Vitamin D, through its intracellular receptor, plays a major role in mineral homeostasis, bone remodeling, cell growth, and differentiation. It also displays immune and anticancer actions. The long term objective of the project is to determine the structure of the vitamin D-3 receptor (VDR), both with and without its natural hormone, 1,25-dihydroxy vitamin D-3 (1,25(OH)2D3). To achieve this goal, recombinant VDR will be expressed and purified by a scheme that will be developed to optimize yield and purity. The expression system of choice is a yeast host with a copper-inducible ubiquitin-VDR vector that has been optimized to produce 0.3 - 3.0 percent of total soluble protein as VDR. This system has been shown to yield intact, nonproteolyzed VDR that retains its native affinity for both 1,25(OH)2D3 and DNA. The plan is to devise a purification scheme utilizing the incorporation of a histidine tag in the VDR to facilitate its purification and yield. Preliminary experiments show that this his- VDR protein functions identically to native VDR. Also, the hormone- binding domain of the VDR (VDR-HBD) will be delineated to determine the lowest molecular weight protein that retains native affinity for the hormone. This molecule also will be tagged with histidine and produced in the yeast system, its expression levels and function assessed, and then purified to homogeneity in milligram amounts by metal affinity chromatography for both crystal structure and NMR solution structure determinations. The project also will include a random screen for VDR agonists and/or antagonists, and compounds that are active without displaying hypercalcemic properties would be candidates for phase II structure determinations with the purified receptors to aid in rational drug design. The 1,25(OH)2D3-like drugs have potential in the treatment of diseases such as osteoporosis, psoriasis, breast and prostatic cancer, and leukemia.
