Generation of a mammalian cell line stably expressing a tetracycline-regulated epitope-tagged human androgen receptor: Implications for steroid hormone receptor research

The androgen receptor (AR) is hormone-activated transcription factor that r egulates the expression of genes involved in differentiation, development, and maintenance of male reproductive functions. To establish a useful model system for studying molecular mechanisms of AR action, we generated a HeLa -derived cell line (termed E19) that stably expresses human AR. Because ove rexpression of AR in cultured cells can be cytotoxic, we placed AR expressi on under the control of a tetracycline-regulated promoter. The stably expre ssed AR also contains an N-terminal FLAG-epitope tag (f:AR) that provides a n advantageous method for immunopurificatio. We show that f:AR expression i n E19 cells can be precisely modulated by varying the concentration of tetr acycline or its chemical derivative doxycycline in the growth media. The fu nctional activity of E19-expressed f:AR is demonstrated in vivo by its abil ity to activate transiently transfected AR reporter genes in an androgen-de pendent manner, and in vitro by its ability to specifically bind AR-respons e elements using DNA-mobility shift assays. We further show that f:AR in an drogen-stimulated E19 cells is markedly phosphorylated and coimmunopurifies with the transcriptional coactivator CREB-binding protein (CBP). The impli cations of these findings on steroid receptor research and the identificati on of receptor coregulatory factors will be discussed. (C) 2001 Academic Pr ess.