Bai M, Trivedi S, Lane C R, Yang Y, Quinn S J, Brown E M
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Aug 14;273(33):21267-75. doi: 10.1074/jbc.273.33.21267.
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 (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), 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 Ca2+i 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 Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC 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 (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 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 Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.
先前在表达G蛋白偶联的细胞外钙敏感受体（CaR）的甲状旁腺细胞中的研究表明，蛋白激酶C（PKC）的激活可减弱高细胞外钙（Ca2+o）诱发的磷脂酶C刺激以及胞质钙（Ca2+i）的相关增加，这表明PKC可能直接调节CaR与细胞内信号系统的偶联。在本研究中，我们检测了PKC在调节CaR与用人类甲状旁腺CaR瞬时转染的fura-2负载的人类胚胎肾细胞（HEK293细胞）中Ca2+i动态变化的偶联中的作用。我们证明，几种PKC激活剂对由于细胞内钙库释放钙而导致的CaR介导的Ca2+i增加具有抑制作用。与该效应由PKC激活介导一致，PKC激活剂对Ca2+释放的抑制作用可被PKC抑制剂阻断。使用定点诱变显示，氨基酸位置888处的苏氨酸是介导PKC激活剂对Ca2+动员抑制作用的主要PKC位点。通过突变三个PKC位点（Thr888、Ser895和Ser915）或所有五个PKC位点，PKC激活的效应可被最大程度地阻断。体外磷酸化显示Thr888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激活对甲状旁腺细胞中Ca2+o诱发的Ca2+i动态变化的影响的分子基础。
