Domain interactions between coregulator ARA70 and the androgen receptor (AR)

The coregulator function of AR-associated protein 70 (ARA(70)) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA(70) deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA(70) amino acids 321-441 of the 614 amino acid ARA(70) protein. Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays. Yeast one-hybrid assays revealed an intrinsic ARA(70) transcriptional activation domain within amino acids 296-441. In yeast assays the ARA(70) domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA(70). Full-length ARA(70) increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA(70) also increased constitutive transcriptional activity of an AR NH2-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays. Binding was independent of the ARA(70) FxxLF motif. The results identify an ARA(70) motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA(70) can interact with the NH2-terminal and carboxyl-terminal regions of AR.