Subcellular distribution of normal and mutant vitamin D receptors in living cells. Studies with a novel fluorescent ligand.
To understand the subcellular localization of the vitamin D receptor (VDR) and to measure VDR content in single cells, we recently developed a fluorescent labeled ligand, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY)-calcitriol. This tagged hormone has intact biological activity, high affinity and specific binding to the receptor, and enhanced fluorescent emission upon receptor binding. Using BODIPY-calcitriol, here we monitored the subcellular distribution of VDR in living cultured cells by microscopy. Time course studies showed that an equilibrium between the cytoplasmic and nuclear hormone binding developed within 5 min and was maintained thereafter. We found a substantial proportion of VDR residing in the cytoplasm, colocalized with endoplasmic reticulum, the Golgi complex, and microtubules. Confocal microscopy clarified the presence of VDR within discrete regions of the nucleus and along the nuclear envelope. There was no VDR in the plasma membrane. Low affinity BODIPY-calcitriol binding sites were in the mitochondria. Mutations in the VDR gene selectively and specifically altered BODIPY-calcitriol distribution. Defects in the hormone binding region of VDR prevented both nuclear and cytoplasmic hormone binding. Defects in the DNA binding region decreased the nuclear retention of VDR and prevented localization to nuclear foci. These results with BODIPY-calcitriol reveal cytoplasmic VDR localization in living cells and open the possibility of studying the three-dimensional architecture of intranuclear target sites.
Barsony
J
J
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. jul@helix.nih.gov
Renyi
I
I
McKoy
W
W
D001896:Boron Compounds / Q000378:metabolism*; D002117:Calcitriol / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D016296:Mutagenesis; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D018167:Receptors, Calcitriol; D019284:Thapsigargin; D002117:Calcitriol