The mineralocorticoid receptor (MR) that mediates signals generated by aldosterone is an important receptor involved in the stress mechanism.Because of the lability of the steroid-receptor complex in tissue extracts, its properties and functions have not been well characterized.Therefore, it is proposed to overexpress the human MR in insect cells using the baculovirus system. This will facilitate the generation of milligram quantities of receptor that can be purified from Spodoptera frugiperda (Sf9) cells. In addition,the first 350 amino acid truncated MR together with the DNA binding domain but lacking the ligand binding domain will be overexpressed as will a full length GR-MR chimeric receptor.These receptors will be purified from Sf9 cells for experiments bearing on activation and transcriptional functions. The oligomeric form of the MR will be reconstituted from overexpressed and purified MR, HSP70 and other purified cytosolic factors (p59, modulator) to study the mechanism of assembly and to define the cytoplasmic activation mechanism in vitro. The specific DNA binding of the MR and its derivatives will be analyzed as will the activation of cell-free transcription. This will entail specifically phosphorylated MR and its different forms that have been overexpressed and purified from Sf9 cells.The MR responsive element will be mapped on the 5' flanking regions of the Na,K-ATPase alpha and beta subunits' DNA by footprinting. Specific transcription factors that interact with the N-terminal domain will be isolated after their binding to the overexpressed N-terminus by chemical (and reversible) cross- linking. A specific anti-MR antibody that will not cross-react with the glucocorticoid receptor will be generated from a predicted peptide sequence in the N-terminus. This antibody will be used in the DNA binding experiments and as a means to purify the MR. This information will impact on the understanding of the stress adaptation mechanism at the molecular level and will apply to human diseases of hyperaldosteronism and hypertension.
