Transcriptional activation and nuclear targeting signals of the human androgen receptor.
Transcriptional activation and nuclear targeting signals of the human androgen receptor.
复制标题
DOI：
10.1016/s0021-9258(18)52466-2
复制
发表时间：
1991-01
期刊：
影响因子：
--
通讯作者：
J. Simental;Madhabananda Sar;Malcolm V. Lane;Frank S. French;E. M. Wilson
中科院分区：
文献类型：
--
作者：
J. Simental;Madhabananda Sar;Malcolm V. Lane;Frank S. French;E. M. Wilson
雄激素受体(AR)是一种信号转导蛋白，在男性表型的性别分化、发育和表达中是必需的。通过使用限制性内切酶的定点突变、聚合酶链式反应，或者对于一系列NH2末端的单向缺失，通过外切酶III酶切，创建了一系列人AR缺失突变体。免疫印迹显示，受体突变体在猴肾COS细胞中以截短的AR蛋白形式表达，截短的AR蛋白大小在20~107 kDa之间，其中野生型AR为114~108 kDa的双重体。免疫细胞化学染色的亚细胞定位显示雄激素依赖的核内AR从细胞质的核周区域摄取。雄激素诱导野生型AR的核摄取需要一个与糖皮质激素受体序列和位置相似、与SV40大T抗原同源的核靶向信号。缺乏NH2末端和/或类固醇结合区的AR突变体是结构性核，转录活性降低。在使用与小鼠乳腺肿瘤病毒启动子相连的氯霉素乙酰转移酶报告基因对CV1细胞的共转染研究中，野生型AR的转录激活依赖于雄激素。缺失突变发现，在NH2末端区域有一个完整转录活性所需的结构域，而在类固醇结合结构域中，有一种抑制功能，删除它会产生一个结构性的活性受体。通过与一个非活性的NH2末端片段共表达抑制野生型AR，表明竞争转录调控所需的核因子。这些研究表明，AR蛋白的结构域在调节基因转录方面存在协调的相互作用。
The androgen receptor (AR) is a signal-transducing protein required for sexual differentiation, development, and expression of the male phenotype. A series of human AR deletion mutants were created either by site-directed mutagenesis using restriction enzyme digestion, the polymerase chain reaction, or, for a series of unidirectional NH2-terminal deletions, exonuclease III digestion. Receptor mutants were expressed in monkey kidney COS cells as truncated AR proteins between 20 and 107 kDa as revealed on immunoblots, where wild type AR was a doublet of 114 and 108 kDa. Subcellular localization by immunocytochemical staining demonstrated androgen-dependent nuclear uptake of AR from a perinuclear region of the cytoplasm. A nuclear targeting signal similar in sequence and position to the glucocorticoid receptor and homologous to the SV40 large T antigen was required for androgen-induced nuclear uptake of wild type AR. AR mutants lacking the NH2-terminal and/or steroid binding domains were constitutively nuclear with reduced transcriptional activity. Transcriptional activation by wild type AR was androgen-dependent in cotransfection studies of CV1 cells using the chloramphenicol acetyltransferase reporter gene linked to the mouse mammary tumor virus promoter. Deletion mutagenesis revealed within the NH2-terminal region a domain required for full transcriptional activity and within the steroid binding domain, an inhibitory function, deletion of which yielded a constitutively active receptor. Inhibition of wild type AR by coexpression with an inactive NH2-terminal fragment suggested competition for nuclear factors required for transcriptional regulation. These studies demonstrate a concerted interplay among the domains of the AR protein in regulating gene transcription.