Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane
Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane
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DOI：
10.1210/en.2006-1605
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发表时间：
2007-07-01
期刊：
影响因子：
4.8
通讯作者：
Thomas, P.
中科院分区：
文献类型：
--
作者：
Filardo, E.;Quinn, J.;Thomas, P.
G 蛋白偶联受体 30 (GPR30) 是一种七次跨膜受体 (7TMR)，与快速雌激素依赖性 G 蛋白信号传导和特异性雌激素结合相关。目前，GPR30作用的亚细胞位点尚不清楚。先前使用抗体和荧光染料标记的雌二醇（E2）的研究未能检测到细胞表面上的GPR30，这表明GPR30可能在7TMR中作为细胞内受体发挥独特的作用。在这里，我们表明可以通过荧光激活细胞分选来选择转染的 HEK-293 细胞表面上可检测的 GPR30 表达。使用凝集素伴刀豆球蛋白 A 作为质膜标记物，通过共聚焦显微镜证实细胞表面 GPR30 的表达。用 17β-E2 刺激表达 GPR30 的 HEK-293 细胞，导致 GPR30 从细胞表面隔离，并导致其与网格蛋白共分布和细胞内钙储备的动员。来自细胞表面的 GPR30 信号的证据是从实验中获得的，该实验表明细胞不可渗透的 E2 蛋白缀合物 E2-BSA 和 E2-辣根过氧化物酶促进 GPR30 依赖性细胞内 cAMP 浓度升高。亚细胞分级分离研究进一步支持质膜作为 GPR30 作用位点，具有与质膜相关的特异性 [H-3]17 β-E2 结合和 G 蛋白激活，但与从 HEK-293 或 SKBR3 乳腺癌细胞制备的微粒体或其他组分无关。这些结果表明，GPR30 与其他 7TMR 一样，具有质膜受体的功能。
G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17 beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [H-3]17 beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK- 293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.