The vitamin D receptor (VDR) is a member of the superfamily of ligand- inducible transcription factors which includes the receptors for steroid and thyroid hormones and retinoic acid. A current model of vitamin D action proposes that the hormone-bound VDR binds to the vitamin D responsive element (VDRE) in DNA as a heterodimer with retinoid X receptors (RXRs), and the resulting VDR-RXR-VDRE complex then interacts with the transcriptional apparatus to regulate target gene expression. However, the molecular details of this model have not been characterized. The major objectives of the proposed research are to identify the structural determinants in VDR required to discriminate the VDRE from the other hormone responsive elements (HREs) and to investigate the consensus recognition sequences for VDR by in vitro binding site selection. For the studies of target gene specificity, a chimeric receptor containing the DNA binding domain of hVDR and the hormone binding domain of the glucocorticoid receptor will be generated to determine whether this construct can activate from the VDRE in the presence of dexamethasone. If this chimera is active, then certain crucial amino acids in the zinc- finger region of hVDR will be changed to the corresponding amino acids in other hormone receptors and the mutant chimera tested for their ability to distinguish between different HREs using a gel retardation assay, in vitro, and VDRE-mediated transcriptional activity, in vivo. To define the consensus recognition sequence of VDR, polymerase chain reaction will be employed to amplify sequences selected from a mixture of randomized oligonucleotides either by the gel mobility-shift assay or by immunoprecipitation. These selected motifs will be cloned into an expression vector containing a human growth hormone reporter gene and cotransfected with an hVDR expression vector into mammalian cells to examine their transcriptional activity in response to 1,25(OH)2D3, the active form of vitamin D. This approach will avoid the bias of conventional methods which involve searching gene sequences with a consensus nucleotide sequence. To facilitate these experiments, a combined mutagenesis and overexpression/purification system will be established. In this system, the hVDR or RXR cDNAs will be cloned first into a shuttle vector containing the fl replication origin to construct the desired mutants. After mutagenesis, the wild-type and mutant hVDRs and RXRs will be transfected into mammalian cells for preliminary analysis and then subcloned into an E. coli expression vector for large-scale production. To evaluate whether RXR interacts with the VDRE according to the proposed heterodimerization model, several mutants in the zinc finger region of RXR will be generated and analyzed. For three-dimensional structure determination, E. coli-expressed, functional fragments of hVDR complexed with/without the VDRE and sterol ligand will be crystallized. The X-ray crystal structure should provide definitive proof regarding the VDR-VDRE and VDR-sterol interactions predicted from the molecular biological experiments. These results will increase our understanding of vitamin D hormone action at the molecular level and may facilitate new therapies for common bone disorders such as renal osteodystrophy and osteoporosis.
