Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation
Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation
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DOI：
10.1681/asn.2008080873
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发表时间：
2009-08-01
影响因子：
13.6
通讯作者：
Hoenderop, Joost G. J.
中科院分区：
文献类型：
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作者：
de Groot, Theun;Lee, Kyupil;Hoenderop, Joost G. J.
低细胞外钙 (Ca2+) 会促进甲状旁腺激素 (PTH) 的释放，甲状旁腺激素作用于多个器官以维持总体 Ca2+ 平衡。在肾单位远端，PTH 通过腺苷酸环化酶-cAMP-蛋白激酶 A (PKA) 途径刺激活性 Ca2+ 重吸收，但该途径的分子靶标尚不清楚。瞬时受体电位香草酸 5 (TRPV5) 通道构成 Ca2+ 进入远曲小管的管腔门，并具有几个假定的 PKA 磷酸化位点。在这里，我们研究了 PTH 诱导的 cAMP 信号对 TRPV5 活性的影响。利用荧光共振能量转移，我们研究了共表达 PTH 受体和 TRPV5 的 HEK293 细胞在 PTH 刺激过程中的 cAMP 和 Ca2+ 动力学。 PTH 增加了 cAMP 水平，随后 TRPV5 介导的 Ca2+ 流入增加。 PTH（1 至 31）和毛喉素可激活 cAMP 通路，模拟 TRPV5 活性的刺激。值得注意的是，TRPV5 的激活仅限于强细胞内 Ca2+ 缓冲的条件。细胞表面生物素化研究表明，毛喉素不会影响细胞表面的 TRPV5 表达，这表明它改变了固定数量的 TRPV5 通道的单通道活性。应用PKA催化亚基磷酸化TRPV5，直接增加TRPV5通道开放概率。苏氨酸-709 的丙氨酸取代消除了 TRPV5 的体外磷酸化和 PTH 介导的刺激。总之，PTH 激活 cAMP-PKA 信号级联，迅速磷酸化 TRPV5 的苏氨酸 709，增加通道的开放概率并促进远端肾单位的 Ca2+ 重吸收。
Low extracellular calcium (Ca2+) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca2+ balance. In the distal part of the nephron, PTH stimulates active Ca2+ reabsorption via the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca2+ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca2+ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca2+ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca2+ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel's open probability and promoting Ca2+ reabsorption in the distal nephron.