Identification of the subdomain in the nuclear receptor for the hormonal form of vitamin D3, 1 alpha,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, 1 alpha,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 1 alpha,25-dihydroxyvitamin D3-3 beta-bromoacetate (1,25(OH)2 D3-3-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 in Escherichia coli as a glutathione S-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.
Swamy
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Department of Physiology, Boston University School of Medicine, Massachusetts, USA.
Kounine
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Ray
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D000345:Affinity Labels; D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites; D001667:Binding, Competitive; D002117:Calcitriol / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D012516:Osteosarcoma / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002117:Calcitriol