An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.
An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.
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DOI：
10.1093/nar/20.6.1355
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发表时间：
1992-03
影响因子：
14.9
通讯作者：
A. Morris;B. Kloss;R. E. McChesney;C. Bancroft;L. Chasin
中科院分区：
文献类型：
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作者：
A. Morris;B. Kloss;R. E. McChesney;C. Bancroft;L. Chasin
尽管选择性剪接已经被证明可以引起一些转录因子的异构体，但以前还没有在POU同源结构域蛋白Pit-1中检测到这样的异构体。通过对大鼠垂体GH3细胞表达文库的筛选，获得了一个编码35.8kD蛋白质(Pit-1a)的克隆，命名为pCMVPit-1a，在Pit-1反式激活结构域插入了26个氨基酸。插入片段的位置，加上Southern杂交分析，表明Pit-1a mRNA是通过Pit-1基因转录本的选择性剪接而产生的。在GH3大鼠垂体瘤细胞中检测到Pit-1a基因，其表达水平约为Pit-1基因的1/7。在大鼠和小鼠脑垂体以及小鼠甲状腺肿瘤TTT细胞中也检测到了PIT-1a的mRNA特异性序列。DNA迁移率改变分析表明，Pit-1a与催乳素启动子近端的Pit-1结合，但产生的DNA-蛋白质复合体的迁移率与Pit-1明显不同。在稳定转染的CHO细胞中，Pit-1反式激活了催乳素启动子驱动的CAT结构，而Pit-1a没有检测到反式激活，这意味着Pit-1a/Pit-1的反式激活比率低于0.05。因此，26个类似组成的氨基酸插入到Pit-1的激活域中，立即影响了该蛋白的结合方式和作为反式激活剂的能力。
Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.