Identification of Amino Acid Sequence in the Hinge Region of Human Vitamin D Receptor That Transfers a Cytosolic Protein to the Nucleus

Abstract

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. GFPtagged 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. The vitamin D receptor (VDR)1 is one of the ligand-dependent transcription factors that make up the nuclear hormone receptor superfamily (1-3). To modulate the transcription of target genes in response to its cognate ligand 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), VDR must be localized in nucleus and then bind to an enhancer designated as the vitamin D-responsive element (VDRE), forming a heterodimer with retinoid X receptor (1-6). In contrast to the case for the glucocorticoid receptor (GR), which translocates from the cytoplasm to the nucleus when exposed to its ligand, VDR does not bind to heat shock protein 90, and both immunocytochemical and biochemical fractionation studies suggested the nuclear localization of VDR even in the absence of 1,25(OH) 2 D 3 (7-10).Several reports, however, demonstrated that VDR was located in cytoplasm in the absence of ligand and transported to nucleus in response to 1,25(OH) 2 D 3 (11-13). Although the reason for conflicting results as to the distribution of VDR is not clear, the fixation and cell permeabilization procedures in immunostaining might influence the subcellular distribution of the subject protein. Consistent with this explanation, Barsony et al. (11), by the fixation of cells using a microwave, revealed the cytoplasmic localization of VDR in contrast to the nuclear localization detected by a conventional fixation method utilizing the same antibody against VDR.To avoid the fixation and cell permeabilization steps required in the immunostaining procedure, in the present study we have taken advantage of fusion with green fluorescent protein (GFP), which has been proven to be a useful tag for monitoring the subcellular distribution and trafficking of various proteins in living cell...

Nuclear Import of the Retinoid X Receptor, the Vitamin D Receptor, and Their Mutual Heterodimer

“…3, compare D with B), the distinct ligand response of VDRnlsm suggests that, unlike RXR whose nuclear translocation relies solely on the DBD-NLS, VDR contains an additional, ligand-responsive NLS. These observations are thus in agreement with previous reports indicating that VDR harbors more than one functional NLS (21,23).…”

“…It was thus reported that the glucocorticoid receptor harbors an additional NLS within its LBD, and that this sequence possesses a different importin selectivity than that displayed by the NL1 (22). VDR appears to harbor a bipartite NLS in the stretch between residues 67 and 108 (21) and another NLS in its hinge region (23).…”

Dimerization with Retinoid X Receptors Promotes Nuclear Localization and Subnuclear Targeting of Vitamin D Receptors

“…We found earlier that the cytoplasmic portion of VDR-GFP also translocates into the nucleus in a ligand-dependent fashion (5,6). The mechanisms controlling VDR translocation, however, remained to be elucidated.…”

“…Recently we and others have used green fluorescent protein chimeras of VDR to study the receptor distribution in living cells (5)(6)(7). Unlike the glucocorticoid receptor (GR), which stays in the cytoplasm without the ligand, the unliganded VDR distributes evenly between the cytoplasm and the nucleus.…”

Nucleo‐cytoplasmic cycling of the vitamin D receptor in the enterocyte‐like cell line, Caco‐2

“…The deletion of VDR in the enterocyte results in a 70% reduction in intestinal calcium absorption efficiency that is coincident with a significant reduction in CaBPD9k, TRPV6, and PMCA1b gene expression [Van Cromphaut et al, 2001;Song et al, 2003a].Previous research shows that binding of 1,25(OH) 2 D 3 to VDR in the cytoplasm of cells stimulates heterodimerization of VDR with RXR and the redistribution of the VDR-RXR-hormone complex to the nucleus [Barsony et al, 1990;Michigami et al, 1999;Racz and Barsony, 1999;Sunn et al, 2001]. The nuclear import of the VDR-RXR-hormone complex is active [Racz and Barsony, 1999;Miyauchi et al, 2005], enhanced by 1,25(OH) 2 D 3 treatment [Michigami et al, 1999;Racz and Barsony, 1999;Sunn et al, 2001], dependent upon the presence of intact nuclear localization sequences (NLSs) in both VDR and RXR [Prufer et al, 2000], and requires various importins [Miyauchi et al, 2005;Yasmin et al, 2005]. Most of the studies examining the mechanism of VDR nuclear import have been conducted in COS-7 kidney cells (characterized by low expression of endogenous VDR) and some characteristics of VDR import have been confirmed in cells from bone, skin, or kidney.…”