Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action.

Limited proteolysis of in vitro produced human androgen receptor was used to probe the different conformations of the receptor after binding of androgens and several antiandrogens. The results provide evidence for five different conformations of the receptor, as detected by the formation of proteolysis resisting fragments: 1) an initial conformation of the unoccupied receptor not resisting proteolytic attack; and receptor conformations characterized by 2) a 35-kDa proteolysis resisting fragment spanning the ligand binding domain and part of the hinge region, obtained with most antagonists, and in an initial step after agonist binding; 3) a 29-kDa proteolysis resisting fragment spanning the ligand binding domain, obtained in the presence of agonists after an activation process; 4 and 5) 30- and 25-kDa fragments, derived from 2 and 3, but missing part of the C terminus, obtained with RU486 (RU486 has antiandrogenic properties, besides its effects as an antiprogestagen/antiglucocorticoid). Concomitantly with the change from 2 to 3 (and of 4 to 5 for RU486), dissociation of the 8 S complex of receptor with associated proteins occurred. With a mutant receptor (LNCaP cell mutation in C-terminal region), some antagonists activated transcription analogous to agonists, and induced the activated receptor conformation 3. A mutant lacking the C-terminal 12 amino acids bound RU486 but not androgens, and formed with RU486 conformation 5. These data imply that, after the initial rapid binding of ligand, androgens induce a conformational change of the receptor, a process that also involves release of associated proteins. RU486 induces an inappropriate conformation of the C-terminal end, similar as found for its effect on the progesterone receptor. In contrast, the other antiandrogens act at a different step in the mechanism of action: they do not induce an abnormal conformation, but act earlier and prevent a conformation change by stabilizing a complex with associated proteins.