Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus.
The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the 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 the 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 VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly 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.
Michigami
T
T
Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
Suga
A
A
Yamazaki
M
M
Shimizu
C
C
Cai
G
G
Okada
S
S
Ozono
K
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D015533:Transcriptional Activation
D017931:DNA Primers; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins