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 regulates 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 overexpression of AR in cultured cells can be cytotoxic, we placed AR expression under the control of a tetracycline-regulated promoter. The stably expressed AR also contains an N-terminal FLAG-epitope tag (f:AR) that provides an advantageous method for immunopurification. We show that f:AR expression in E19 cells can be precisely modulated by varying the concentration of tetracycline or its chemical derivative doxycycline in the growth media. The functional activity of E19-expressed f:AR is demonstrated in vivo by its ability to activate transiently transfected AR reporter genes in an androgen-dependent manner, and in vitro by its ability to specifically bind AR-response elements using DNA-mobility shift assays. We further show that f:AR in androgen-stimulated E19 cells is markedly phosphorylated and coimmunopurifies with the transcriptional coactivator CREB-binding protein (CBP). The implications of these findings on steroid receptor research and the identification of receptor coregulatory factors will be discussed.