Abstract 1903 Epidermal growth factor receptor interactions with phosphatidylinositol 4,5-bisphosphate

Protein-lipid interaction plays a crucial role in the regulation of the activity of membrane proteins such as the epidermal growth factor receptor (EGFR). Most of the reported lipid interactions involved the transmembrane and juxtamembrane domains of the protein, but little is known about the function effects of lipid interactions with EGFR. Here we investigate the interaction of anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to a poly-His-tagged EGFR intracellular domain (ICD) in vitro. Liposomes were prepared using a modified lipid film hydration technique and were extruded in a filter membrane with 100 nm size pores. N-terminal His-tagged proteins with a C-terminal mCherry fluorescent tag were incubated with anionic phospholipid containing liposomes. The binding of the EGFR-ICD to the liposomes were evaluated using a pulsed interleaved excitation fluorescent cross-correlation spectroscopy (PIE-FCCS). The effect of anionic lipids on the kinase activity of EGFR-ICD was observed over a range of lipid concentrations. Inhibition of kinase activity was observed for lipid-bound EGFR-ICD compared to EGFR-ICD without lipids. PIP2-containing liposomes had a much more significant inhibitory effect on EGFR-ICD kinase activity to phosphatidylserine (PS)-containing liposomes at similar concentrations. The decrease of the kinase activity of the EGFR-ICD was also more pronounced in low ionic strength environment compared to a higher ionic strength suggesting that the inhibition is mediated by electrostatic lipid-protein interactions. From the results, PIP2 shows an inhibitory effects on the kinase activity of EGFR-ICD. Overall, our work helps to reveal the molecular level details of anionic lipid inhibition of EGFR kinase activity, which is critical for the design of EGFR inhibitors for cancer drug discovery. We would like to acknowledge National Science Foundation, the main funding agency for the project, and our collaborator, Vanderbilt University. We would also to acknowledge Texas Tech University for the additional funding support.