MOLECULAR PROBING OF VITAMIN D RECEPTOR

这项研究计划的直接目标是探索和批判 研究促钙蛋白质的结构/功能关系， 激素，1 α，25-二羟维生素D3（1，25（OH）2D 3）及其受体 维生素D受体（vitamin D receptor，VDR），特别关注维生素D受体的结合。 VDR的域结构。 我们建议利用我们最近开发的 亲和标记法共价标记酶结合口袋 人重组VDR（hVDR），蛋白水解片段，并分离 所述肽携带大部分标记。 序列测定 该肽将描绘hVDR的结合口袋以及 该地区的联络点。 我们还将发展替代亲和力 类似物，在1，25（OH）2D 3的不同部分具有亲和探针， 分子，以确定标记的特异性。 功能测试 将通过开发突变hVDR来提供该标记过程 蛋白质，在接触点突变（由我们的亲和力描绘 研究），并确定1.25（OH）2D 3和 维生素D3的其他代谢物对这些突变蛋白质。 对酶结合结构域结构的了解对于 正确理解人类维生素D缺乏的病理生理学 佝偻病II型综合征，涉及激素的自然突变， hVDR的DNA结合区。 关于联络点的信息 （在粘合剂结合口袋内）也将有助于识别 1，25（OH）2D 3分子中的重要识别标记， 这将有助于化学合成1.25（OH）2D 3的类似物。 这些类似物将被赋予最近发现的抗癌作用 1，25（OH）2D 3的性质，毒副作用小。 亲和 标记方法在识别和 对假定的“膜结合维生素D受体”的表征， 已知1.25（HO）2D 3可诱导非常快速的生物学效应的组织 （非基因组效应）。 概括地说， 应用程序将提供有关各种重要信息 1，25（OH）2D 3激素的生理特性。

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.