We recently found that the spatial organization of the receptor tyrosine kinase EphA2 plays a key role in signal transduction in breast epithelial cancer cells. Upon stimulation from its native ligand ephrinA1 bound to a supported lipid membrane, EphA2 is spatially reorganized into large clusters at the cancer cell and supported membrane junction. Patterning nanostructures on the supported membrane substrate, using a spatial mutation technique, imposes physical restrictions to ligands bound to the supported membrane (i.e. ephrinA1). We used this strategy to restrict the movement of EphA2-ephrinA1 clusters and discovered alterations in downstream signaling events as a function of EphA2 organization. In particular, recruitment of the disintegrin and metalloprotease 10 (ADAM10) to the clusters is inhibited. ADAM10 has been implicated in the cleavage and subsequent endocytosis of other ephrin ligands, and may play a role in the endocytosis of EphA2-ephrinA1. Here we report that dynamin2, a large GTPase involved in the scission of endocytic vesicles, is recruited to the EphA2-ephrinA1 clusters. Preliminary evidence suggests that clathrin-mediated endocytosis (CME) is involved in the initial endocytosis of EphA2-ephrinA1. Finally, we have also found that the function of ADAM10 is necessary for ephrinA1 endocytosis. The implications of these observations for spatial regulation of EphA2 signaling will be discussed.