CAR2 displays unique ligand binding and RXRα heterodimerization characteristics
CAR2 displays unique ligand binding and RXRα heterodimerization characteristics
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DOI：
10.1124/dmd.106.012641
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发表时间：
2007-03-01
影响因子：
3.9
通讯作者：
Omiecinski, Curtis J.
中科院分区：
文献类型：
--
作者：
Auerbach, Scott S.;DeKeyser, Joshua G.;Omiecinski, Curtis J.
组成型雄烷受体（CAR; NR 1 I3）调节参与异生物质代谢的基因的表达。人CAR基因的选择性剪接产生编码结构上不同的蛋白质的mRNA阵列。CAR的一种形式，称为CAR 2，含有额外的四个氨基酸（SPTV），预计这些氨基酸将重塑配体结合口袋。目前的研究显示，含有最佳DR-3、DR-4和DR-5反应元件的报告基因以及来自天然CYP 2B 6和CYP 3A 4基因启动子的报告基因具有显著的配体非依赖性CAR 2介导的反式激活。RXR α配体结合结构域的过表达对于实现这些效应至关重要。CAR 2与SRC-1的相互作用类似地依赖于RXR α的共表达。Ser 233（SPTV）突变为丙氨酸残基产生的受体具有较高的组成型活性。或者，将Ser 233突变为天冬氨酸残基大大降低了CAR 2的反式激活能力。这些诱变形式的CAR 2反式激活DR 4 x 3报告元件的相应能力与它们与RxR α相互作用并以配体调节的方式募集SRC-1的能力相关。总之，这些结果证明了CAR 2对共激活因子的稳健的RXR α依赖性募集和反式激活。此外，与受体的参考形式相比，CAR 2显示出对克霉唑和雄甾烷醇的新剂量反应，同时保留了结合CITCO的能力。这一结果支持了一种假设，即CAR 2中的四个氨基酸插入在结构上改变了其配体结合口袋，表明CAR 2受一组配体调节，这些配体与控制参考CAR活性的配体不同。
The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXR alpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXR alpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR4 x 3 reporter element correlated with their ability to interact with RxR alpha and to recruit SRC-1 in a ligand- regulated manner. Together, these results demonstrate a robust RXR alpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.