Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de Santé, Luxembourg, Grand-Duchy of Luxembourg.
Mol Cell Proteomics. 2013 Jul;12(7):1764-79. doi: 10.1074/mcp.M112.022947. Epub 2013 Jan 22.
Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.
糖皮质激素通过多种机制发挥快速非基因组效应，包括激活膜结合糖皮质激素受体（mGR）。在这里，我们报告了第一个关于 mGR 激活的蛋白质组学研究，该研究使用 BSA 结合皮质醇（Cort-BSA）。蛋白质组学数据集中的一组靶蛋白通过 Western blot 进行了验证，我们发现它们在另外三种细胞系中对 BSA 结合皮质醇激活 mGR 有反应，这表明在不同组织来源的细胞中存在保守效应。BSA 结合皮质醇处理的 CCRF-CEM 白血病细胞的蛋白质组变化与早期和快速的促凋亡、免疫调节和代谢效应相关，与细胞质糖皮质激素受体（cGR）的经典活性相吻合，并可能“启动”这些效应。研究主要信号通路靶基因的 PCR 阵列表明，mGR 不会通过这些白血病细胞中任何最常见激酶的转录活性发挥其作用，而是 RhoA 信号从我们的通路分析中显现出来。所有测试的细胞系在其表面都显示出非常低水平的 mGR。非常敏感和特异的原位邻近连接测定法甚至在以前被认为是 mGR 阴性的细胞中也能检测到低数量的 mGR。当使用三种针对 cGR N 端的不同抗-GR 单克隆抗体时，我们获得了类似的结果。这强烈表明 mGR 和 cGR 具有高度的序列同源性，并且很可能源自同一基因。此外，mGR 似乎位于 caveolae 中，并且在使用双识别邻近连接测定法研究的四个细胞系中的两个中，与 caveolin-1（Cav-1）的关联被清楚地检测到。然而，我们的结果表明，Cav-1 对于 GR 的膜定位不是必需的，因为 CCRF-CEM 和 Jurkat 细胞具有功能性 mGR，但不表达这种 caveolar 蛋白。然而，如果表达，这种膜蛋白会与 mGR 二聚化，从而调节其功能。
