Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer.

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10(-6) m CORT than at 10(-9) m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

Real-time imaging of green fluorescent protein-tagged beta 2-adrenergic receptor distribution in living cells.

In an attempt to investigate the subcellular trafficking of beta(2)-adrenergic receptor (beta(2)AR) in living cells, we performed real-time imaging of beta(2)AR tagged with green fluorescent protein (GFP). We transiently transfected a chimera construct of beta(2)AR and GFP (beta(2)AR-GFP) into HEK 293 cells, primary cultured rat hippocampal neurons and cortical neuronal cells, and then compared the dynamic changes in subcellular localization of beta(2)AR-GFP in these live cells. In the absence of ligands, beta(2)AR-GFP fluorescence was detected predominantly on the plasma membrane in HEK 293 cells as well as on the surface of cell somata and dendrites in cortical neuronal cells. In contrast, in hippocampal neurons, beta(2)AR-GFP was diffusely distributed not only on the surface of cells but in the whole cell somata and dendrites. In HEK 293 cells, cortical neuronal cells and cortical glial cells, time-lapse images showed the rapid appearance of a punctate distribution pattern that became more numerous over the 15-min course of agonist exposure. Semiquantitative analysis revealed the time-course internalization of beta(2)AR-GFP in a single living cell. In hippocampal neurons, beta(2)AR-GFP distribution became scattered both in cell somata and dendrites following agonist exposure. Three-dimensional analysis of time-lapse images revealed a significant portion of beta(2)AR-GFP was distributed in endosomal compartments, along with Alexa 546-labeled transferrin, in all types of cells. Our results demonstrate spatial and temporal redistribution pattern of beta(2)AR in living non-neuronal cells and neuronal cells.