We examined the intracellular trafficking of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in living neurons and non-neural cells using a fusion protein of green fluorescent protein (GFP). 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. We also showed that microtubules are not involved in the nuclear translocation of these receptors
but hsp 90 plays important roles for nuclear translocation of these receptors. 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
YFP MR chimera was accumulated in the nucleus faster than CFP-GR chimera in the presence of 10^<-9>M CORT
while there is 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 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. Furthermore
we investigated the relationships of importin α and these corticosteroid receptors using the same fusion protein methods. When YFP-importin α is singly transfected in COS cells
they reside in both cytoplasm and nucleus in major populations of the transfected cells
and YFP importin α is translocated into the nucleus without any stimulation. However
YFP-importin α and CFP-GR are co transfected into COS cells
YFP-importin α is more strongly accumulated in the nucleus after ligand treatment. These results demonstrate that this system successfully detects a nuclear import process of carrier proteins and nuclear receptors in living intact cells.
