Protein kinase C phosphorylation of threonine at position 888 in Ca2+0-sensing receptor (CaR) inhibits coupling to Ca2+ store release
Protein kinase C phosphorylation of threonine at position 888 in Ca2+0-sensing receptor (CaR) inhibits coupling to Ca2+ store release
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DOI：
10.1074/jbc.273.33.21267
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发表时间：
1998-08-14
影响因子：
4.8
通讯作者：
Brown, EM
中科院分区：
文献类型：
--
作者：
Bai, M;Trivedi, S;Brown, EM
先前对表达 G 蛋白偶联胞外钙敏感受体 (CaR) 的甲状旁腺细胞的研究表明，蛋白激酶 C (PKC) 的激活会减弱高细胞外钙 (Ca-o(2+)) 诱发的磷脂酶 C 刺激以及相关的胞质钙 (Ca-i(2+)) 增加，表明 PKC 可能直接调节 CaR 与细胞内信号系统的偶联。 PKC 在瞬时转染人甲状旁腺 CaR 的负载 fura-2 的人胚胎肾细胞（HEK293 细胞）中调节 CaR 与 Ca-i(2+) 动力学耦合的作用。我们证明，由于细胞内储存的 Ca2+ 释放，多种 PKC 激活剂对 CaR 介导的 Ca-i(2+) 增加发挥抑制作用。与 PKC 激活介导的作用一致，PRC 激活剂对 Ca2+ 释放的抑制作用可以被 PKC 抑制剂阻断。定点诱变的使用表明，888位氨基酸的苏氨酸是介导PKC激活剂对Ca2+动员的抑制作用的主要PKC位点。通过突变三个PKC位点（Thr（888）、Ser（895）和Ser（915））或全部五个PKC位点可以最大程度地阻断PKC激活的作用。体外磷酸化显示 Thr(888) 很容易被 PKC 磷酸化。我们的结果表明 CaR 的磷酸化是先前描述的 PKC 激活对 Ca-o(2+) 引起的甲状旁腺细胞中 Ca-i(2+) 动力学变化的影响的分子基础。
Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca-o(2+))-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca-i(2+)), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems, In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca-i(2+) dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR, We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca-i(2+) due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PRC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization, The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr(888), Ser(895), and Ser(915)) or all five PKC sites. In vitro phosphorylation shows that Thr(888) is readily phosphorylated by PKC, Our results suggest that phosphorylation of the CaR, is the molecular basis for the previously described effect of PKC activation on Ca-o(2+)-evoked changes in Ca-i(2+) dynamics in parathyroid cells.