Functional analysis of thyroid hormone receptor interacting protein

为了从分子水平了解甲状腺激素的作用，我们利用酵母双杂交系统克隆了甲状腺激素受体TR相互作用蛋白。用Gal 4 DNA结合域载体（pGBT 9）中的人甲状腺激素受体β（TRbeta）筛选Gal 4激活域载体（pGAD-GH）构建的Hela细胞cDNA文库。分离出一个与TRbeta相互作用的片段（命名为B1），并通过核苷酸测序认为其是人核受体辅阻遏物（hN-CoR）的部分片段。利用菌落杂交技术和5 '-RACE技术，我们克隆了野生型和其它三种hN-CoRs变异体。一种变体（hN-CoRvl）是56个氨基酸（a.a.）插入a. a.第二个（hN-CoRv 2）是野生型N-CoR的120 a.a. 1857年和1977年。第三个是羧基末端变体，在120 a.a.用不同的50个a.a.替换野生型序列。为研究hN-CoR的染色体定位，应用荧光原位杂交技术， ...更多信息 R cDNA作为探针。将hN-CoR基因定位于染色体17p11.2。由于hN-CoR定位于一个位点，因此认为变异体是通过选择性剪接产生的，由于mN-CoR通过其羧基端区域与TR和视黄酸受体（RAR）相互作用，因此N-CoR变异体（克隆H3）羧基端的改变可能影响受体相互作用的特异性。N-CoR异构体的存在为不同表达的N-CoR异构体参与甲状腺激素的转录调控提供了可能。在所有研究的细胞系中，与野生型相比，检测到两种N-CoR变体（hN-CoRv 2和v3）的mRNA表达最低。然而，hN-CoRv 1在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-CoRv 1的表达均高于野生型。hN-CoR变异体的不同表达可能改变了辅阻遏物的阻遏水平，调节了激素信号传导。少

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