MONOCLONAL ANTIBODIES AS PROBES TO VITAMIN D3 ACTION

1，25-二羟基维生素D3(1，25(OH)2D3)的多种生物学作用如下 被认为是由细胞内受体蛋白介导的，似乎 在细胞核水平上运作。我们打算继续我们的研究 这些来自鸟类和哺乳动物来源的独特多肽 利用免疫学和常规探针。调查将会 关注对我们理解分子的四个基本领域 1，25(OH)2D3作用机理：1)生理生化性质 受体，2)受体的细胞内生物学及其作用 1，25(OH)2D3)受体的功能表征，以及4)分子 赋予细胞对1，25(OH)2D3抗性的受体缺陷。 物理和生化特征将包括评估 禽类受体的两种抗原性相关形式，拓扑图 鸟类和哺乳动物的受体，哺乳动物的测定 1，25(OH)2D3依赖的受体磷酸氨基酸种类，以及分离和 鸟类和哺乳动物受体的序列分析。的各个方面。 1，25(OH)2D3受体的生物学将包括对 未被占用形式的细胞内起源，受体合成和 降解，1，25(OH)2D3对细胞受体和受体水平的影响 其受体失活的性质和机制。功能性 研究将包括潜在受体酶的测定 受体的活性、功能意义的评价 受体DNA序列偏好性的修饰和分析 克隆维生素D依赖性钙结合蛋白基因和受体基因片段。 分子异常的评估将涉及到应用 从上述技术到检测异常人类受体 1，25(OH)2D3耐药患者的成纤维细胞 来自开发的非人类变种。总而言之，这些研究建议 在这里，正常的受体代表了一种综合的方法来 受体在多种生物学调节中的作用阐明 受1，25(OH)2D3调控的现象。此外，它们还为 解释受体中的分子缺陷可能与 抗维生素D3的疾病状态。

The diverse biologic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are thought to be mediated byintracellular receptor proteins which appear to operate at the level of the cell nucleus. We propose to continue our study of these unique polypeptides derived from both avian and mammalian sources utilizing immunologic as well as conventional probes. Investigation will focus upon four areas essential to our understanding of the molecular mechanism of 1,25(OH)2D3 action: 1) physical and biochemical properties of receptors, 2) intracellular biology of receptors and the role of 1,25(OH)2D3 3) functional characterization of receptors, and 4) molecular defects in receptors which confer cellular resistance to 1,25(OH)2D3. Physical and biochemical characterization will include evaluation of the two antigenically related forms of the avian receptor, topological mapping of both avian and mammalian receptors, determination of mammalian 1,25(OH)2D3-dependent receptor phosphoamino acid species, and isolation and sequence analysis of both avian and mammalian receptors. Aspects of the biology of 1,25(OH)2D3 receptors will include further evaluation of the intracellular origins of unoccupied forms, receptor synthesis and degradation, the effects of 1,25(OH)2D3 on cellualr levels of receptor and its mRNA, the nature and mechanism of receptor inactivation. Functional studies will include determination of potential receptor enzymatic activities, evaluation of teh functional significance of receptor modification, and analysis of receptor DNA sequence preference utilizing cloned vitamin D-dependent calbindin gene and receptor gene fragments. Evaluation of molecular abnormalities will involve the application of the above technology to the examination of abberrant human receptors from fibroblasts obtained from patients with reisitance to 1,25(OH)2D3 as well as from developed nonhuman variants. Collectively, the studies proposed here on normal receptors represent an integrated approach to the elucidation of the role of receptor in mediating various bilogical phenomena regulated by 1,25(OH)2D3. In addtion, they provide the basis for interpreting molecular defects in receptors which may be associated with vitamin D3-resistant disease states.