR protein (molecular mass = 91 kD a) that is not recognized by antibodies specific for the transcriptionally inactive and dominant negative, non-hormone-binding hGR beta isoform. We re port here that when a single GR cDNA is transfected in COS-1 cells, or tran scribed and translated in vitro, two forms of the receptor are observed, si milar to those seen in cells that contain endogenous On. These data suggest that two forms of the hGR alpha are produced by alternative translation of the same gene and are henceforth termed GR-A and GR-B. To test this hypoth esis, we have investigated the role of an internal ATG codon corresponding to methionine 27 (M27) as a potential alternative translation initiation si te for the On. Mutagenesis of this ATG codon to ACG in human, rat, and mous e GR cDNA results in generation of a single 94-kDa protein species. On-A. M oreover, mutagenesis of the initial ATG codon to ACG (Met 1 to Thr) also re sulted in production of single, shorter protein species (91 kDa), GR-B. Mut agenesis of the Kozak translation initiation sequence strongly indicates th at a leaky ribosomal scanning mechanism is responsible for generating the G R-A and -B isoforms. Western blot analysis using peptide-specific antibodie s show both the A and B receptor forms are present in human cell lines. Bot h receptors exhibit similar subcellular localization and nuclear translocat ion after ligand activation. Functional analyses of hGR-A and hGR-B under v arious glucocorticoid- responsive promoters reveal the shorter hGR-B to be nearly twice as effective as the longer hGR-A species in gene transactivati on, but not in transrepression.