Redox-dependent DNA binding of the purified androgen receptor: evidence for disulfide-linked androgen receptor dimers.
Full-length histidine-tagged, dihydrotestosterone-bound human androgen receptor (AR) was purified to homogeneity by affinity and gel-filtration chromatography for antibody production and analysis of AR dimerization and DNA binding properties. A monoclonal antibody was raised that recognized human and rat AR epitope (360)ArgAspTyrTyrAsnPheProLeuAla(368) in the NH(2)-terminal domain and slowed migration of AR-DNA complexes in mobility shift assays. AR binding to androgen response element DNA had a K(d) of 2.0 nM and a Hill coefficient of 2.1, indicating high-affinity, cooperative binding. AR solution dimerization was detected only at >/=0.2 microM AR, and DNA binding increased dimerization up to 30-fold. Slow- and fast-migrating AR-DNA complexes were detected under different reducing conditions that differed 5-fold in their dissociation rates from DNA. Treatment with the sulfhydryl oxidizing reagent diamide formed the faster migrating, slower dissociating complex, indicating it represents disulfide-linked AR dimers bound to DNA. The results indicate that high concentrations of purified AR are required for solution dimerization and that cooperative DNA binding stabilizes two dimer forms that differ in redox state.
Liao
M
M
Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill 27599, USA.
Zhou
Z x
Zx
Wilson
E M
EM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000235:genetics; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012996:Solutions; D018411:Spodoptera / Q000235:genetics
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D004220:Disulfides; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D012996:Solutions; D013196:Dihydrotestosterone; D006639:Histidine