Dynamic changes in subcellular localization of mineralocorticoid receptor in living cells: in comparison with glucocorticoid receptor using dual-color labeling with green fluorescent protein spectral variants.
Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors. Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively. In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10(-9) M CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10(-6) M CORT. On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.
Nishi
M
M
Department of Anatomy and Neurobiology Kyoto Prefectural University of Medicine Kawaramachi Hirokoji, Kamigyo-ku Kyoto 602-8566, Japan. nmayumi@koto.kpu-m.ac.jp
Ogawa
H
H
Ito
T
T
Matsuda
K I
KI
Kawata
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D016227:Benzoquinones; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003345:Corticosterone / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004622:Embryo, Mammalian; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000648:ultrastructure; D007202:Indicators and Reagents; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins / Q000235:genetics*; D009474:Neurons / Q000648:ultrastructure; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018161:Receptors, Mineralocorticoid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry*; D014158:Transcription, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D047029:Lactams, Macrocyclic; D008164:Luminescent Proteins; D011809:Quinones; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D005576:Colforsin; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D003345:Corticosterone; C001277:geldanamycin