Expression level-dependent contribution of glucocorticoid receptor domainsfor functional interaction with STAT5

The action of the glucocorticoid receptor (GR) on beta -casein gene transcr iption serves as a well-studied example of a case where the action of the G R is dependent on the activity of another transcription factor, STAT5. We h ave investigated the domain-requirement of the GR for this synergistic resp onse in transfection experiments employing GR mutants and CV-1 or COS-7 cel ls, The results were influenced by the expression levels of the GR construc ts. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and t he antiglucocorticoid RU486 exhibited a weak agonistic activity When the N- terminal region of the GR was exchanged with the respective domain of the p rogesterone receptor, STAT5 dependent transactivation was reduced at low an d high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta -casein gene promoter. Furthermore, at high GR expressi on levels RU186 was nearly as efficient as dexamethasone in activating tran scription via the STAT5 dependent beta -casein gene promoter. The results r econcile the controversial issue regarding the DNA binding-independent acti on of the GR together with STAT5 and provide evidence that the mode of acti on of the GR depends not only on the type of the particular promoter at whi ch it acts but also on the concentration of the GR. GR DNA bin;ling functio n appears to be mandatory for beta -casein gene expression in mammary epith elial cells, since the promoter function is completely dependent on the int egrity of GR binding sites in the promoter.