CHARACTERIZATION OF THE INTERACTION OF THE HUMAN MINERALOCORTICOSTEROID RECEPTOR WITH HORMONE RESPONSE ELEMENTS

Although the mineralocorticosteroid receptor (MR) belongs to the super family of hormone-dependent transcription factors, the molecular mecha nism by which it regulates gene expression is poorly understood. Bindi ng of the MR to target gene promoters has never been characterized, an d specific mineralocorticosteroid response elements (MREs) remain to b e identified. The human MR (hMR) was overexpressed in Sf21 insect cell s using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examin e the DNA-binding properties of the recombinant MR with glucocorticost eroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perf ect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and ident ified the MR in Western blot analysis. In vitro DNAase I protection as says with the hormone-regulated murine mammary tumour virus promoter s howed that recombinant hMR generated four footprints whose limits enco mpassed the GRE motifs. By means of these two complementary approaches , no difference between the interaction of free, agonist- or antagonis t-bound MR and DNA was detected. We provide evidence that hMR function s as a sequence-specific DNA-binding protein.