Molecular modeling and in vitro investigations of the human androgen receptor DNA-binding domain: Application for the study of two mutations

Jean Marc Lobaccaro, Nicolas Poujol, Laurent Chiche, Serge Lumbroso, Terry R. Brown, Charles Sultan

Research output: Contribution to journalArticlepeer-review

Abstract

In two families with complete androgen insensitivity, we have identified naturally occurring point mutations in the human androgen receptor gene that encode amino acid substitutions within the DNA-binding domain. The two amino acid substitutions, a valine to phenylalanine and a leucine to proline, occur at positions 581 and 616, respectively, of the androgen receptor. The mutations were recreated by site-directed mutagenesis. Expression of the mutants androgen receptors in COS 7 and CV 1 cells revealed a normal size 110-kDa receptor protein on Western blots, normal androgen binding capacities and affinities, but absence of binding to target DNA on electrophoretic mobility shift assays. In cotransfection assays, the mutant ARs failed to activate transcription of an androgen-responsive reporter gene. To study the possible structural impact of these mutations, three-dimensional models of the normal androgen receptor and of each mutant were built by homology with the glucocorticoid receptor. Analysis of the models predicts that mutation Val581Phe 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 experimental investigations and the molecular modeling studies of the mutant androgen receptors observed in families with complete androgen insensitivity syndrome, highlight the role of Val-581 and Leu-616 in receptor structure and function.

Original languageEnglish (US)
Pages (from-to)137-147
Number of pages11
JournalMolecular and Cellular Endocrinology
Volume116
Issue number2
DOIs
StatePublished - Feb 5 1996

Keywords

  • Androgen receptor
  • Glucocorticoid receptor
  • Modelization
  • Structure-function relationship
  • Testicular feminization

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology

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