RFG (ARA70, ELE1) interacts with the human androgen receptor in a ligand-dependent fashion, but functions only weakly as a coactivator in cotransfection assays

Abnormalities of the human androgen receptor (hAR) cause a range of clinica l defects in male development. A large proportion of these mutations are si ngle amino acid substitutions in the hormone-binding domain (HBD) that alte r AR function by interfering with the capacity of the AR to bind androgen o r to form stable hormone-receptor complexes. Prior studies have suggested t hat the formation of such stable, active hormone-receptor complexes is a cr ucial step in the modulation of genes by the AR. It is presumed that these hormone-receptor complexes interact with other proteins to participate in t he formation of active transcription complexes at the initiation sites of a ndrogen-responsive genes. Using a yeast two-hybrid screening method, we isolated a partial cDNA encod ing the carboxy terminus of a protein that interacts with the hAR-HBD (amin o acid residues 623-917) in a ligand-dependent fashion in a yeast two-hybri d assay. Sequence analysis of this clone revealed that it encoded a portion of a protein that had been previously characterized as RFG (RET Fused Gene ). Using glutathione-S-transferase (GST) fusions of the hAR HBD and immunop recipitation of the in vitro translated proteins, we have demonstrated that this interaction can be reproduced in vitro. To determine the capacity of this protein to modulate the activity of the AR in transfection assays, we expressed full-length RFG in the CV1 and DU145 cell lines, in combination w ith an AR expression vector and model androgen-responsive genes [mouse mamm ary tumor virus (MMTV) and PRE2-tk luciferase]. Our results demonstrate tha t RFG alters the induction of these reporter genes very weakly (no greater than 2-fold compared with transfections without the RFG expression plasmid) . Thus, while our findings are in agreement with published reports which in dicate that RFG interacts with AR-HBD in a ligand-dependent fashion, in our assays RFG does not exert major effects on the activity of the hAR in resp onse to androgen or to other steroid hormones.