Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors.
Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors.
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酵母激素反应元件测定可检测并表征甲状腺和类视黄醇核受体对 GRIP1 共激活剂依赖性转录的激活。
DOI：
10.1073/pnas.94.8.3697
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发表时间：
1997
影响因子：
11.1
通讯作者：
Stallcup,MR
中科院分区：
文献类型：
--
作者：
Walfish,PG;Yoganathan,T;Yang,YF;Hong,H;Butt,TR;Stallcup,MR
小鼠糖皮质激素受体相互作用蛋白(GRIP1)是核受体(NR)共激活子(包括SRC-1和TIF2)ERAP160家族中的一员，在与同源激素反应元件(HRE)结合的配体激活的NR和转录起始器(TIA)之间起着桥梁蛋白的作用。尽管这些共激活子与几个NRs结合，但在哺乳动物细胞中过度表达这些共激活子和这些NRs的研究并没有一致地观察到相应的配体依赖反式激活的增强。在这里，我们发现GRIP1以配体依赖的方式与甲状腺受体、维甲酸受体和维甲酸X受体相互作用。此外，在酵母(酿酒酵母)中，GRIP1共激活蛋白显著增加了这些全长II类NR反式激活含有同源HRE的β-半乳糖苷酶报告基因的能力。连接的NR同源二聚体对GRIP1的增强程度取决于NR亚型和HRE构型。对于大多数HRE构型，甲状腺受体和维甲酸受体同源二聚体在缺乏GRIP1的情况下基本上没有反应或非常弱的活性，但GRIP1显著恢复了这些NRs的配体依赖功能。虽然GRIP1在没有同源配体的情况下对NR同源二聚体没有显著影响，但它增加了未连接的NR异源二聚体的反式激活。GRIP1是否将异源二聚体的配体依赖反式激活增加到高于同源同源二聚体的水平，取决于HRE构型和拷贝数。与酵母双杂交系统和哺乳动物共表达系统的局限性相比，本报告中描述的酵母HRE检测系统既有助于检测哺乳动物的NR辅助激活功能，也有助于阐明其反式激活增强的机制。
The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interactsin vitroin a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate β-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.