Jääskeläinen J, Deeb A, Schwabe J W, Mongan N P, Martin H, Hughes I A
University Department of Paediatrics, Addenbrooke's Hospital, University of Cambridge, Hills Road CB2 2QQ, UK.
J Mol Endocrinol. 2006 Apr;36(2):361-8. doi: 10.1677/jme.1.01885.
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.
雄激素受体（AR）配体结合域（LBD）中的大多数突变会破坏天然配体：双氢睾酮和睾酮的结合。一些AR LBD突变不影响配体结合，但会破坏雄激素诱导的N端基序FXXLF与C端激活功能2（AF2）之间的相互作用。由于N端/ C端相互作用需要体内具有雄激素活性的激动剂结合，因此与表型密切相关。为了进一步研究这一点，我们在剑桥双性人数据库中搜索了AR LBD中检测到错义突变且配体结合正常的患者。选择了六个突变（D695N、Y763C、R774H、Q798E、R855H和L907F），并通过定点诱变将其引入pSVAR和pM-LBD质粒中。使用pGRE-LUC作为报告载体，通过双荧光素酶测定法检测野生型和突变型雄激素受体（pSVAR）的反式激活潜力。使用野生型和突变型AR LBD（pM-LBD）、pVP16-rAR-（5-538）（编码大鼠N端AR）和pCMX-UAS-TK-LUC作为报告，通过哺乳动物双杂交测定法研究N端/ C端相互作用。AR LBD突变D695N、R774H和L907F的反式激活能力最小，N端/ C端相互作用完全被破坏。突变Y763C和R885H具有一些残留的剂量依赖性反式激活潜力，N端/ C端相互作用最小。Q798E具有良好的反式激活潜力，并且其N端/ C端相互作用仅显示轻度降低。对于所选突变，N端/ C端相互作用与AR反式激活和表型密切相关。在大多数LBD突变不破坏配体结合的情况下，N端/ C端相互作用的破坏能够为雄激素不敏感综合征提供机制。此外，导致N端/ C端相互作用破坏的突变可定位于AR LBD三维结构中的某些关键区域。我们的研究表明，除了先前报道的区域外，螺旋3之前、螺旋5和6之间以及螺旋10处的区域对于AR N端/ C端相互作用也很重要。
