THYROXINE-PROTEIN INTERACTION

甲状腺素-蛋白质相互作用

• 批准号: 3226541
• 负责人: JACK H OPPENHEIMER
• 金额: $ 43.28万
• 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/3226541
• 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 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.

拟议的研究旨在调查两个密切相关的 问题：（A）各种细胞的形式、细胞内容和功能 甲状腺激素受体和受体变体以及 (B) 分子 甲状腺激素确保机体正常发育的机制 中枢神经系统。 (A) T3 受体：过去四年的研究表明 至少有四种独特的受体，其编码是 两个基因，指定为 alpha1、alpha2、beta1、beta1。 而 beta1 受体似乎对垂体具有特异性，α1 和 α2 具有 更广泛的分布。 与其他形式相比，α2 形式不结合 T3，因为配体结合结构域是从初级剪接出来的 成绩单。 特异性多克隆抗体的开发 α2和β1蛋白以及Western blot技术的应用 已经显示出这些受体大小的进一步多样性。 研究是 计划确定这种异质性的基础是否是由于 翻译后修饰、多个翻译起始位点或其他 因素。 还提出了将这些研究扩展到分析的方法 α1 受体并定量各种浓度 组织和细胞系中的同工型。 具体的可用性 抗体应该能够精确测量特定的甲状腺 同二聚体和异二聚体中的激素受体成分已被证明 在甲状腺激素的作用中发挥重要作用。 拟议的研究 应该有助于阐明各种受体的具体作用 启动激素生理作用的异构体。 (B) 中枢神经系统的甲状腺激素依赖性发育。 甲状腺激素在大脑正常发育中起着决定性作用。 最近的 研究表明，β1 mRNA 水平激增 40 倍 发生在大鼠新生后的前 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 中枢神经系统发育的变化。 免疫沉淀研究表明 beta1 mRNA 的增加伴随着 beta1 mRNA 的类似增加 受体水平。 此外，免疫细胞化学研究表明， 小脑浦肯野细胞中β1受体的浓度，辅音 甲状腺激素在正常发育中起着重要作用 这种细胞类型。 在浦肯野细胞中选择性表达的三个基因 研究表明，T3 直接或间接调节 发展过程。 瞬时转染实验旨在 定义靶基因TRE的性质，辅助基因的潜在作用 反式和顺式作用因子，并确定β1受体是否 在表达调节中发挥着独特的作用。 这些研究应该提供 不仅对甲状腺的具体机制有新的见解 激素控制大脑发育，但控制大脑发育的一般模式 其他核作用激素发挥其个体发育作用。