Structural functional analysis of the human androgen receptor

The androgen receptor (AR) mediates the various actions of androgens and is responsible for the male reproductive system and male phenotype. AR mutations result in variable degrees of androgen insensitivity in XY individuals, ranging from complete to mild androgen insensitivity syndrome (AIS). Most of the AR-mutations are located in the ligand-binding-domain (LBD) and result in a complete or near complete loss of ligand binding. As revealed by several AR-LBD crystal structures, most of these mutations are located far from the bound-ligand, therefore their severe ligand-binding abnormalities must reflect more widespread LBD structural aberrations rather than local distortions at the mutated residues.To better understand the structural/functional relationship of such mutations, I have characterized the abnormalities associated with alternate substitutions at two identical positions in the AR-LBD (R855H and R855C) and (P892A and P892L) identified in unrelated AIS patients. Results of these analyses have explained the mechanism by which the AR-R855H and AR-R855C resulted in significantly different AIS phenotypes. These studies also revealed that the AR-P892 residue is critical for the dynamic properties of the C-terminal helix (helix 12) of the LBD. Helix 12 dynamic properties are crucial for ligand-binding and for the transactivational properties of the receptor.To further analyse the properties of the AR-LBD within the context of multiple domains, I have developed a high-yield bacterial-expression method for the production of soluble AR fragment that contains the DNA-binding-domain, the hinge region and the LBD (AR-DBDLBD). This will allow for detailed analyses of the structural and functional properties of these domains and their intra-molecular communications in the presence and absence of DNA and/or ligand. This method is likely to be generally applicable to other nuclear receptors and may resolve the low solubility, instability, and toxicity problems associated with their overexpression.Furthermore, I have reached the final stages in generating a knockout mouse for a newly identified AR-coregulator, the Androgen Receptor N-terminal- Interacting Protein (ARNIP)

Androgen receptor mutation in breast cancer

Normal breast growth and development depends on functional androgen:estrogen (A:E) balance. Androgen actions are mediated by the androgen receptor (AR), a DNA-binding, transcriptional-regulatory protein. Decreased AR transactivational. activity lowers A:E balance and may result in functional hyperestrogenicity: this could promote the pathogenesis of breast cancer (BC). The present study is the first to seek AR mutations in female BC. The length of the polymorphic CAG-repeat in exon 1 of the AR correlates inversely with the transactivational activity of the AR. Using 10% polyacrylamide gels, I found a significant (p < 0.0001) shift to greater CAG-repeat lengths in BC samples. This suggests a role for ARs with long polyglutamine tracts in the initiation and/or progression of BC. Exons 2--8 of the AR in 81 fresh frozen BC tumor tissues were screened for mutations using SSCP analysis. I did not detect any mutations in these exons

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity

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 simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. 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 because of 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, whereas 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.NRC publication: Ye