Screening of a ligand for hypoxia-induced receptor by negative selection method

近年来，GPR30被报道为一种新的雌激素受体；然而，其在细胞内的定位仍然存在争议。为了研究GPR30在体内的细胞内定位，我们针对GPR30不同的表位制备了四种多克隆抗体。利用抗gpr30抗体和抗flag抗体进行免疫细胞化学观察，发现FLAG-GPR30在转染后24小时定位于质膜。在表达FLAG-GPR30的HeLa细胞中，雌激素（17β-雌二醇或E2）在10秒内引起细胞内Ca^<2+>浓度升高（[Ca^<2+>]_i）。此外，E2在刺激后1小时诱导GPR30从质膜转移到细胞质。免疫组化分析表明，GPR30存在于CA2锥体神经元细胞的细胞表面。透射电镜图像显示，GPR30定位于锥体细胞质膜相关的特定区域。这些数据表明，雌激素的跨膜受体GPR30定位于大鼠脑海马CA2锥体神经元细胞的质膜。

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 hr after transfection.Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) within 10 sec in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 hr 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 on the 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.