Alen P, Claessens F, Schoenmakers E, Swinnen J V, Verhoeven G, Rombauts W, Peeters B
Division of Biochemistry, Faculty of Medicine, Campus Gasthuisberg, University of Leuven, Belgium.
Mol Endocrinol. 1999 Jan;13(1):117-28. doi: 10.1210/mend.13.1.0214.
Steroid-regulated gene transcription requires the coordinate physical and functional interaction of hormone receptors, basal transcription factors, and transcriptional coactivators. In this context ARA70, previously called RFG and ELE1, has been described as a putative coactivator that specifically enhances the activity of the androgen receptor (AR) but not that of the glucocorticoid receptor (GR), the progesterone receptor, or the estrogen receptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA70 by RT-PCR from RNA derived from different cell lines (HeLa, DU-145, and LNCaP). In accordance with the previously described sequence, we obtained a 1845-bp PCR product for the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp PCR product was obtained. This shorter variant results from an internal 985-bp deletion and is called ELE1beta; accordingly, the longer isoform is referred to as ELE1alpha. The deduced amino acid sequence of ELE1alpha, but not that of ELE1beta, differs at specific positions from the one previously published by others, suggesting that these two proteins are encoded by different nonallelic genes. ELE1alpha is expressed in the three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examined, ELE1alpha expression is highest in the testis. This is also the only tissue in which we could demonstrate ELE1beta expression. Both ELE1alpha and ELE1beta interact in vitro with the AR, but also with the GR and the ER, in a ligand-independent way. Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had only minor effects on the transcriptional activity of the human AR. ELE1alpha has no intrinsic transcription activation domain or histone acetyltransferase activity, but it does interact with another histone acetyltransferase, p/CAF, and the basal transcription factor TFIIB. The interaction with the AR occurs through the ligand-binding domain and involves the region corresponding to the predicted helix 3. Mutation in this domain of leucine 712 to arginine greatly reduces the affinity of the AR for ELE1alpha but has only moderate effects on its transcriptional activity. Taken together, we have identified two isoforms of the putative coactivator ARA70/ELE1 that may act as a bridging factor between steroid receptors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Further experiments will be required to highlight the in vivo role of ELE1 in nuclear receptor functioning.
类固醇调节的基因转录需要激素受体、基础转录因子和转录共激活因子在物理和功能上的协同相互作用。在这种情况下，ARA70（以前称为RFG和ELE1）已被描述为一种假定的共激活因子，它能特异性增强雄激素受体（AR）的活性，但不能增强糖皮质激素受体（GR）、孕激素受体或雌激素受体（ER）的活性。在这里，我们描述了通过RT-PCR从不同细胞系（HeLa、DU-145和LNCaP）来源的RNA中克隆ELE1/ARA70的cDNA。与先前描述的序列一致，我们从HeLa和LNCaP RNA中获得了一个1845 bp的PCR产物。然而，从T-47D RNA开始，获得了一个860 bp的PCR产物。这个较短的变体是由内部985 bp的缺失导致的，被称为ELE1β；相应地，较长的异构体被称为ELE1α。ELE1α推导的氨基酸序列在特定位置与其他人先前发表的序列不同，而ELE1β的则没有，这表明这两种蛋白质由不同的非等位基因编码。ELE1α在检测的三种前列腺来源的细胞系（PC-3、DU-145和LNCaP）中表达，并且这种表达不受雄激素处理的影响。在所有检测的大鼠组织中，ELE1α在睾丸中的表达最高。这也是我们能够证明ELE1β表达的唯一组织。ELE1α和ELE1β在体外均以配体非依赖的方式与AR相互作用，也与GR和ER相互作用。在DU-145、HeLa或COS细胞中过表达任何一种ELE1异构体对人AR的转录活性只有轻微影响。ELE1α没有内在的转录激活结构域或组蛋白乙酰转移酶活性，但它确实与另一种组蛋白乙酰转移酶p/CAF以及基础转录因子TFIIB相互作用。与AR的相互作用通过配体结合结构域发生，涉及对应于预测的螺旋3的区域。该结构域中亮氨酸712突变为精氨酸大大降低了AR对ELE1α的亲和力，但对其转录活性只有中等影响。综上所述，我们鉴定出了假定的共激活因子ARA70/ELE1的两种异构体，它们可能作为类固醇受体与转录起始复合物成分之间的桥梁因子，但缺乏经典核受体共激活因子的一些基本特性。需要进一步的实验来突出ELE1在核受体功能中的体内作用。
