The coregulator function of AR-associated protein 70 (ARA70) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA70 deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA70 amino acids 321-441 of the 614 amino acid ARA70 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 ARA70 transcriptional activation domain within amino acids 296-441. In yeast assays the ARA70 domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA70. Full-length ARA70 increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA70 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 ARA70 FxxLF motif. The results identify an ARA70 motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA70 can interact with the NH2-terminal and carboxyl-terminal regions of AR.
ASJC Scopus subject areas
- Molecular Biology