Interaction of the putative androgen receptor-specific coactivator ARA(70)/ELE1 alpha with multiple steroid receptors and identification of an internally deleted ELE1 beta isoform

Steroid-regulated gene transcription requires the coordinate physical and f unctional interaction of hormone receptors, basal transcription factors, an d transcriptional coactivators. In this context ARA(70), previously called RFG and ELE1, has been described as a putative coactivator that specificall y enhances the activity of the androgen receptor (AR) but not that of the g lucocorticoid receptor (GR), the progesterone receptor, or the estrogen rec eptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA(70) 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, howev er, an 860-bp PCR product was obtained. This shorter variant results from a n internal 985-bp deletion and is called ELE1 beta; accordingly, the longer isoform is referred to as ELE1 alpha. The deduced amino acid sequence of E LE1 alpha, but not that of ELE1 beta, differs at specific positions from th e one previously published by others, suggesting that these two proteins ar e encoded by different nonallelic genes. ELE1 alpha is expressed in the thr ee prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examin ed, ELE1 alpha expression is highest in the testis. This is also the only t issue in which we could demonstrate ELE1 beta expression. Both ELE1 alpha a nd ELE1 beta 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 activ ity of the human AR. ELE1 alpha has no intrinsic transcription activation d omain or histone acetyltransferase activity, but it does interact with anot her histone acetyltransferase, p/CAF, and the basal transcription factor TF IIB. The interaction with the AR occurs through the ligand-binding domain a nd 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 ELE1 alpha but has only moderate effects on its transcriptional acti vity. Taken together, we have identified two isoforms of the putative coact ivator ARA(70)/ELE1 that may act as a bridging factor between steroid recep tors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Furt her experiments will be required to highlight the in vivo role of ELE1 in n uclear receptor functioning.