| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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, respectiv
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ely. 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. Less
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