Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation
Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation
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DOI：
10.1074/jbc.m117.806604
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发表时间：
2018-01-05
影响因子：
4.8
通讯作者：
Negishi, Masahiko
中科院分区：
文献类型：
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作者：
Shizu, Ryota;Min, Jungki;Negishi, Masahiko
核蛋白组成活性/雄甾烷受体（CAR或NR1I3）调节多种肝脏功能，如药物和能量代谢以及细胞生长或死亡，这些功能通常参与糖尿病和肝细胞癌等疾病的发展。CAR通过类视黄醇X受体（RXR）从无活性的同型二聚体转化为有活性的异源二聚体，CAR中Thr-38位点dna结合域（DBD）的磷酸化调节了这一转化。在这里，我们揭示了这种磷酸化调节CAR的DBD和配体结合域（LBD）之间的分子内相互作用的分子机制，从而实现了同二聚体-异二聚体的转化。在Huh-7细胞中，用Asp代替Thr-38可增加CAR - DBD与CAR - LBD的共免疫沉淀。等温滴定量热分析还显示，重组CAR - DBD- t38d与CAR - LBD肽结合，而非磷酸化CAR - DBD与CAR - LBD肽结合。这种DBD-LBD相互作用掩盖了CAR的二聚体界面，阻止了CAR同二聚体的形成。值得注意的是，EGF信号传导减弱了CAR - DBD - T38D与CAR - LBD的相互作用，将CAR转化为同二聚体形式。DBD-T38D-LBD相互作用也阻止了CAR与RXR形成异源二聚体。然而，这种相互作用打开了CAR表面，允许与蛋白磷酸酶2A相互作用。然后，Thr-38去磷酸化解离了DBD-LBD相互作用，使CAR与RXR形成异源二聚体。我们得出结论，磷酸化的DBD与LBD的分子内相互作用使CAR适应一种瞬时单体构型，可以转化为无活性的同二聚体或活性的异二聚体。
The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor (RXR), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXR. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXR. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.