Identification of amino acid sequence in the hinge region of human vitaminD receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its li gand 1,25-dihydroxyvitamin D-3 was investigated using chimera proteins fuse d to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fu sion proteins were transiently or stably introduced into COS7 cells, and th e subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout th e cells. 10(-8) M 1,25-dihydroxyvitamin D-3 promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDR s fused to GFP. Mutant VDRs that did not bind to DNA were also localized pr edominantly in:nuclei, while the deletion of the hinge region resulted in t he loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to transl ocate to nuclei. These results indicate that 1) VDR is located predominantl y in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.