Transmembrane and Juxtamembrane Interactions of EphA2 with Lipid Membranes in the Active and Inactive States

Abstract

Aberrant signaling of the receptor tyrosine kinase EphA2 is linked to tumor aggressiveness in many types of cancers. Upon binding of an extracellular ephrin ligand EphA2 undergoes oligomerization and phosphorylation inducing an intracellular signaling cascade. Little else is known of the mechanisms by which the signal is conferred across the membrane to drive phosphorylation or how in the absence of ligand the receptor remains inactive. Evidence suggests that prior to activation EphA2 dimers exhibit a distinct 15° interhelical crossing angle between the transmembrane domains (TMD) and that upon activation a rotation of the TMDs occurs and it widens to 45°. It is hypothesized that positively charged residues on the intracellular juxtamembrane (JM) segment interact with negatively charged Phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on the inner leaflet of the cell membrane inhibiting activation in the absence of bound ligand. Here we use a method of studying a peptide comprised of the TMD and first five JM residues of EphA2 in the putative active and inactive conformations by varying membrane thickness using 14:1 PC and 22:1 PC. We established that the TMD orientation is more tilted in thin bilayers and a less tilted in thick bilayers. We used this system to investigate the interaction of JM residues with membranes containing PIP2 in thin and thick bilayers. Fluorescence experiments indicate that JM residues experience different environments in thin vs. thick bilayers in the presence of PIP2. FRET results suggest that the JM is buried deeper into the membrane in the presence of PIP2 in 22:1 PC but not 14:1 PC. The extent of burial has been further examined by quenching experiments using brominated lipids. These results provide new mechanistic insights into the inhibition and activation of EphA2 signaling.