DEMONSTRATION OF NUCLEAR TRANSLOCATION OF THE MINERALOCORTICOID RECEPTOR (MR) USING AN ANTI-MR ANTIBODY AND CONFOCAL LASER-SCANNING MICROSCOPY

Using a synthetic peptide that corresponds to a unique region of the N -terminal domain of the human mineralocorticoid receptor (MR), amino a cids 87-96, we have generated a polyclonal antibody, human (h) MRsN. T his sequence shares no homology with the corresponding sequences of th e glucocorticoid receptor or other steroid/thyroid hormone receptor su perfamily members. Antibody hMRsN cross-reacts with MR from human, rat , and mouse cells and recognizes denatured MR from either crude prepar ations or partially purified rat kidney cytosol, rat colon, or recombi nant hMR overexpressed in baculovirus-infected Sf9 cells. Immunoprecip itation of the native MR from either partially purified or crude prepa rations of rat kidney cytosol with hMRsN, followed by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis and silver stain, demonstrat ed a major protein band with a mol wt of 116 kilodaltons. In addition, using confocal laser scanning microscopy and digital image analysis, the immunocytochemical localization of the recombinant hMR overexpress ed in Sf9 cells 24 h posttransfection was determined. In the absence o f ligand, the MR was detected solely in the cytoplasm, after a 30-min exposure to 100 nm aldosterone the MR was perinuclear, and after 60 mi n, the MR was predominantly nuclear. To ascertain that this phenomenon was not unique to insect cells, aldosterone induced MR nuclear transl ocation in mouse macrophage cells was also demonstrated immunocytochem ically, clearly indicating a role for nuclear translocation in MR func tion.