Funakoshi Takeshi, Yanai Akie, Shinoda Koh, Kawano Michio M, Mizukami Yoichi
Center for Gene Research, Yamaguchi University, Yamaguchi 755-8505, Japan.
Biochem Biophys Res Commun. 2006 Aug 4;346(3):904-10. doi: 10.1016/j.bbrc.2006.05.191. Epub 2006 Jun 9.
Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17beta-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.
最近，GPR30被报道为一种新型雌激素受体；然而，其细胞内定位仍存在争议。为了研究GPR30在体内的细胞内定位，我们针对GPR30上不同的表位制备了四种多克隆抗体。使用抗GPR30抗体和抗FLAG抗体的免疫细胞化学观察表明，FLAG-GPR30在转染后24小时定位于质膜。在用雌激素（17β-雌二醇或E2）处理后，表达FLAG-GPR30的HeLa细胞内的细胞内Ca2+浓度（[Ca2+]i）在10秒内升高。此外，E2在刺激后1小时诱导GPR30从质膜向细胞质转运。免疫组织化学分析表明，GPR30存在于CA2锥体神经元细胞的细胞表面。透射电子显微镜图像显示，GPR30定位于与锥体细胞质膜相关的特定区域。这些数据表明，作为雌激素跨膜受体的GPR30定位于大鼠脑海马体CA2锥体神经元细胞的质膜。
