The signaling lipid PIP 2 promotes specific dimerization of the membrane region of the EphA2 receptor

The influence of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 can induce anti‐oncogenic signaling after ligand binding, but ligand‐independent activation is pro‐oncogenic. It is believed that the transmembrane (TM) domain of EphA2 can adopt two alternate conformations in the ligand‐dependent and the ligand‐independent states. However, it is poorly understood how the different TM helical crossing angles that characterize the two conformations, impact the activity and regulation of EphA2. Here, we devise a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations show different TM crossing angles that agree with the ligand‐dependent and ligand‐independent states of EphA2. We developed a single‐molecule technique that uses SMALP nanodiscs to measure dimerization in membranes. We observed that the signaling lipid PIP 2 (phosphatidylinositol 4,5‐bisphosphate) promotes TM dimerization, but only in the conformation with a small crossing angle, which we propose corresponds to the ligand‐independent state. In this conformation the two TM are almost parallel, and the positively charged JM segments are expected to be close to each other and cause electrostatic repulsion. We propose that that PIP 2 uses its high density of negative charges to alleviate this repulsion, which promotes EphA2 dimerization. Our results indicate a conformational coupling between the TM and JM regions of EphA2, and suggest that PIP 2 directly exerts a regulatory effect on EphA2 activation in cells that is specific to the ligand‐independent conformation of the receptor.