Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation
Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation
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DOI：
10.1042/bj20061826
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发表时间：
2007-05-15
影响因子：
4.1
通讯作者：
Conigrave, Arthur D.
中科院分区：
文献类型：
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作者：
Lee, Heather J.;Mun, Hee-Chang;Conigrave, Arthur D.
钙敏感受体（CaR）介导Ca-0（2+）（细胞外Ca 2+）浓度的反馈控制。尽管其机制尚未完全了解，但CaR与几种重要的细胞内信号传导酶偶联，包括PI-PLC（磷酸肌醇特异性磷脂酶C），导致Ca 1（2+）（细胞内Ca 2+）动员，以及ERK 1/2（细胞外信号调节激酶1/2）。除了Ca-0（2+）之外，CaR还被L-氨基酸的几个亚类变构激活，包括芳香族L-苯丙氨酸和L-色氨酸。这些氨基酸增强了表达CaR的HEK-293（人胚肾）细胞和正常人甲状旁腺细胞中Ca-O（2+）动员的Ca-O（2+）敏感性。此外，在生理性空腹血清L-氨基酸混合物的背景下，它们诱导了PTH（甲状旁腺激素）分泌的Ca-o（2+）依赖性抑制的小的但生理上显著的增强。然而，氨基酸对CaR刺激的ERK 1/2的影响尚未确定。在本研究中，我们通过Western印迹和一种新开发的定量分析（SureFire）来检测L-氨基酸对Ca-o（2+）刺激的ERK 1/2磷酸化的影响。L-氨基酸诱导了一个小的，但显着的，增强钙o（2+）-刺激的ERK 1/2。在表达CaR的HEK-293细胞中，10 mM L-苯丙氨酸将Ca-o（2+）的EC 50从约100 mg/L降低至约100 mg/L。2.3在Western印迹分析中为2.0 mM，在SureFire分析中为3.4 - 2.9 mM。该效应是立体选择性的（L > D），并且另一种芳香族氨基酸L-色氨酸也是有效的。在表达CaR突变体S169 T的HEK-293细胞中进一步研究了氨基酸的作用。L-苯丙氨酸使Ca-0（2+）刺激的Ca-1（2+）的EC 50标准化，从约0.000000动员。1/2 mM至5.0 mM，ERK 1/2磷酸化从约4.6总之，数据表明，L-苯丙氨酸和其他氨基酸增强CaR刺激的ERK 1/2磷酸化的Ca-o（2+）敏感性;然而，该效应相对较小，并且以微调机制的形式起作用。
The calcium-sensing receptor (CaR) mediates feedback control of Ca-0(2+) (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca-i(2+) (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca-0(2+), the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca-0(2+),-sensitivity of Ca-i(2+) mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca-o(2+)- dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca-o(2+)-stimulated ERK1/2 phosphorylation as deterurnined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca-o(2+)-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca-o(2+) from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L > D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylaianine normalized the EC50 for Ca-o(2+)-stimulated Ca-i(2+), mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca-o(2+) sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.