Characterization of the activation function-2 domain of the human 1,25-dihydroxyvitamin D3 receptor1
Introduction
Most of the biological effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated through the 1,25(OH)2D3 receptor (VDR) located in the nucleus of target cells (Haussler et al., 1988a). The VDR belongs to the steroid/thyroid/retinoid receptor superfamily (Baker et al., 1988), and regulates the transcription of target genes in a ligand-dependent manner. The primary event in the cascade leading to the transcriptional regulation of target genes via VDR is hormone binding (Haussler et al., 1988b). Presumably, the binding of ligand initiates a conformational change in the VDR protein. The ligand-bound VDR then forms a heterodimer with retinoid X receptor (RXR) (Liao et al., 1990, Yu et al., 1991, Kliewer et al., 1992, MacDonald et al., 1993) on distinct sequences of nucleotides, termed vitamin D responsive elements (VDREs), located upstream of target genes, and modulates transcription (Ozono et al., 1991). The VDR consists of a central DNA binding domain (C domain) and a carboxyl (C)-terminal ligand binding domain (E domain). Unlike other nuclear hormone receptors, either the VDR does not possess any, or at most has a very short N-terminal A/B domain (Baker et al., 1988) containing an activation function domain 1 (AF-1) which contributes to ligand-independent transactivation (Pham et al., 1992). The DNA binding domain contains eight cysteine residues forming two zinc fingers to recognize the VDRE in the promoter region of the target genes. The E domain contributes to the ligand binding (Nakajima et al., 1996) and to the protein/protein interactions (Nakajima et al., 1994, Whitfield et al., 1995). We demonstrated that the mutant hVDR, truncated with C-terminal 25 amino acids (Δ403), is able to bind 1,25(OH)2D3 and form a heterodimer with RXR, but does not exhibit ligand-dependent transcription activation (Nakajima et al., 1994). These 25 amino acids include the region corresponding to the activation function domain 2 (AF-2) previously reported in thyroid hormone receptors (TR) (Barettino et al., 1994, Baniahmad et al., 1995) and retinoic acid receptors (RAR) (Durand et al., 1994). Crystallographical analysis of the ligand binding domain of TR (Wagner et al., 1995) and RAR (Renaud et al., 1995) revealed that the extreme C-terminal AF-2 domain forms one of the protein/protein interacting surfaces because it is occupied with their cognate ligands. In addition, mutations at the AF-2 domain of the estrogen receptor (ER) (Ince et al., 1993) and the RAR (Damm et al., 1993) confer a strong dominant negative effect on the ligand-dependent transactivation mediated through wild type receptors. Taken together, these findings suggest that the AF-2 domain binds coactivator(s) to bridge between the nuclear receptors and the basal transcriptional factors by binding to the ligands.
In order to delineate the molecular mechanism of the VDR’s regulation of gene transcription, it is important to determine the precise sites of the hVDR responsible for the ligand-dependent transactivation (AF-2) and to elucidate the functional roles of the AF-2 region. In this study, we identified the AF-2 sites in the hVDR and examined whether the AF-2 mutant VDR was able to exhibit a dominant negative effect on the wild type VDR in mammalian cells. We also examined the functional role of lysine-264, which is presumed to be vital in forming a salt bridge within the ligand binding domain, based on the crystallographical analysis of retinoic acid receptor (Renaud et al., 1995).
Section snippets
Preparation of expression and reporter plasmids
The hVDR expression vector, pSG5hVDR (Nakajima et al., 1994), was utilized to create truncated or point-mutant hVDR plasmids by polymerase chain reaction-based site-directed mutagenesis, replacing Glu-420 with Gln, Leu-417 with Ala, Cys-79 with Ser, Lys-264 with Ala or Cys-403 with stop codon. Four copies of the CT4 oligonucleotide, which contains the rat osteocalcin VDRE, were subcloned upstream of the viral thymidine kinase promoter-growth hormone reporter gene construct creating (CT4)4-TKGH (
Expression and biochemical analysis of mutant vitamin D receptors
Fig. 1A depicts a schematic representation of the mutant hVDRs in which point mutations or deletion were introduced in the extreme C-terminal and/or DNA binding domain. Each VDR was overexpressed in COS-7 cells, and the molecular sizes of the expressed proteins were confirmed by Western blotting (Fig. 1B). The binding affinities for 1,25(OH)2D3 of the expressed hVDRs in COS-7 cells were determined in two separate experiments. Normal ligand binding affinities were observed with E420Q and
Discussion
1,25(OH)2D3, the active form of vitamin D, plays a key role in calcium homeostasis and regulates the differentiation/proliferation of normal cells as well as malignant cells. The VDR, the nuclear receptor for 1,25(OH)2D3, mediates most of the cellular effects of vitamin D in its target organs. Therefore, it is very important to understand the molecular mechanism by which the VDR regulates the ligand-dependent transcriptional activities of its target genes. Here, we determined the essential
Acknowledgements
We thank Dr Mark R. Haussler of the University of Arizona for the generous gifts of the human VDR expression vector and the rat osteocalcin VDRE-GH reporter gene, and Dr Masami Horikoshi of the University of Tokyo for bacterial expression vector pET11d-hTFIIB. We also gratefully acknowledge Chika Shimizu, Akira Suzuki, Tomoko Hayashi and Noriko Tsuda for their excellent technical assistance, and Satsuki Nozu for her secretarial help. This work was supported in part by grants from the Ministry
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- 1
A preliminary report of some of the data presented in this manuscript was presented at the annual meeting of the American Society for Bone and Mineral Research, Baltimore, September, 1995.
- 2
Present address: Department of Pediatrics, Faculty of Medicine, Osaka University, Suita, Osaka 565, Japan.