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Human Molecular Genetics Advance Access published online on January 31, 2006
Human Molecular Genetics, doi:10.1093/hmg/ddl009
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© The Author 2006. Published by Oxford University Press. All rights reserved
Received November 20, 2005
Revised January 27, 2006
Accepted January 27, 2006

Article

Impaired Helix 12 Dynamics due to Proline 892 Substitutions in the Androgen Receptor are Associated with Complete Androgen Insensitivity

Youssef A. Elhaji 1 *, Ileana Stoica 2, Sheldon Dennis 2, Enrico O. Purisima 2, and Mark A. Trifiro 3

1 Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Department of Human Genetics, McGill University, 3755 Cote-Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2; Department of Human Genetics, McGill University, Montreal, Quebec, Canada
2 Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada
3 Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec, Canada; Department of Human Genetics, McGill University, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada

* To whom correspondence should be addressed.
Youssef A. Elhaji, E-mail: youssef.elhaji{at}mail.mcgill.ca


  Abstract

Structural studies of the ligand binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand-binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic (MD) simulations to analyse two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome (CAIS).

Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand-binding and transactivational potential due to an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, while the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR but also in other nuclear receptors where this proline is conserved.


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