Pip2 Promotes Conformation-Specific Dimerization Of The Epha2 Transmembrane Domain Through Juxtamembrane Interactions

The impact on cancer of the EPHA2 receptor hinges on the two different ways it can be activated. EPHA2 induces anti-tumorigenic signaling after ligand binding, but ligand-independent activation of EPHA2 is pro-tumorigenic. It is believed that the transmembrane (TM) domain of EPHA2 adopts two alternate conformations in the ligand-independent and ligand-dependent states. However, it is poorly understood how the change in helical crossing angles found in the two conformations impacts the activity and regulation of EPHA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EPHA2 TM domain and a part of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different tm crossing angles, resembling the ligand-independent and ligand-dependent states. We developed a Single-Molecule TIRF technique using SMALPS to measure dimerization in the membrane. We observed that the signaling lipid PIP2 promotes tm dimerization, but only in the low crossing angle conformation, corresponding to the ligand-independent form. The mechanism PIP2 uses to promote dimerization might involve alleviating inter-JM repulsion, which is expected only for a tm dimer with a low crossing angle. Our data indicate conformational coupling between the TM and JM regions, and suggest that PIP2might directly exert a regulatory effect on EPHA2 that is specific to the ligand-independent conformation of the receptor.