Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains
Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains
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DOI：
10.1677/jme.1.01885
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发表时间：
2006-04-01
影响因子：
3.5
通讯作者：
Hughes, IA
中科院分区：
文献类型：
--
作者：
Jääskeläinen, J;Deeb, A;Hughes, IA
雄激素受体（AR）配体结合结构域（LBD）中的大多数突变会破坏天然配体（二氢睾酮和睾酮）的结合。一些AR LBD突变不影响配体结合，但它们破坏雄激素诱导的N-末端基序FXXLF和C-末端激活功能2（AF 2）的相互作用。由于N-/C-末端相互作用需要结合体内具有雄激素活性的激动剂，因此它与表型密切相关。为了进一步研究，我们在剑桥两性数据库中搜索了在AR LBD中检测到错义突变的患者，这些患者表现为正常的配体结合。选择六个突变（D 695 N、Y 763 C、R774 H、Q798 E、R855 H和L907 F），并通过定点诱变引入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来检查野生型和突变型雄激素受体（pSVAR）的反式激活潜力。以野生型和突变型AR LBD（pM-LBD）、pVF`16-rAR-（5-538）（编码大鼠氨基末端AR）和pCMX-UAS-TK-LUC作为报告基因，通过哺乳动物双杂交试验研究N-/C-末端相互作用。AR LBD突变D 695 N、R774 H和L907 F表现出最小的反式激活能力，N-/C-末端相互作用被完全破坏。突变Y 763 C和R885 H具有一定的残留剂量依赖性反式激活潜力和最小的N-/C-末端相互作用。Q798 E具有良好的反式激活潜力，其仅显示N-/C-末端相互作用的轻度降低。对于选定的突变，N-/C-末端相互作用与AR反式激活和表型相关性良好。在LBD突变不破坏配体结合的大多数情况下，破坏的N-/C-末端相互作用能够提供雄激素不敏感综合征的机制。此外，导致破坏的N-/C-末端相互作用的突变可以定位于AR LBD的三维结构中的某些关键区域。我们的研究表明，除了以前报道的区域，区域前螺旋3，螺旋5和6之间，并在螺旋10也是重要的AR N-/C-末端相互作用。
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), pVF`16-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.