Kang Lianguo, Zhang Xintian, Xie Yan, Tu Yaping, Wang Dong, Liu Zhenming, Wang Zhao-Yi
Department of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, Nebraska 68178, USA.
Mol Endocrinol. 2010 Apr;24(4):709-21. doi: 10.1210/me.2009-0317. Epub 2010 Mar 2.
Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.
越来越多的证据表明，孤儿G蛋白偶联受体（GPR）30介导雌激素的非基因组反应。本研究旨在探讨GPR30功能的分子机制。我们发现，在乳腺癌SK-BR-3细胞中敲低GPR30的表达会下调雌激素受体（ER）-α36（ER-α的一种变体）的表达水平。将GPR30表达载体导入不表达GPR30的细胞可诱导内源性ER-α36的表达，共转染实验表明，GPR30通过激活蛋白1结合位点激活ER-α36的启动子活性。17β-雌二醇（E2）和G1（一种据报道为选择性GPR30激动剂的化合物）均可增加SK-BR-3细胞中MAPK/ERK1/2的磷酸化水平，而针对其配体结合域的抗ER-α36特异性抗体可阻断这种增加。G1诱导由ER-α36介导的活性，如VP16-ER-α36融合蛋白的转录激活活性以及在表达ER-α36的细胞中激活MAPK/ERK1/2。表达ER-α36的细胞而非不表达的细胞表现出高亲和力、特异性的E2和G1结合，且E2和G1诱导的细胞内Ca(2+)动员仅发生在表达ER-α36的细胞中。综上所述，我们的结果表明，先前报道的GPR30对雌激素的反应活性是通过其诱导ER-α36表达的能力实现的。选择性G蛋白偶联受体（GPR）30激动剂G1实际上与ER-α36相互作用。因此，参与非基因组雌激素信号传导的是ER-α变体ER-α36，而非GPR30。
