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Functional analysis of thyroid hormone receptor interacting protein

甲状腺激素受体相互作用蛋白的功能分析

基本信息

批准号：
09671045
负责人：
NAGAYA Takashi
金额：
$ 2.05万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671045/
关键词：
Thyroid hormone receptor TR interacting protein Yeast two hybrid system Co-repressor Thyroid hormone Cloning 甲状腺ホルモン受容体共役因子コレプレッサー分子生物学

项目摘要

To understand the molecular aspect of thyroid hormone action, we performed to clone TR interacting protein by yeast two hybrid system. Hela cell cDNA library constructed in Gal4 activation domain vector (pGAD-GH) was screened by human thyroid hormone receptor beta (TRbeta) in Gal4 DNA binding domain vector (pGBT9). One done (named B1) to interact with TRbeta was isolated and considered to be a partial fragment of human nuclear receptor co-repressor (hN-CoR) by nucleotide sequencing. By colony hybridization technique and 5'-RACE, we cloned wild type and other three variants of hN-CoRs. One variant (hN-CoRv1) is 56 amino acids (a.a.) insertion in to a.a. 1018 of wild type N-CoR.The second one (hN-CoRv2) is a deletion of 120 a.a between a.a. 1857 and 1977. The third one is a carboxyterminal variant, in that 120 a.a. of wild type sequence is replaced with distinct 50 a.a..To study the chromosomal localization of hN-CoR, fluorescence in situ hybridization technique was performed using hN-Co … More R cDNA as a probe. Then the locus of hN-CoR was assigned to chromosome 17p11.2. Since hN-CoR localizes at one locus, the variants are considered to be generated by alternative splicings.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.Expression profile of N-CoR variants was analyzed in 8 human cell lines by RT-PCR method. The extracted RNA was reverse-transcribed and followed by the amplification of N-CoR variants with PCR.In all cell lines studied, mRNA expression of two N-CoR variants (hN-CoRv2 and v3) were detected minimally in comparison with Wild type. However, the expression of hN-CoRv1 was different in the cell lines. In three cell lines (NB-1 neuroblastoma cell line, Hela cervival carcinoma cell line and HOS osteosarcoma cell line), hN-CoRv1 was expressed predominant than wild type. The diffrent expression of hN-CoR variants might modify repression level by co-repressor and modulate hormonal signalings. Less

为了了解甲状腺激素作用的分子机制，我们利用酵母双杂交系统克隆了TR相互作用蛋白。利用人甲状腺激素受体β (trβ)在Gal4 DNA结合域载体（pGBT9）上筛选构建在Gal4激活域载体（pGAD-GH）上的Hela细胞cDNA文库。其中一个与TRbeta相互作用的分子（命名为B1）被分离出来，通过核苷酸测序认为是人类核受体共抑制因子（hN-CoR）的部分片段。利用集落杂交技术和5′-RACE技术，克隆了野生型和其他3种变异的hN-CoRs。其中一个变异（hN-CoRv1）是在野生型N-CoR的a.a.1018中插入56个氨基酸（a.a.a）。第二个（hN-CoRv2）是1857年至1977年之间的120 a.a的缺失。为了研究hN-CoR的染色体定位，以hN-Co . More R cDNA为探针，采用荧光原位杂交技术进行了研究。然后将hN-CoR定位在染色体17p11.2上。由于hN-CoR定位于一个位点，变体被认为是由备选剪接产生的。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。采用RT-PCR方法分析了N-CoR变异在8株人细胞系中的表达谱。提取的RNA逆转录，然后用PCR扩增N-CoR变异。在所研究的所有细胞系中，与野生型相比，两种N-CoR变体（hN-CoRv2和v3）的mRNA表达量最低。然而，hN-CoRv1在不同细胞系中的表达是不同的。在NB-1神经母细胞瘤细胞系、Hela宫颈癌细胞系和HOS骨肉瘤细胞系中，hN-CoRv1的表达高于野生型。hN-CoR变体的不同表达可能通过共抑制因子改变抑制水平并调节激素信号。少