Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP
Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP
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DOI：
10.1016/j.bbrc.2012.11.046
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发表时间：
2013-01-04
影响因子：
3.1
通讯作者：
Fujita, Norihisa
中科院分区：
文献类型：
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作者：
Sugita, Kazuya;Yamamura, Chiaki;Fujita, Norihisa
我们利用CHO细胞建立了稳定表达孤儿GPCR GPR 174的细胞系，并研究了该受体的生理和药理学特征。表达GPR 174的细胞表现出细胞-细胞粘附，肌动蛋白丝定位于细胞膜，并显示出细胞增殖的显著延迟。由于GPR 174-细胞的形态学变化与用霍乱毒素处理的模拟CHO细胞非常相似，我们测量了细胞内cAMP的浓度。结果显示，在GPR 174-细胞中浓度显著升高。通过测量GPR 174-细胞中的细胞内cAMP浓度，我们筛选脂质和核苷酸以鉴定GPR 174的配体。我们发现，溶血磷脂酰丝氨酸（LysoPS）刺激增加细胞内cAMP的剂量依赖性的方式。此外，在GPR 174细胞中，Erk的磷酸化被LysoPS升高。这些LysoPS反应被NF 449抑制，NF 449是G α（s）蛋白的抑制剂。这些结果表明，GPR 174是一个假定的LysoPS受体与G α（s）结合，其表达诱导CHO细胞的形态学变化，通过组成性激活腺苷酸循环伴随着细胞连接和增殖延迟。(C)2012 Elsevier Inc. All rights reserved.
We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G alpha(s) protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G alpha(s), and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation. (C) 2012 Elsevier Inc. All rights reserved.