Ichijo Yuta, Mochimaru Yuta, Azuma Morio, Satou Kazuhiro, Negishi Jun, Nakakura Takashi, Oshima Natsuki, Mogi Chihiro, Sato Koichi, Matsuda Kouhei, Okajima Fumikazu, Tomura Hideaki
Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan.
Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan.
Biochem Biophys Res Commun. 2016 Jan 1;469(1):81-86. doi: 10.1016/j.bbrc.2015.11.075. Epub 2015 Nov 22.
Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.
人类G2A可被多种刺激激活，如溶血磷脂酰胆碱（LPC）、9-羟基十八碳二烯酸（9-HODE）和质子。该受体与多种细胞内信号通路偶联，包括Gs蛋白/cAMP/CRE、G12/13蛋白/Rho/SRE和Gq蛋白/磷脂酶C/NFAT通路。在本研究中，我们检测了斑马鱼G2A同源物（zG2A-a和zG2A-b）是否能对这些刺激作出反应并激活多种细胞内信号通路。我们还检测了如果这些同源物感知质子，其N端的组氨酸残基和碱性氨基酸残基是否也发挥与人类G2A残基类似的作用。我们发现，zG2A-a在pH 8.0时表现出高CRE、SRE和NFAT活性，然而，zG2A-b仅表现出高SRE活性。从pH 7.4到6.3的细胞外酸化改善了表达zG2A-a的细胞中的这些活性。另一方面，酸化改善了表达zG2A-b的细胞中的SRE活性，但没有改善CRE和NFAT活性。LPC或9-HODE未改变任何一种同源物的活性。将zG2A-a N端第174位的组氨酸残基替换为天冬酰胺残基可减弱质子诱导的CRE和NFAT活性，但不影响SRE活性。将zG2A-a N端第32位的精氨酸残基替换为丙氨酸残基也减弱了其高活性以及质子诱导的CRE和NFAT活性。相反，该替换未减弱SRE活性。将zG2A-b N端第10位的精氨酸残基替换为丙氨酸残基也未减弱其高活性或质子诱导的SRE活性。这些结果表明，斑马鱼G2A同源物被质子激活，但不被LPC和9-HODE激活，且这些同源物的激活机制与人类G2A相似。
