Characterization and identification of a steroid receptor-binding protein, SRB-RGS
Characterization and identification of a steroid receptor-binding protein, SRB-RGS
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DOI：
10.1248/bpb.30.1056
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发表时间：
2007-06-01
影响因子：
2
通讯作者：
Minatogawa, Yohsuke
中科院分区：
文献类型：
--
作者：
Ikeda, Mitsunori;Inoue, Satoshi;Minatogawa, Yohsuke
我们克隆了一种新的类固醇受体结合蛋白SRB-RGS的cDNA，该蛋白抑制雌激素受体（ER）α介导的和其他启动子驱动的转录活性。这项研究通过免疫共沉淀试验揭示了全长SRB-RGS与全长ER α或ER β之间的相互作用。全长SRB-RGS和全长ER α在COS-7细胞中通过哺乳动物双杂交系统相互作用。用凝胶位移法观察了大鼠子宫内质网提取液中内源性SRB-RGS与内质网的相互作用。这些结果有力地表明，SRB-RGS与结合到细胞核中的DNA（雌激素反应元件）的ER相互作用。SRB-RGS非常有效地抑制ER α、ER β和ER α +ER β介导的转录活性。增强型绿色荧光蛋白（EGFP）标记的SRB-RGS的绿色荧光定位于细胞核和细胞质中。HeLa细胞的胞核和胞浆中均可见内源性SRB-RGS免疫染色。用抗SRB-RGS抗体进行免疫染色和Western blotting，对推测的SRB-RGS进行鉴定。过表达SRB-RGS可诱导HeLa细胞死亡。我们以前克隆的SRB-RGS cDNA的核苷酸序列与新分离的RGS 3 cDNA的核苷酸序列完全相同。SRB-RGS可与细胞核内的ERs结合DNA相互作用，抑制ERs介导的转录活性。
We cloned the cDNA of a novel steroid receptor-bin ding protein, SRB-RGS, which suppressed the estrogen receptor (ER)alpha-mediated and other promoter-driven transcriptional activities. This study revealed the interaction between the full-length SRB-RGS and full-length ER alpha or ER beta by a coimmunoprecipitation assay. The full-length SRB-RGS and full-length ER alpha interacted in COS-7 cell by a mammalian two-hybrid system. The interaction between intrinsic SRB-RGS and ERs in the nuclear ER extract from the rat uteri was observed by the gel-shift assay. These results strongly suggested that SRB-RGS interacts with ERs bound to DNA (estrogen response element) in the nuclei of the cells. SRB-RGS suppressed very efficiently the ER alpha-, ER beta-, and ER alpha+ER beta-mediated transcriptional activities. Green fluorescence of enhanced green fluorescence protein (EGFP)-tagged SRB-RGS was localized both in the nucleus and in the cytoplasm. Intrinsic SRB-RGS was immunostained in the nucleus and the cytoplasm of HeLa cells. The putative SRB-RGS deduced from cDNA sequence was identified by the immunostaining and Western blotting by using the anti-SRB-RGS antibody. Overexpression of SRB-RGS induced the cell death in the HeLa cells. The nucleotide sequence of SRB-RGS cDNA that we cloned previously is identical with that of the newly isolated RGS3 cDNA. SRB-RGS could interact with ERs bound DNA in the nuclei of the cells and suppressed the ERs-mediated transcriptional activities.