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.
Wang
Q
Q
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Fondell
J D
JD
D015153:Blotting, Western; D050882:CREB-Binding Protein; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011944:Receptors, Androgen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013752:Tetracycline / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D000939:Epitopes; D009687:Nuclear Proteins; D011944:Receptors, Androgen; D015534:Trans-Activators; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D013752:Tetracycline