Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains

Abstract

Most mutations in the androgen receptor (AR) ligand-binding domain (LBD) disrupt binding of the natural ligands: dihydrotestosterone and testosterone. Some AR LBD mutations do not affect ligand binding but they disrupt androgen-induced interaction of the N-terminal motif FXXLF and C-terminal activation function 2 (AF2). As N-/C-terminal interaction requires binding of agonists that have androgen activity in vivo, it correlates well with the phenotype. To study this further, we searched the Cambridge intersex database for patients with a detected missense mutation in the AR LBD presenting with normal ligand binding. Six mutations (D695N, Y763C, R774H, Q798E, R855H and L907F) were selected and introduced by site-directed mutagenesis into the pSVAR and pM-LBD plasmids. The transactivational potential of the wild-type and mutant androgen receptors (pSVAR) was examined by dual-luciferase assay using pGRE-LUC as a reporter vector. N-/C-terminal interaction was studied by mammalian two-hybrid assay using wild-type and mutated AR LBD (pM-LBD), pVP16-rAR-(5-538) (encoding rat amino-terminal AR) and pCMX-UAS-TK-LUC as a reporter. AR LBD mutations D695N, R774H and L907F presented with minimal transactivational capacity and N-/C-terminal interaction was totally disrupted. Mutations Y763C and R885H had some residual dose-dependent transactivational potential and minimal N-/C-terminal interaction. Q798E presented with good transactivational potential and it showed only mild reduction in N-/C-terminal interaction. With the selected mutations, N-/C-terminal interaction correlated well with AR transactivation and the phenotype. Disrupted N-/C-terminal interaction is capable of providing the mechanism for androgen-insensitivity syndrome in most cases where the mutation in the LBD does not disrupt ligand binding. Furthermore, mutations leading to the disrupted N-/C-terminal interaction can be localized to certain critical regions in the three-dimensional structure of the AR LBD. Our study shows that apart from the previously reported regions, regions just before helix 3, between helices 5 and 6, and at helix 10 are also important for AR N-/C-terminal interaction.

Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor

“…The AR N/C interaction depends on high-affinity binding of testosterone, DHT, or synthetic anabolic steroids, which are agonists in vivo (36,62). The functional importance of the AR N/C interaction in androgen-dependent gene regulation is supported by naturally occurring AR AF2 site mutations that cause the androgen insensitivity syndrome, reduce AR FXXLF motif binding, and increase the dissociation rate of bound androgen without altering the apparent equilibrium androgen binding affinity (15,22,34,49,57).…”

“…The AR N/C interaction slows the dissociation rate of bound androgen (62), stabilizes AR binding to androgen response element DNA (60), and appears to be involved in domain swapping between an intramolecular AR monomer in the cytoplasm and an intermolecular antiparallel AR dimer in the nucleus (39, 51). The importance of the N/C interaction for AR function is supported by studies on the human androgen insensitivity syndrome, in which naturally occurring single amino acid mutations in AF2 reduce binding of the AR FXXLF and SRC/p160 LXXLL motifs without altering the apparent equilibrium androgen binding affinity (15,22,34,49). The different physiological potencies of testosterone and DHT have been linked to ligand-specific effects transmitted to the AF2 surface, where the weaker potency of testosterone, a more polarized steroid than DHT, derives from its inability to fully stabilize the AF2 binding surface for AR FXXLF and SRC/p160 coactivator LXXLL motif binding (1).…”

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation

“…Mutations D695N, Y763C, E772A, R774H, R774C and Q798E from AIS subjects are all located on the surface of the LBD at a relatively large distance from AF2, but surprisingly all mutants display a defective NC-TDI (Ghali et al, 2003;Jaaskelainen et al, 2006). Three of these residues (D695, Y763 and R774) together with residues R752 and F754 have been suggested to form a new region for protein-protein interactions, although this is not supported by experimental data (Jaaskelainen et al, 2006). Another mutation, R855H, found in an AIS individual is located also at a large distance from the AF2 region, within helices 10/11 which contain residues of the ligand binding pocket (Matias et al, 2000).…”

“…In addition, the AR mutants F725L and I737T have a defective interaction with SRC1 (Quigley et al, 2004). AR mutations L907F and R885H, which both can result in an defective NC-TDI, are also found in close proximity of the AF2 (Jaaskelainen et al, 2006).…”

A novel mutation (c.T3816 > C) in the androgen receptor gene in a 46,XY female patient with androgen insensitivity syndrome

“…Furthermore, binding of proteins to the cofactor-binding groove might affect binding of the ligand [11]. There is evidence indicating that disrupted N/C interaction can serve as a mechanism for AIS also in cases where ligand binding is normal [12][13][14].…”