Using the combination of a cDNA library prepared from membrane glucocorticoid (mGR)-enriched S-49 cells and a mouse leukocyte genomic library, we have cloned a 7.3 kb full-length glucocorticoid receptor 1A cDNA. Primer extension, 5'RACE, and long distance PCR identified the transcription start site as being located at 1026 bp from the ATG codon. The first 1,013 nucleotides (nts) of the full length sequence constitute 5' UTR sequence (exon 1), the next 2349 bp, the coding region, and the last 3,907 bp, the 3'UTR. The entire 5'UTR sequence is unique to transcript 1A. The 3'UTR sequence is approximately 88.5 % conserved with the rat 3'UTR. Western blot analysis compared the molecular weight of in vitro translation products from the cloned 1A cDNA with partially purified cellular mGR. Both preparations contained the novel 150 KD and the 94 KD classical GR peptides, suggesting that transcript 1A encodes both receptor forms. Transfection of mGR-less and glucocorticoid lysis-resistant AtT-20 and HL-60 cells with full-length GR 1A cDNA imparted both mGR expression and glucocorticoid lysis-sensitivity to these cells.
Copyright 1999 Wiley-Liss, Inc.