Restricting EphA2 Receptor Movement Affects Internalization and Signaling in Living Cells

The importance of spatial reorganization of receptors and ligands that occurs at the cell-cell interface is becoming increasingly evident in a variety of cell systems. Our lab has developed a unique experimental platform in which cells are interfaced with supported lipid membranes displaying ligands creating a controllable and laterally mobile ligand presentation system that recapitulates essential aspects of the cell-cell contact. To probe how protein organization in the membrane influences cell signaling events, we use a spatial mutation strategy in which we pattern supported lipid bilayers into microscale corrals to restrict protein movement within the corrals and then measure alterations in cell signaling. When MDAMB231 breast cancer cells expressing the receptor tyrosine kinase EphA2 are interfaced with a supported lipid bilayer containing the ephrinA1 ligand, EphA2 binds to ephrinA1 and the complex reorganizes into large clusters. When receptor-ligand movement is disrupted with the spatial mutation, the reorganization of EphA2-ephrinA1 clusters results in alterations of downstream signaling. In particular, we found that recruitment of the metalloprotease ADAM10 to the clusters is inhibited, indicating that EphA2 signaling is sensitive to the spatial organization of ephrinA1. ADAM10 has been implicated in the cleavage and endocytosis of other ephrins, suggesting that ADAM10 may be necessary for efficient endocytosis of EphA2-ephrinA1. Therefore, our research is probing how endocytosis is affected by the spatial organization of EphA2. Here, we report the development of a novel and highly quantitative endocytosis assay. Using this assay, we found that the spatial organization of EphA2-ephrinA1 does alter ephrinA1 endocytosis. We also found that Pitstop2, a small molecular inhibitor of clathrin-mediated endocytosis (CME), negatively effects ephrinA1 endocytosis, indicating that CME is important in ephrinA1 internalization. The implications of these observations for spatial regulation of EphA2 signaling will be further discussed.