Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly.

Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic ERK and Akt protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the N terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions driving the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis.