Redox-dependent DNA binding of the purified androgen receptor: Evidence for disulfide-linked androgen receptor dimers

Full-length histidine-tagged, dihydrorestosterone-bound human androgen rece ptor (AR) was purified to homogeneity by affinity and gel-filtration chroma tography for antibody production and analysis of AR dimerization and DNA bi nding properties. A monoclonal antibody was raised that recognized human an d rat AR epitope (360)ArgAspTyrTyrAsnPheProLeuAla(368) in the NH2-terminal domain and slowed migration of AR-DNA complexes in mobility shift assays. A R binding to androgen response element DNA had a K-d of 2.0 nM and a Hill c oefficient of 2.1, indicating high-affinity, cooperative binding. AR soluti on dimerization was detected only at greater than or equal to 0.2 mu M AR, and DNA binding increased dimerization up to 30-fold. Slow- and fast-migrat ing AR-DNA complexes were detected under different reducing conditions that differed 5-fold in their dissociation rates from DNA. Treatment with the s ulfhydryl oxidizing reagent diamide formed the faster migrating, slower dis sociating complex, indicating it represents disulfide-linked AR dimers boun d to DNA. The results indicate that high concentrations of purified AR are required for solution dimerization and that cooperative DNA binding stabili zes two dimer forms that differ in redox state.