DIFFERENTIAL ACTIVATION OF THE VITAMIN D RECEPTOR

描述(改编自申请者摘要)：降钙素 激素1，25-二羟基维生素D3[1，25(OH)2D3]也调节细胞 生长和分化，所以它被认为是一种治疗 恶性肿瘤和牛皮癣。1，25(OH)2D3的几个类似物最高可达10,000 比1，25(OH)2D3更强的生长抑制和分化作用 药物，但它们增强的活性与更强的亲和力无关 核内维生素D受体(VDR)或钙离子增强 动员。相反，这些活动与增强的 VDR的转录激活。这项研究的目的是 通过这些分析导致VDR最大激活的机制 类似物，以便更有效、更适用于临床的化合物可以 发展起来的。假设与配体相互作用的方式 VDR可以通过化学和立体化学修饰来改变 1，25(OH)2D3；这些配体的结构变化也调节水平 以及通过改变VDR介导的转录活性的谱 VDR/配体络合物的二聚化和DNA结合偏好。 为了检验这一假设，1，25(OH)2D3结合位点将通过 定点突变及其与VDR的接触点将是 通过1，25(OH)2D3的配基结合部位的共价标记确认 可被光激活的衍生物。1，25(OH)2D3的结合活性 将野生型和突变型VDR与类似物进行比较，因此 化学和立体化学修饰对配体的影响 对其约束的要求可以确定。 确定类似物是否可以修改VDR与核的相互作用 我们将研究受体对VDR二聚化偏好的影响。 在试管中。细菌表达的VDR或RXR将用于捕获 配体激活的VDR络合物，后者将被量化。这个 1，25(OH)2D3及其类似物对二聚界面的影响 通过配体依赖的二聚化结构域的突变分析进行检测。 研究差异配基相互作用对核糖核酸类型的影响 用于VDR作用的反应元件，细胞或合成VDR配体 复合体将与随机寡核苷酸孵育。特定的DNA 结合的序列将从受体/DNA复合体中提取， 通过聚合酶链式反应扩增，测序和检测 DNA转移法的转录活性。

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.