G-Protein-Coupled Receptor-Mediated MAPK and PI3-Kinase Signaling Is Maintained in Chinese Hamster Ovary Cells after γ-Irradiation

Abstract

To expedite G-protein-coupled receptor (GPCR) drug screening studies, cell lines amenable to transfection (e.g. CHO cells) have been widely used as cellular models. These cells can be frozen in a ready-to-use format, allowing screening of a single batch of cells and validation of the cellular material prior to the screening run. A common method used to deliver frozen cells to screening programs is to γ-irradiate the cells, abrogating cell division after thawing and ensuring consistency in the number of cells analyzed per well. With the recognition that signaling proteins such as ERK and Akt are important markers of GPCR activation, along with the availability of suitable assays for their measurement, these outputs have become important for GPCR screening programs. Here we show that several γ-irradiated and frozen CHO-K1 cell lines expressing transfected GPCRs, initially optimized for performing cAMP or AequoScreen calcium flux assays, can be used for the measurement of GPCR-mediated ERK and Akt phosphorylation. Furthermore, CHO-K1 cells transfected with NOP or GAL 1 receptors show pharmacology for a number of agonists and antagonists that is consistent with nonirradiated cultured lines. These data indicate that γ-irradiated CHO-K1 cells can be reliably used for the measurement of GPCR-mediated kinase signaling outputs.

Regulation of Connexin-Based Channels by Fatty Acids

“…There is evidence in support of the participation of membrane receptors in the short-term effect on Cx-based channels (both GJCs and HCs). In some cases, the FA-mediated effect requires the participation of protein kinases that could be activated downstream of different GPCRs (Osmond et al, 2012; Suire et al, 2012; Liang et al, 2015), such as FA receptors (Itoh et al, 2003; Hirasawa et al, 2005). In agreement with such evidence, several protein kinases have been identified to modify Cxs (Solan and Lampe, 2014; Pogoda et al, 2016).…”

Allantoin activates imidazoline I-3 receptors to enhance insulin secretion in pancreatic β-cells

“…CCL-61) were purchased from the Culture Collection and Research Center of the Food Industry Institute (Hsin-Chiu City, Taiwan). Following a previous method [24,25], Min 6 cells were maintained in RPMI 1640 medium, while CHO-K1 cells were maintained in F-12 K medium supplemented with 10% fetal bovine serum. The cells were sub-cultured once every three days by trypsinization (Gibco), and the culture medium was refreshed every 2-3 days.…”

Transforming growth factor-β, MAPK and Wnt signaling interactions in colorectal cancer

“…This complex crosstalk in CRC, we propose, can be investigated by implementing a combination of sophisticated informatics, -omics technologies and in vivo studies in a spatio-temporal manner, coupled with larger protein tracking and interaction studies. Emerging multiplexed technologies such as SOMAmer 1 [170], proximity extension assays [35,171] and/or SureFire 1 assays [172] will be crucial in the coming years to perform more elaborate experiments in order to elucidate complex cell signaling behaviors within a matrix of different pathways and the crosstalk between them. It is crucial to remember that in cancer and various diseases, we cannot study these pathways in isolation but instead must transition into a matrix-oriented systems approach that more comprehensively models the spatio-temporal ramifications of signaling activities within the complexity of living cells and tissues.…”