Cross-talk among nuclear receptor heterodimers

甲状腺激素受体（TR）是一种配体依赖性、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，TR与维甲酸X受体（RXR）以异二聚体的形式与顺式作用的DNA元件结合。这些helerodimer显示不同的特异性，以介导靶基因转录的激素反应。我们利用酵母双杂交系统研究了TRbal和RXR α通过其配体结合域（LBD）的相互作用以及配体对相互作用的影响。与TRalpha的LBD融合的酵母Ga 14的DNA结合结构域（BD）本身没有转录活性，但是当与与RXR "a"的LBD融合的酵母Ga 14的激活结构域（AD）共表达时，赋予携带Ga 14结合位点的报告基因激活，表明TRalpha 1和RXR alpha的LBD在溶液中彼此相互作用。此外，T3和9cis-RA增加了报告活性，并且当添加两种配体时观察到协同效应，表明TR α/RXR α异源二聚化在体内被它们各自的配体增强。使用体外下拉实验，我们证实了在酵母系统中观察到的配体依赖性相互作用。基质结合的GST-RXR α特异性共沉淀的1S标记的TR α 1高于对照组，并且通过加入T3和9cis RA增加了相关的1S标记的TR α 1。<35><35>这些结果意味着一个复杂的，敏感的串扰在体内核受体和它们各自的配体通过不同的激素信号通路。

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.