title: Hormone-dependent translocation of vitamin D receptors is linked to transactivation.
abstract: Vitamin D receptor (VDR) acts as a transcription factor mediating genomic actions of calcitriol. Our earlier studies suggested that calcitriol induces translocation of cytoplasmic VDR, but the physiologic relevance of this finding remained uncertain. Previous studies demonstrated that the activation function 2 domain (AF-2) plays an essential role in VDR transactivation. To elucidate hormone-dependent VDR translocation and its role, we constructed green fluorescent protein (GFP) chimeras with full-length VDR (VDR-GFP), AF-2-truncated VDR (AF-2del-VDR-GFP), and ligand-binding domain (LBD)-truncated VDR (LBDdel-VDR-GFP). COS-7 cells were transiently transfected with these constructs. Western blot analysis, fluorescent microscopy, and transactivation assays showed that the generated chimeras are expressed and fluoresce and that VDR-GFP is transcriptionally active. After hormone treatment, cytoplasmic VDR-GFP translocated to the nucleus in a concentration-, time-, temperature-, and analog-specific manner. Hormone dose-response relationships for translocation and for transactivation were similar. Truncation of LBD and truncation of AF-2 each abolished hormone-dependent translocation and transactivation. Our data confirm a hormone-dependent VDR translocation, demonstrate that an intact AF-2 domain is required for this translocation, and indicate that translocation is part of the receptor activation process.
authors.affiliation: Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: A
authors.initials: A
authors.lastname: Racz
authors.forename: J
authors.initials: J
authors.lastname: Barsony
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002117:Calcitriol / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003199:Computer Systems; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D008024:Ligands; D008164:Luminescent Proteins; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; C074279:KH 1060; D049452:Green Fluorescent Proteins; D002117:Calcitriol