title: Importin 4 is responsible for ligand-independent nuclear translocation of vitamin D receptor.
abstract: Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4-427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.
authors.affiliation: Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Miyauchi
authors.forename: Toshimi
authors.initials: T
authors.lastname: Michigami
authors.forename: Naoko
authors.initials: N
authors.lastname: Sakaguchi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Sekimoto
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
authors.forename: John Wesley
authors.initials: JW
authors.lastname: Pike
authors.forename: Masayo
authors.initials: M
authors.lastname: Yamagata
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ozono
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D015233:Models, Statistical; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D014162:Transfection; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: C106306:KPNA3 protein, human; D010446:Peptide Fragments; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D004247:DNA; D005982:Glutathione Transferase