MOLECULAR MODELING AND IN-VITRO INVESTIGATIONS OF THE HUMAN ANDROGEN RECEPTOR DNA-BINDING DOMAIN - APPLICATION FOR THE STUDY OF 2 MUTATIONS

In two families with complete androgen insensitivity, we have identifi ed naturally occurring point mutations in the human androgen receptor gene that encode amino acid substitutions within the DNA-binding domai n. The two amino acid substitutions. a valine to phenylalanine and a l eucine to proline, occur at positions 581 and 616, respectively, of th e androgen receptor. The mutations were recreated by site-directed mut agenesis. Expression of the mutants androgen receptors in COS 7 and CV 1 cells revealed a normal size 110-kDa receptor protein on Western bl ots, normal androgen binding capacities and affinities, but absence of binding to target DNA on electrophoretic mobility shift assays. In co transfection assays, the mutant ARs failed to activate transcription o f an androgen-responsive reporter gene. To study the possible structur al impact of these mutations, three-dimensional models of the normal a ndrogen receptor and of each mutant were built by homology with the gl ucocorticoid receptor. Analysis of the models predicts that mutation V al581Phe would affect interaction between the protein and DNA, whereas the Leu616Pro mutation would more likely be involved in destabilizing the protein structure or protein dimerization. Taken together, the ex perimental investigations and the molecular modeling studies of the mu tant androgen receptors observed in families with complete androgen in sensitivity syndrome, highlight the role of Val-581 and Leu-616 in rec eptor structure and function.