MOLECULAR PROBING OF VITAMIN D RECEPTOR

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The immediate goal of this research proposal is to probe and critically investigate the structure/function relationship between the calciotropic hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), and its receptor (vitamin D receptor, VDR) with a particular focus on the hormone-binding domain-structure of VDR. We propose to utilize our recently developed affinity labelling method to covalently label the hormone-binding pocket of human recombinant VDR (hVDR), fragment it proteolytically, and isolate the peptide bearing majority of the label. Sequence-determination of this peptide will delineate the binding pocket of hVDR as well as the contact points within this area. We will also develop alternate affinity analogs, with affinity probes at different part of the 1,25(OH)2D3, molecule, to determine the specificity of labelling. Functional tests for this labelling process will be provided by developing mutant hVDR proteins, point-mutated at contact points (delineated by our affinity studies), and determining the binding characteristics of 1.25(OH)2D3 and other metabolites of vitamin D3 towards these mutant proteins. Knowledge of the hormone-binding domain structure will be important to properly understand the pathophysiology of human vitamin D deficient rickets type II syndromes which involve natural mutations at hormone- and DNA-binding regions of hVDR. Information about the contact points (within the hormone-binding pocket) will also be useful in identifying the important recognition markers in the 1,25(OH)2D3 molecule, which, in turn will be useful in chemically synthesizing analogs of 1.25(OH)2D3. These analogs will be endowed with recently discovered anti-cancer properties of 1,25(OH)2D3 with less toxic side effects. The affinity labelling method will also be very useful in the identification and characterization of the putative 'membrane-bound vitamin D receptor' in tissues where 1.25(HO)2D3 is known to induce very rapid biologic effects (non-genomic effects). In summary the projects outlined in the application will provide important information regarding various physiologic properties of 1,25(OH)2D3 hormone.