Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4α dimerisation and transcriptional activity
Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4α dimerisation and transcriptional activity
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DOI：
10.1093/nar/gkg850
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发表时间：
2003-11-15
影响因子：
14.9
通讯作者：
Laine, B
中科院分区：
文献类型：
--
作者：
Eeckhoute, J;Oxombre, B;Laine, B
肝细胞核因子4α(HNF4α，NR2A1)是肝细胞和胰岛β细胞功能的中枢。与维甲酸X受体α(RXRpha)一样，HNF4α属于核受体亚家族2(NR2)，其特征是在配体结合区(LBD)的螺旋7(H7)上有一个保守的精氨酸残基和一个谷氨酸残基。结晶学研究表明，RXRalphaH7中的R348和E352残基参与了电荷驱动的相互作用，促进了二聚反应。与这些发现一致，我们表明去除HNF4αH7、R258和E262中相应残基的电荷会损害溶液中的二聚作用。此外，我们的结果提供了一个新的概念，根据HNF4Alpha LBD二聚化界面的螺旋对DNA结合所需的二聚体的贡献不同；与H9和H10不同，H7不参与DNA结合。E262的替换减少了显性负HNF4α突变体对HNF4α转录活性的抑制，突显了该残基在细胞环境中二聚的重要性。E262插入对HNF4pha的功能至关重要，因为它的缺失取消了HNF4pha的转录活性和辅激活子招募。H7的谷氨酸残基插入和保守的精氨酸残基很可能代表核受体NR2亚家族的特征。
Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.