MOLECULAR PROBING OF VITAMIN D RECEPTOR

这项研究提案的直接目标是探索和批判性地 探讨促钙剂之间的结构/功能关系 激素，1α，25-二羟基维生素D3(1，25(OH)2D3)及其受体 (维生素D受体，VDR)，特别关注激素结合 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激素结合口袋 重组人维生素D受体(HVDR)，用蛋白水解法将其片段，并分离 带有大部分标记的多肽。顺序.测定 该多肽将描绘hVDR的结合口袋以及 这一区域内的联络点。我们还将发展替代亲和力 类似物，在1，25(OH)2D3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 对于这个标记过程，将通过开发突变体hVDR来提供 在接触点发生点突变的蛋白质(由我们的亲和力描述 研究)，并测定了1.25(OH)2D3和 维生素D3对这些突变蛋白的其他代谢物。 激素结合域结构的知识对于 正确认识人类维生素D缺乏的病理生理学 II型软骨病综合征，涉及荷尔蒙和 HVDR的DNA结合区。关于联系点的信息 (在荷尔蒙结合口袋内)也将有助于识别 1，25(OH)2D3分子中的重要识别标记，在 TURN在化学合成1.25(OH)2D3类似物方面将是有用的。 这些类似物将被赋予最近发现的抗癌作用。 1，25(OH)2D3的特性，毒副作用较小。亲和力 标签方法也将非常有用的识别和 膜结合型维生素D受体的特性研究 已知1.25(HO)2D3可引起非常迅速的生物效应的组织 (非基因组效应)。总括而言， 应用程序将提供有关各种 1，25(OH)2D3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.