IN-VITRO TRANSLATION OF ANDROGEN RECEPTOR CRNA RESULTS IN AN ACTIVATED ANDROGEN RECEPTOR PROTEIN

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate an d subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot an alysis revealed a single binding component with high affinity for R188 1 (K(d) = 0.3 nM). All AR molecules synthesized specifically bound ste roid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [H-3]R1881 and analysed on sucrose-d ensity gradients, a complex of approx. 6 S was observed. The complex w as shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-bindi ng domain. In the presence as well as the absence of hormone, AR molec ules were able to bind to DNA-cellulose without an activation step. Ge l retardation assays revealed that the AR forms complexes with a DNA e lement containing glucocorticoid-responsive element/androgen-responsiv e element sequences. Receptor-DNA interactions were stabilized by diff erent polyclonal antibodies directed against either the N- or C-termin al part of the AR and were abolished by an antibody directed against t he DNA-binding domain of the receptor. In conclusion, translation of A R cRNA in vitro yields an activated AR protein which binds steroid wit h high affinity. It is proposed that AR antibodies enhance AR-DNA bind ing by stabilizing AR dimers when bound to DNA.