Identification of the Subdomain in the Nuclear Receptor for the Hormonal Form of Vitamin D3, 1α,25-Dihydroxyvitamin D3, Vitamin D Receptor, That Is Covalently Modified by an Affinity Labeling Reagent

Multiple physiological actions of the hormonal form of vitamin D3, 1α,25-dihydroxyvitamin D3(1,25(OH)2D3), are mediated by a genomic pathway which is initiated by the highly specific recognition and binding by its cognate receptor (vitamin D receptor, VDR) in the target cells. Thus, knowledge of the three-dimensional geometries of the ligand, i.e., 1,25(OH)2D3, and the 1,25(OH)2D3-binding domain of VDR is crucial for a better understanding of diverse physiological roles of this hormone. Recently our laboratory has developed 1α,25-dihydroxyvitamin D3-3β-bromoacetate (1,25(OH)2D3-3-BE) as an affinity labeling reagent for covalently modifying the hormone binding domain of native VDRs from calf thymus and rat osteosarcoma cells and baculovirus-expressed recombinant human VDR (hVDR). In the present report, we report affinity labeling of the hormone binding domain of hVDR, expressed inEscherichia colias a glutathioneS-transferase fusion partner, site-specific cleavage of the affinity-labeled VDR with 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole, and identification of the C-terminal subdomain of human VDR containing the putative hormone binding site.