Sousa Cátia, Ribeiro Madalena, Rufino Ana Teresa, Leitão Alcino Jorge, Mendes Alexandrina Ferreira
a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.
b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
J Recept Signal Transduct Res. 2017 Apr;37(2):181-188. doi: 10.1080/10799893.2016.1203943. Epub 2016 Jul 12.
CONTEXT/OBJECTIVE: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30.
The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals.
None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1.
Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.
背景/目的：用于研究G蛋白偶联受体30（GPR30）或G蛋白偶联雌激素受体（GPER）作为雌激素反应介质作用的细胞系产生了相互矛盾的结果。这项研究确定了一种简单的检测方法，用于预测细胞系对GPR30进行药理学研究的适用性。
评估了ERK1/2、Akt、c-Fos和eNOS的磷酸化或表达水平，以评估MCF-7和T-47D乳腺癌细胞系以及牛主动脉内皮细胞中已知激动剂（17β-雌二醇和G-1）刺激下的GPR30激活情况。通过qRT-PCR和使用针对其羧基和氨基末端的两种不同抗体进行的蛋白质印迹分析GPR30的表达。
在所测试的任何浓度下，这些激动剂均未激活任何一种靶蛋白。进一步的实验排除了信号通路破坏、培养基中酚红的干扰以及GPR30的组成型蛋白酶体降解作为这三种细胞系缺乏反应的可能原因。受体表达分析显示，先前在对17β-雌二醇和G-1有反应的细胞系中鉴定出的44 kDa和50 - 55 kDa的GPR30蛋白在这些细胞系中未被清晰检测到。
不表达44 kDa和50 - 55 kDa蛋白的细胞对GPR30激动剂无反应。因此，这些GPR30蛋白的有无是一种简单快速的方法，可用于确定给定的细胞系是否适合进行GPR30调节的药理学或分子研究。
