DEXAMETHASONE NEGATIVELY REGULATES THE ACTIVITY OF A CHIMERIC DIHYDROFOLATE-REDUCTASE GLUCOCORTICOID RECEPTOR PROTEIN

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [H-3]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.