Intragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms.

Glucocorticoid receptors (GR) are ligand-dependent transcription factors that play a critical role in the endocrine control of cell growth, differentiation, and death. These steroid receptors are widely recognized to undergo down-regulation after exposure to ligand in cell cultures and animals, including humans. This reduction in cellular receptor levels leads to insensitivity to subsequent hormone administration. The mechanisms controlling homologous down-regulation of the GR are, however, poorly understood. We have previously shown (1) that a transfected human GR (hGR) complementary DNA (cDNA) contains sequences that are sufficient to recapitulate the down-regulation of both hGR messenger RNA (mRNA) and protein seen in vivo. We have now evaluated potential mechanisms involved in the hormonal regulation of the hGR mRNA and, further, have identified an intragenic domain of the hGR cDNA that contains the down-regulatory signal. Glucocorticoid treatment of COS-1 cells expressing a transfected hGR cDNA resulted in down-regulation of the hGR mRNA in the presence of cycloheximide or actinomycin-D, suggesting that a glucocorticoid-inducible protein was not essential for down-regulation. We show that prolonged receptor occupation by ligand leads to increased GR mRNA turnover, and furthermore, that either the agonist dexamethasone or the antagonist RU486 decreased transcription of the hGR cDNA. To resolve which receptor cDNA sequences are critical in down-regulation, a cotransfection strategy was employed in which a series of hGR cDNA deletion mutants was transfected in conjunction with the full-length hGR cDNA. The effects of glucocorticoid on the regulation of receptor mRNAs encoded by the mutant receptor cDNAs were examined. Deletions within the 5' half of the receptor cDNA produced transcripts that were susceptible to glucocorticoid-mediated down-regulation, whereas deletion of sequences located in the 3'-end of the receptor-coding sequence (corresponding to amino acids 550-697) resulted in receptor transcripts that were only minimally down-regulated by glucocorticoid. Together these studies indicate that multiple mechanisms control GR mRNA abundance, and an intragenic element within the ligand-binding domain is critical for this down-regulation.