THYROXINE-PROTEIN INTERACTION

甲状腺素-蛋白质相互作用

• 批准号: 2137381
• 负责人: JACK H OPPENHEIMER
• 金额: $ 47.53万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-03-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137381
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; cerebellar Purkinje cell; developmental genetics; developmental neurobiology; dimer; gel mobility shift assay; gene expression; genetic regulatory element; genetic translation; genetically modified animals; hormone receptor; hormone regulation /control mechanism; immunologic assay /test; immunoprecipitation; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protein isoforms; protein sequence; protein structure function; receptor binding; receptor expression; transfection; triiodothyronine; western blottings

The studies proposed are designed to investigate two closely related issues: (A) the form, cellular content, and function of the various thyroid hormone receptors and receptor variants and (B) the molecular mechanisms by which thyroid hormone assures normal development of the central nervous system. (A) T3 receptors: Studies within the past four years have established that there are at least four distinctive species of receptors which are coded by two genes, designated alpha1, alpha2, beta1, beta1. Whereas the beta1 receptor appears to be specific for the pituitary, alpha1 and alpha2 have a wider distribution. In contrast to the others, alpha2 forms do not bind T3 since the ligand-binding domain is spliced out of the primary transcript. The development of specific polyclonal antibodies to the alpha2 and beta1 proteins and the application of Western blot techniques have shown even further diversity in size of these receptors. Studies are planned to determine the basis of this heterogeneity whether due to posttranslational modification, multiple translational start sites or other factors. Methods are also proposed to extend these studies to an analysis of the alpha1 receptor and to quantitate the concentration of the various isoforms in tissues and cell lines. The availability of specific antibodies should allow precise measurements of the specific thyroid hormone receptor components in homo- and heterodimers which have been shown to play an important role in thyroid hormone action. The proposed studies should be helpful in clarifying the specific role of the various receptor isoforms in initiating the physiological action of the hormones. (B) Thyroid hormone-dependent development of the central nervous system. Thyroid hormones play a decisive role in normal brain development. Recent studies have shown that a 40-fold surge in the level of the beta1 mRNA occurs in the first 10 neonatal days of rats. This period is coincident with the rise in plasma and tissue T3 and the timing of T3-dependent changes in CNS development. Immunoprecipitation studies have shown that the rise in beta1 mRNA is accompanied by a comparable increase in the beta1 receptor level. Further, immunocytochemical studies have shown a high concentration of beta1 receptor in cerebellar Purkinje cells, consonant with the essential role of thyroid hormones in the normal development of this cell type. Three genes expressed selectively in Purkinje cells have been shown to be regulated either directly or indirectly by T3 in the developmental process. Transient transfection experiments are designed to define the nature of the target gene TREs, the potential role of auxiliary trans- and cis-acting factors, and to determine whether the beta1 receptor plays a unique role in regulating expression. These studies should provide fresh insights not only into the specific mechanism by which thyroid hormones govern brain development but into the general patterns by which other nuclear-acting hormones exert their ontogenic effects.

提出的研究旨在研究两个密切相关的 问题：（a）各种形式，蜂窝内容和功能 甲状腺激素受体和受体变体，（b）分子 甲状腺激素确保正常发展的机制 中枢神经系统。 （a）T3受体：过去四年内的研究确定 至少有四种独特的受体物种由 两个基因，分别为alpha1，alpha2，beta1，beta1。 而beta1 受体似乎特定于垂体，alpha1和alpha2具有 更广泛的分布。 与其他人相反，alpha2形式不绑定 T3由于配体结合域是从主要的 成绩单。 特定多克隆抗体的开发 alpha2和beta1蛋白以及蛋白质印迹技术的应用 在这些受体的大小上显示了进一步的多样性。 研究是 计划确定这种异质性的基础 翻译后修改，多个翻译起始站点或其他 因素。 还提出了将这些研究扩展到分析的方法 α1受体的量子，并定量各种浓度 组织和细胞系中的同工型。 特定的可用性 抗体应允许对特定甲状腺进行精确测量 同二聚体和异性二聚体中的激素受体成分已显示 在甲状腺激素作用中发挥重要作用。 提出的研究 应该有助于阐明各种受体的特定作用 同工型启动激素的生理作用。 （b）中枢神经系统的甲状腺激素依赖性发育。 甲状腺激素在正常的大脑发育中起决定性作用。 最近的 研究表明，beta1 mRNA水平有40倍的激增 发生在大鼠的前10天。 这个时期是一致的 随着血浆和组织T3的升高以及T3依赖性的时间 中枢神经系统开发的变化。 免疫沉淀研究表明 beta1 mRNA的升高伴随着beta1的可比增加 受体水平。 此外，免疫细胞化学研究表明 小脑Purkinje细胞中β1受体的浓度，辅音 甲状腺激素在正常发育中的重要作用 该单元格类型。 在Purkinje细胞中选择性表达的三个基因具有 显示在T3中直接或间接地调节 发展过程。 瞬态转染实验设计为 定义靶基因TRE的性质，辅助的潜在作用 反式和顺式作用因子，并确定beta1受体是否是否 在调节表达中起着独特的作用。 这些研究应提供 新见解不仅是甲状腺的特定机制 激素控制大脑的发育，但进入了一般模式 其他核作用激素发挥了其个体作用。