Monitoring the Dynamic Change of Inositol Lipid Pools upon EGFR and M3R Activation in Live Cells

Abstract

Phosphoinositides (PI) in the plasma membrane (PM) can dynamically change upon hormonal stimulation which can influence several cellular processes thus measuring the level of PIs can help us to better understand their distinct functions. We developed a highly sensitive method which enables us to follow the dynamic change of these lipids in living cells. We performed BRET measurements between various luciferase‐labeled PI‐binding domains and a PM‐targeted Venus in HEK 293T cells. To monitor the inositol lipid pools the followings were used: the 2xP4M domain of SidM for PI(4)P, the PH domain of PLCδ1 for PI(4,5)P2, the PH domain of BTK for PI(3,4,5)P3 and an intramolecular IP3 BRET sensor based on the ligand binding domain of the type‐1 IP3 receptor. Stimulation of cells with 100 ng/ml EGF resulted in a sustained increase of PI(3,4,5)P3, a slowly developing IP3 signal a transiently decreased PI(4,5)P2 level and an unexpected increase of PI(4)P. We also tested the lipid changes in case of M3R activation and we found that 100 µM carbachol causes more robust increase in the IP3, and decrease in the PI(4,5)P2 and PI4P levels however decreasing the carbachol concentration to 100 nM resulted in a very similar lipid changes as were seen in case of the EGF stimulation. Using different PI4K inhibitors we found that the increase in the PI(4)P level was caused by the activation of PI4KIIIα, and could be prevented by PKC inhibitors. With the help of our highly sensitive approach we were able to follow the dynamic inositol lipid changes upon stimulation of the cells with various compounds, and we could identify a possible signaling route how the cells can resynthesize PIs.