Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5
Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5
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DOI：
10.1152/ajprenal.00007.2008
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发表时间：
2008-05-01
影响因子：
4.2
通讯作者：
Huang, Chou-Long
中科院分区：
文献类型：
--
作者：
Cha, Seung-Kuy;Wu, Tao;Huang, Chou-Long
瞬时受体电位香草酸5（TRPV 5）是肾脏跨上皮钙重吸收的顶端进入途径。许多激素至少部分地通过调节TRPV 5来改变肾Ca 2+重吸收。磷脂酶C偶联激素对TRPV 5的急性调节机制在很大程度上是未知的。在这里，我们发现蛋白激酶C（PKC）激活剂1-油酰基-乙酰基-sn-甘油（OAG）增加TRPV 5电流密度和表面丰度在培养的细胞。OAG介导的TRPV 5增加可通过与特异性PKC抑制剂预孵育来阻止。与显性负性发动蛋白共表达增加了基础TRPV 5电流密度，并阻止了OAG的增加。通过小干扰RNA（siRNA）敲低caveolin-1可阻止OAG引起的TRPV 5的增加。相比之下，网格蛋白重链的敲低没有影响。OAG对来自caveolin-1敲除小鼠的caveolin-1无效细胞中表达的TRPV 5没有影响。在caveolin-1基因敲除细胞中，重组caveolin-1的强制表达恢复了OAG对TRPV 5的调节。TRPV 5的丝氨酸-299和/或丝氨酸-654（PKC磷酸化的共有残基）的突变取消了OAG的调节。甲状旁腺激素（PTH）增加TRPV 5和1型PTH受体共表达细胞中TRPV 5电流密度。由PTH引起的增加被PKC抑制剂、丝氨酸-299/丝氨酸-654突变或caveolin-1敲低所阻止。因此，TRPV 5经历组成型小窝介导的内吞作用。PKC的激活通过抑制内吞作用增加TRPV 5的细胞表面丰度。PKC的这种调节机制可能与PTH对TRPV 5的急性刺激和肾钙重吸收有关。
Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca2+ reabsorption in kidney. Many hormones alter renal Ca2+ reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca2+ reabsorption by PTH.