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Cross-talk among nuclear receptor heterodimers

核受体异二聚体之间的串扰

基本信息

批准号：
08671145
负责人：
MIYAMOTO Takahide
金额：
$ 1.6万
依托单位：
Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

The thyroid hormone receptors (TR) are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related steroid/thyroid hormnone receptor superfamily, TRs bind to cis-acting DNA elements as heterodimers with the retinoid X receptors (RXR). These helerodimers display distinct specificities to mediate the hormonal response to target gene transcription. We characterized the interaction between TRalphal and RXRalpha via their ligand binding domains (LBD) and the effect of ligands on the interaction using a yeast two-hybrid system. The DNA binding domain (BD) of yeast Ga14 fusion to the LBD of TRalpha had no transcriptional activity on its own but, when co-expressed with the activation domain (AD) of yeast Ga14 fusion to LBD of RXR"a" conferred activation to a reporter gene harboring Ga14 binding site, indicating that LBDs of TRalpha1 and RXRalpha interact each other in solution. Furthermore, T3 and 9cis-RA increased the reporter activity and synergistic effect was observed when both ligands were added, indicating that the TRalpha/RXRalpha heterodimerization is augmented by their respective ligands in vivo. Using an in vitro pull down experiment, we confirmed the ligand dependent interaction observed in the yeast system. Matrix bound GST-RXRalpha specifically co-precipitated the ^<35>S-labeled TRalpha1 above the control and associated ^<35>S-labeled TRalpha1 was increased by addition of T3 and 9cis RA.This suggests that T3 induced -confrontational changes of TR required for the ligand dependent heterodimerization. These results imply a complex, sensitive cross-talk in vivo among nuclear receptors and their respective ligands through distinct hormonal signaling pathways.

甲状腺激素受体是一类配体依赖、DNA结合、反式作用的转录因子，属于ERBA相关的类固醇/甲状腺激素受体超家族，与顺式作用的DNA元件作为异源二聚体与维甲酸X受体结合。这些分子二聚体表现出明显的特异性，以调节荷尔蒙对靶基因转录的反应。我们利用酵母双杂交系统，通过配体结合域(LBD)表征了Tralphal和RXRpha之间的相互作用，以及配体对相互作用的影响。酵母Ga14与RXRαLBD的DNA结合域(BD)本身没有转录活性，但当与RXR“a”LBD的激活域(AD)共表达时，激活了一个含有Ga14结合位点的报告基因，表明TRAlpha1和RXRpha的LBD在溶液中相互作用。此外，T3和9cis-RA增加了报告基因的活性，并且两种配体的加入都有协同作用，说明它们各自的配体在体内都增强了Trpha/RXRpha的异源二聚作用。通过体外下拉实验，我们证实了在酵母系统中观察到的配体依赖的相互作用。与基质结合的GST-RXRα特异性地共沉淀了~&lt；35&gt；S标记的TRalpha1和相关的^&lt；35&gt；S标记的TRalpha1，随着T_3和9cis RA的加入而增加。这表明T_3诱导的对抗性改变是配体依赖的异源二聚反应所需的。这些结果表明，在体内，核受体和它们各自的配体之间通过不同的激素信号通路存在复杂而敏感的串扰。