Determining if spatio-mechanosensitivity of EphA2 signaling stems from physical impedance of endocytosis

Juxtacrine cell-cell communication is mediated by receptor ligand interactions on opposing cell surfaces. It has recently become evident that the spatial organization of receptors on the cell surface plays a key role in signal transduction. Our lab has found that upon stimulation from membrane-bound ephrinA1, the EphA2 receptor tyrosine kinase (RTK) is spatially reorganized into higher order clusters in breast epithelial cancer cells. Applying spatiomechanical forces to disrupt the reorganization of EphA2-ephrinA1 clusters does not affect the activation (phosphorylation) of EphA2 but results in alterations of downstream signaling events. In particular, recruitment of the disintegrin and metalloprotease 10 (ADAM10) to the clusters is inhibited, indicating that EphA2 signaling is sensitive to spatial organization. It has been suggested that ADAM10 plays a role in the cleavage and subsequent endocytosis of ephrinA2, a homologous ephrin ligand to ephrinA1. Little, however, is known about the molecular mechanism of EphA2-ephrinA1 endocytosis, which likely includes an ADAM10 cleavage-induced endocytosis mechanism. Thus, my research is probing the role of endocytosis in the spatio-mechnosensitivity EphA2. Preliminary evidence suggests that clathrin-mediated endocytosis (CME) is important in the initial endocytosis of EphA2-ephrinA1. We have found that dynamin2, a large GTPase involved in the scission of endoycytic vesicles is also recruited to the large clusters. However, recruitment is altered when spatiomechanical forces are applied. These results indicate that endocytosis of EphA2-ephrinA1 is likely one mechanism contributing to the spatiomechanical sensitivity of EphA2 signaling.