Physiological Significance of the extracellular Ca2+ Sensitivity of the Metabotropic Glutamate Receptor

我们曾报道，代谢型谷氨酸受体R1 α（mGluR1 α）不仅可被谷氨酸激活，而且可被细胞外Ca^<2+>（Ca^<2+>_0）浓度升高激活，Ca^<2+>_0的持续刺激可引起转染CHO细胞的形态学改变。然而，mGluR1 α的Ca^<2+>_0感应功能的生理作用还不完全清楚，特别是因为Ca^<2+>与其他神经递质不同，组成性地存在于细胞外空间。本研究旨在阐明mGluR1 α的Ca^<2+>_0敏感功能的生理意义。用forskolin模拟Ca^<2+>_0激活mGluR 1 α对CHO细胞形态学的影响。腺苷酸环化酶、蛋白激酶A（PKA）和MAP激酶（MAPKK）抑制剂可抑制mGluR 1 a激活对细胞形态的影响，PKA和MAPKK抑制剂可减弱forskolin的作用，说明cAMP、PKA、MAPKK、MARK通路参与了细胞形态的改变。我们实际上证实了mGluR 1 α的Ca^<2+>_0刺激增加了转染CHO细胞的基础cAMP水平。即使仅使用mGluR1 a转染的CHO细胞的膜级分，也观察到cAMP的这种增加，并且这种增加被抗Gs α抗体抑制。因此，我们认为mGluR1 a的Ca^<2+>_0敏感功能和Ca^<2+>_0的持续刺激通过与Gs的直接偶联引起基础cAMP水平的升高，并触发随后的PKA、MAPKK和MAPK级联反应的激活，导致转染CHO细胞的形态学改变。

We previously reported that the metabotropic glutamate receptor R1 a (mGluR1 α) can be activated not only by applying glutamate but also by raising extracellular Ca^<2+> (Ca^<2+>_0) concentration, and that the constant stimulation by Ca^<2+>_0 causes morphological change of transfected Chinese Hamster Ovary (CHO) cells. The physiological role of the Ca^<2+>_0-sensing function of mGluR1 α, however, is not fully clear yet, especially because Ca^<2+> is constitutively present an the extracellular space unlike other neurotransmitters. In this work, we aimed to elucidate the physiological significance of the Ca^<2+>_0-sensing function of mGluR1 α. The effect of mGluR1 α activation by Ca^<2+>_0on the morphological change of CHO cells was mimicked by forskolin. The effect of mGluR1 a activation on the morphological change was suppressed by the inhibitors of adenylate cyclase, protein kinase A (PKA) and MAP kinase kinase (MAPKK), and the effect of forskolin was also decreased by the inhibitors of PKA and MAPKK.These results demonstrate the involvement of cAMP, PKA, MAPKK, MARK pathway in the morphological change. We actually confirmed that the Ca^<2+>_0stimulation of mGluR1 α increased the basal cAMP level of transfected CHO cells. This increase in cAMP was observed even when only the membrane fraction of mGluR1 a transfected CHO cells were used, and the increase was inhibited by anti-Gsα antibody. Taken together, we concl uded that the Ca^<2+>_0-sensing function of mGluR1 a and the continuous stimulation by Ca^<2+>_0 caused the increase in the basal cAMP level by direct coupling with Gs, and triggered the subsequent activation of PKA, MAPKK, and MAPK cascade which resulted in the morphological change of transfected CHO cells.