We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A-1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5'UTR. Computer analysis of the entire1026 bp comprising the 5'UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno-captured with BUGR-2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon-2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF-2 and uORF-5 protein products were used to perform Western blot analysis on cytosolic proteins from S-49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of approximately 8.5 kDa recognized only with Ab to the uORF-2 peptide; this size is consistent with the computer-predicted size of the uORF-2 product, suggesting that the uORF-2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF-5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF-2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF-2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF-2. Immuno-captured uORF-2 peptide levels correlated with the concentrations of several salt-wash-sensitive cellular proteins, suggesting that protein-protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF-2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno-capture between these two proteins. In summary, our results show that cells can synthesize the uORF-2 peptide, blocking of the synthesis of the uORF-2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF-2 interacts with other cellular factors which might be involved in translation of GR.
Copyright 2001 Wiley-Liss, Inc.