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.
Nishi
Mayumi
M
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. nmayumi@koto.kpu-m.ac.jp
Tanaka
Masayuki
M
Matsuda
Ken-ichi
K
Sunaguchi
Masataka
M
Kawata
Mitsuhiro
M
D000818:Animals; D019556:COS Cells / Q000166:cytology; D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D009474:Neurons / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000378:metabolism*; D014162:Transfection
D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid