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

• Published in: Archives of biochemistry and biophysics 1997-12, Vol.348 (1), p.91-95
• Main Authors: Swamy, Narasimha, Kounine, Melissa, Ray, Rahul
• Format: Article
• Language: English
• Subjects: Affinity Labels; Animals; Baculoviridae; Binding Sites; Binding, Competitive; Calcitriol - chemistry; Calcitriol - metabolism; Cattle; Cell Line; Cell Nucleus - metabolism; Cloning, Molecular; Escherichia coli; Humans; Kinetics; Osteosarcoma - metabolism; Rats; Receptors, Calcitriol - chemistry; Receptors, Calcitriol - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Thymus Gland - metabolism; Transfection; Tumor Cells, Cultured
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1997.0389

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.