Imaging analysis of mineralocorticoid receptor and importins in single living cells by using GFP color variants

Mineralocorticoid receptor (MR) is a ligand-dependent transcription factor involved in gene regulation in association with another corticosteroid receptor, glucocorticoid receptor. In the absence of ligand, MR resides both in the cytoplasm and in the nucleus. Agonists increase the number of MRs residing in the nucleus. Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway. To investigate the interactions between MR and importins, we analyzed the subcellular distribution of MR and importins in response to ligand in living COS-1 cells, which do not express endogenous MR, by using fusion proteins labeled with different spectral variants of green fluorescent protein. In the cells coexpressing fluorescent protein-tagged (FP)-MR and FP-importin alpha, the proteins simultaneously moved into the nucleus from the cytoplasm upon activation with ligand treatment. In the cells coexpressing FP-MR and FP-importin beta, FP-MR moved into the nucleus from the cytoplasm, but the distribution of FP-importin beta was little changed upon ligand treatment. Analysis of a mutant of MR, in which nuclear localization signal (NLS) is inactivated, demonstrated that the intact NLS is necessary for the trafficking of MR related to importin alpha. This is the first visual evidence of the nuclear import of MR in association with importin alpha in single living cells.