Zhu X G, Hanover J A, Hager G L, Cheng S Y
Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1998 Oct 16;273(42):27058-63. doi: 10.1074/jbc.273.42.27058.
Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors that regulate growth, differentiation, and development. To understand the role of the hormone, 3,3', 5-triiodo-L-thyronine (T3), in the nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we appended green fluorescent protein (GFP) to the human TR subtype beta1 (TRbeta1). The fusion of GFP to the amino terminus of TRbeta1 protein did not alter T3 binding or transcriptional activities of the receptor. The subcellular localization of GFP-TRbeta1 in living cells was visualized by laser-scanning confocal microscopy. In the presence of T3, the expressed GFP-TRbeta1 was predominately localized in the nucleus, exhibiting a nuclear/cytoplasmic ratio of approximately 5.5. No GFP-TRbeta1 was detected in the nucleolus. In the absence of T3, more GFP-TRbeta1 was present in the cytoplasm, exhibiting a nuclear/cytoplasmic ratio of approximately 1.5. In these cells, cytoplasmic GFP-TRbeta1 could be induced to enter the nucleus by T3. The T3-induced translocation was blocked when Lys184-Arg185 in domain D of TRbeta1 was mutated to Ala184-Ala185. Furthermore, the inability of the mutant TR to translocate to the nucleus correlated with the loss of most of its transcriptional activity. These results suggest that TR functions may, in part, be regulated by T3-induced nuclear entry.
甲状腺激素核受体（TRs）是依赖配体的转录因子，可调节生长、分化和发育。为了了解激素3,3',5-三碘-L-甲状腺原氨酸（T3）在TRs向染色质调控位点的核转位和靶向中的作用，我们将绿色荧光蛋白（GFP）附加到人TR亚型β1（TRβ1）上。GFP与TRβ1蛋白的氨基末端融合并不改变受体的T3结合或转录活性。通过激光扫描共聚焦显微镜观察活细胞中GFP-TRβ1的亚细胞定位。在T3存在的情况下，表达的GFP-TRβ1主要定位于细胞核，核/质比约为5.5。在核仁中未检测到GFP-TRβ1。在没有T3的情况下，更多的GFP-TRβ1存在于细胞质中，核/质比约为1.5。在这些细胞中，细胞质中的GFP-TRβ1可被T3诱导进入细胞核。当TRβ1的D结构域中的Lys184-Arg185突变为Ala184-Ala185时，T3诱导的转位被阻断。此外，突变型TR无法转位至细胞核与其大部分转录活性的丧失相关。这些结果表明，TR的功能可能部分受T3诱导的核进入调控。
