Regulation of TRPP3 Channel Function by N-terminal Domain Palmitoylation and Phosphorylation.
Regulation of TRPP3 Channel Function by N-terminal Domain Palmitoylation and Phosphorylation.
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通过 N 端结构域棕榈酰化和磷酸化调节 TRPP3 通道功能。
DOI：
10.1074/jbc.m116.756544
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Chen Xing-Zhen
中科院分区：
文献类型：
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作者：
Zheng Wang;Yang JungWoo;Beauchamp Erwan;Cai Ruiqi;Hussein Shaimaa;Hofmann Laura;Li Qiang;Flockerzi Veit;Berthiaume Luc G;Tang Jingfeng;Chen Xing-Zhen
瞬时受体电位多囊蛋白-3（TRPP 3）是一种由钙离子和质子激活的阳离子通道，参与刺猬信号传导、肠道发育和酸味。TRPP 3通道的功能是如何调节的，目前还知之甚少。通过N-末端截短突变、电生理学和异种卵母细胞表达，我们首次鉴定了具有重要功能的片段Asp-21-Ser-42。然后我们发现缺失突变体Δ1-36（TRPP 3缺失片段Met-1-Arg-36）具有与野生型TRPP 3相似的功能，而Δ1-38在功能上是死亡的，这表明瓦尔-37或Cys-38的重要性。进一步的研究发现，Cys-38，而不是瓦尔-37，是功能关键的。Cys-38是棕榈酰化的预测位点，事实上，TRPP 3通道活性被棕榈酰化抑制剂2-溴棕榈酸酯抑制，并被棕榈酰化底物棕榈酸拯救。TRPP 3 N末端（TRPP 3 NT，Met-1-Leu-95）位于HEK 293细胞的质膜上，沿着，但在2-溴棕榈酸处理或C38 A突变后仍留在细胞质中，表明TRPP 3 NT通过Cys-38的棕榈酰化锚定在细胞膜表面。通过酰基-生物素交换试验，我们发现TRPP 3，而不是突变体C38 A，确实是棕榈酰化的。当Cys-38附近的假定磷酸化位点突变为Asp或Glu以模拟磷酸化时，只有T39 D和T39 E降低TRPP 3功能。此外，TRPP 3 NT显示双条带，其中上条带被λ磷酸酶处理或T39 A突变所消除。然而，棕榈酰化在Cys-38和磷酸化在Thr-39独立调节TRPP 3通道的功能，与以前的报告有关的棕榈酰化与邻近的磷酸化。Cys-38的棕榈酰化代表了TRPP 3功能调节的新机制。
Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, andXenopusoocyte expression, we first identified fragment Asp-21–Ser-42 to be functionally important. We then found that deletion mutant Δ1–36 (TRPP3 missing fragment Met-1–Arg-36) has a similar function as wild-type TRPP3, whereas Δ1–38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1–Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.