Novel nuclear localization signal between the two DNA-binding zinc fingers in the human vitamin D receptor.
The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3 may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK.
Hsieh
J C
JC
Department of Biochemistry, The University of Arizona Health Sciences Center, Tucson 85724, USA. jchsieh@medbioc.arizona.edu
Shimizu
Y
Y
Minoshima
S
S
Shimizu
N
N
Haussler
C A
CA
Jurutka
P W
PW
Haussler
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D018167:Receptors, Calcitriol