Regulation of Ras p21 and RalA GTPases activity in mammary epithelial cells by quinine

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Esmati, Laya
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G protein-coupled receptors (GPCRs), a group of 7 transmembrane proteins, are the most targeted molecules in drug discovery studies. These proteins consist of an extracellular amino- and an intracellular carboxy-terminus and regulate various physiological processes in humans. Quinine as a bitter taste receptor agonist can activate G protein-coupled receptor family of proteins. One of the most extensively studied small G protein is Ras p21. The Ras p21 proteins (H-ras, K-ras and N-ras) are important in cell proliferation and upon mutations can be oncogenic. There are other small G proteins in the Ras superfamily that have ~30-50% sequence similarity with Ras p21. Ral G proteins (RalA and RalB) have high homology (~50%) with Ras p21 and participate in various cellular functions. Results from a previous study by our group showed that quinine causes activation of RalA in CHRF cells and, that these cells express the bitter taste receptor, T2R4. Ral proteins can be activated directly or through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using MCF10A (normal mammary epithelial cells) and MCF7 (non-invasive mammary epithelial cancer cells) cell lines we investigated the effect of quinine, a bitter compound, in the regulation of Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF10A and MCF7 cells; however, RalA was inhibited in MCF10A cells and no effect was observed in the case of MCF7 cells. The downstream effector for Ras p21, MAP kinase, was activated in both the MCF10A and MCF7 cells. Western blot analysis confirmed expression of RalGDS in MCF10A cells that was higher than that observed in MCF7 cells. Although RalGDS was detected in MCF10A and MCF7 cells, it did not result in RalA activation due to Ras p21 activation suggesting that Ras p21-RalGDS-RalA pathway is not active. The possibility exists that quinine has a direct effect in the regulation of RalA activity in the MCF10A cells. Preliminary results of protein modeling showed that quinine can interact with RalA through R79 amino acid which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change which results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that play a role in regulating Ral activity in mammary epithelial cells.
G Protein-Coupled Receptors