Molecular cloning of thyroid hormone receptor interacting protein using yeast two hybrid system

利用酵母二杂交系统分子克隆甲状腺激素受体相互作用蛋白

• 批准号: 07671126
• 负责人: NAGAYA Takashi
• 金额: $ 1.47万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671126/
• 关键词: Thyroid hormone receptor; TR interacting protein; Yeast two hybrid system; Co-repressor; Thyroid hormone; Cloning; Two hybrid system; 分子生物学

Thyroid hormone is essential for normal growth and development as well as several metabolic pathways. This effect is mediated through its nuclear receptors, which bind to the DNA sequences mainly as a heterodimer with 9-cis retinoic acid receptors (RXR_s). This complex on DNA can activate or repress thyroid hormone responsive genes in a ligand dependent manner. Recent studies indicated that TR complexe can interact with components of basal transcriptional machinery and that these interaction is critical for transcriptional regulation. Furthemore, the intermediating co-factors between TRs and basal transcriptional machinery are speculated to be important for transducing hormonal signals.1. CLONING OF THYROID HORMONE RECEPTOR INTERACTING PROTEINMolecular cloning of thyroid hormone receptor interacting proteins (co-factors) was performed using yeast two hybrid system. Hela cell cDNA library constructed in Ga14 activation domain vector (pGAD-GH) was screened by human thyroid hormone recept … More orbeta (TRbeta) in Ga4 DNA binding domain vector (pGBT9). One clone (named B1)to interact with TRbeta was isolated and considered to be a human homologue of mouse nuclear receptor co-repressor (N-CoR) by nucleotide sequencing. As Hela cDNA library was screened by plaque hybridization method to obtain full length of this clone, another clone (named H3) which is 80% homology with clone B1 was isolated. The amino acids (a.a.) deduced by clone H3 were different in its carboxy-terminus, in that 120a.a.of clone B1 was replaced with distinct 50a.a.. Since mN-CoR interacts with TR and retinoic acid receptor (RAR) through its carboxy-terminal region, the alteration of carboxy-terminus in N-CoR variant (clone H3) might affect specificity of receptor interactions. The existence of N-CoR isoforms will support the possibility that the different expression of these isoforms may modulate transcriptional regulation by thyroid hormone.2. EXPRESSION OF N-COR ISOFORMS IN THE CELL LINESGene expression of N-CoR isoforms in the cells was analyzed by RT-PCR method. The extracted RNA from 10 different cell lines was reverse-transcribed and followed by the amplification of N-CoR isoforms with PCR.In all cell lines studied, mRNA expression of two N-CoR isoforms were detected. It indicates that N-CoR isoforms are expressed ubiquitously. Less

甲状腺激素是人体正常生长发育以及多种代谢途径所必需的。这种作用是通过其核受体介导的，核受体主要以异源二聚体的形式与9-顺式维甲酸受体（RXR_s）结合。这种DNA上的复合体可以以配体依赖的方式激活或抑制甲状腺激素应答基因。最近的研究表明，TR复合物可以与基础转录机制的组分相互作用，这些相互作用对转录调控至关重要。此外，据推测，TRs和基础转录机制之间的中介辅助因子对激素信号的转导很重要。甲状腺激素受体相互作用蛋白的克隆利用酵母双杂交系统对甲状腺激素受体相互作用蛋白（辅因子）进行了分子克隆。利用人甲状腺激素受体（TRbeta）在Ga4 DNA结合域载体（pGBT9）上筛选构建在Ga14激活域载体（pGAD-GH）上的Hela细胞cDNA文库。一个与TRbeta相互作用的克隆（命名为B1）被分离出来，通过核苷酸测序认为是小鼠核受体共抑制因子（N-CoR）的人类同源物。通过斑块杂交法筛选Hela cDNA文库获得该克隆的全长，分离出与克隆B1同源性80%的另一个克隆（命名为H3）。克隆H3推断出的氨基酸（a.a.a）在其羧基端不同，在120a.a。克隆B1用不同的50a.a代替。由于mN-CoR通过其羧基末端区域与TR和视黄酸受体（RAR）相互作用，因此N-CoR变体（克隆H3）羧基末端的改变可能会影响受体相互作用的特异性。N-CoR异构体的存在将支持这些异构体的不同表达可能调节甲状腺激素的转录调节的可能性。N-COR亚型在细胞系中的表达用RT-PCR方法分析细胞中N-COR亚型的基因表达。从10个不同细胞系中提取RNA进行逆转录，然后用PCR扩增N-CoR亚型。在所研究的所有细胞系中，检测到两种N-CoR亚型的mRNA表达。这表明N-CoR亚型普遍表达。少

项目成果

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Nagaya T,Kopp P,Kitajima K,Jameson, JL,Seo H: "Second zinc finger mutants of thyroid hormone receptor selectively preserve DNA binding and heterodimerization but eliminate transcriptional activation." Biochemical and Biophysical Research Communications. 2

Nagaya T、Kopp P、Kitajima K、Jameson、JL、Seo H：“甲状腺激素受体的第二个锌指突变体选择性地保留 DNA 结合和异二聚化，但消除转录激活。”