Testosterone (T) and dihydrotestosterone (DHT) are natural ligands for the androgen receptor (AR), an intracellular transcription factor and nuclear receptor. DHT is a more potent androgen than T in vivo. In this study, the mechanistic basis for the differential effects of T and DHT on AR activity was investigated. Dissociation kinetics, motif binding affinity and activation function 2 (AF2) transactivation measurements reveal that the slow dissociation of DHT relative to T from AR results from weaker T-induced AR FXXLF motif binding to the AF2 site. T acquires DHT-like activity when the AR ligand binding domain (LBD) contains the H874Y somatic prostate cancer mutation that results in the formation of direct hydrogen bonds between external and core helices 4 and 5, improving AF2 motif binding. The studies reveal that DHT better stabilizes the AR LBD core from the ligand binding pocket to the AF2 surface for maximal AR transactivation. To further define the mechanisms whereby the AR specific coregulator melanoma antigen gene protein-A11 (MAGE-11) modulates AR activity, we pursued observations that MAGE-11 increases AR transcriptional activity independent of AF2. We sought to characterize the effects of MAGE-11 and the coactivators transcription intermediary factor 2 (TIF2) and p300 on AR transcriptional activity. The site of interaction in MAGE-11 that binds the AR FXXLF motif is an F-box within the MAGE homology domain. MAGE-11 Ser-174 is phosphorylated by MAP kinase which influences the interaction of the MAGE-11 F-box with AR. MAGE-11 forms a complex with TIF2 and p300 to modulate AR transactivation independent of AF2. This research provides evidence for a novel function for an F-box protein in which F-box/FXXLF like motif interactions modulate AR transcriptional activity in the absence and presence of ligand
