DIFFERENTIAL ACTIVATION OF THE VITAMIN D RECEPTOR

说明（改编自申请人的摘要）： 激素1，25-二羟维生素D3 [1，25（OH）2D 3]也调节细胞 生长和分化，因此它被认为是治疗 恶性肿瘤和银屑病。 1，25（OH）2D 3的几种类似物高达10，000 比1，25（OH）2D 3的生长抑制和分化能力强一倍 试剂，但其增强的活性与更大的亲和力无关 核维生素D受体（VDR）或增强钙 动员。 相反，这些活动与增强的 VDR的转录激活。 本研究的目的是 剖析导致VDR最大激活的机制， 类似物，以便可以 开发 假设是配体与 VDR可以通过化学和立体化学修饰来改变， 1，25（OH）2D 3;配体中的这些结构变化也调节 以及通过改变VDR介导的转录活性谱， VDR/配体复合物的二聚化和DNA结合偏好。 为了检验假设，将通过以下方法对1，25（OH）2D 3结合位点进行定位： 定点诱变及其与VDR的接触点将 通过用1，25（OH）2D 3共价标记配体结合位点证实 可以被光活化的衍生物。 1，25（OH）2D 3的结合活性 将野生型和突变VDR的类似物进行比较， 配体中的化学和立体化学修饰的效果 可以确定其约束力要求。 为了确定类似物是否可以改变VDR与核的相互作用， 受体的二聚化偏好的影响VDR将被检查 体外 细菌表达的VDR或RXR将用于捕获 配体激活的VDR复合物，后者将被定量。 的 1，25（OH）2D 3和类似物对二聚化界面的影响将 通过配体依赖性二聚化结构域的突变分析来检查。 为了研究不同配体相互作用对分子类型的影响， 用于VDR作用的反应元件，细胞或合成VDR配体 将复合物与随机寡核苷酸一起孵育。 特异性DNA 将从受体/DNA复合物中提取结合的序列， 通过聚合酶链反应扩增，测序并检测 通过DNA转移方法测定转录活性。

DESCRIPTION (Adapted from the Applicant's Abstract): The calcitropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] also regulates cellular growth and differentiation, and so it has been considered as a treatment for malignancy and psoriasis. Several analogs of 1,25(OH)2D3 are up to 10,000 times more potent that 1,25(OH)2D3 as growth-inhibitory and differentiating agents, but their enhanced activity is not correlated with greater affinity to the nuclear vitamin D receptor (VDR) or with enhanced calcium mobilization. Instead, these activities are correlated with enhanced transcriptional activation of VDR. The objective of this study is to dissect the mechanisms that lead to maximal activation of the VDR by these analogs so that more effective, clinically applicable compounds can be developed. The hypothesis is that the mode of ligand interaction with the VDR can be changed by chemical and stereochemical modifications of 1,25(OH)2D3; these structural changes in the ligand also modulate the levels and the spectrum of VDR-mediated transcriptional activities by changing dimerization and DNA-binding preferences of VDR/ligand complexes. To test the hypothesis the 1,25(OH)2D3-binding site will be mapped by site-directed mutagenesis and its contact points with the VDR will be confirmed by covalent labeling of the ligand-binding site with a 1,25(OH)2D3 derivative that can be photoactivated. The binding activity of 1,25(OH)2D3 to wild-type and mutated VDR will be compared with that of the analogs, so that the effect of chemical and stereochemical modifications in the ligand on its binding requirements can be determined. To determine whether or not analogs can modify VDR interaction with nuclear receptors their effect on dimerization preferences of VDR will be examined in vitro. Bacterially-expressed VDR or RXR will be used to capture ligand-activated VDR complexes, and the latter will be quantified. The effect of 1,25(OH)2D3 and analogs on dimerization interfaces will be examined by mutation analysis of the ligand-dependent dimerization domain. To study the effect of differential ligand interaction on the type of response element used for VDR action, cellular or synthetic VDR-ligand complexes will be incubated with random oligonucleotides. The specific DNA sequences that bind will be extracted from the receptor/DNA complexes, amplified by the polymerase chain reaction, sequenced and tested for transcriptional activity by DNA-transfer methods.