The SAM Domain of EphA2 Inhibits Ligand-Independent Clustering and Activation

Abstract Eph receptors are the largest family of receptor tyrosine kinases (RTKs). They play a role in the pathogenesis of various diseases including cancer, atherosclerosis, fibrosis, infectious diseases, diseases of the central nervous system and age-related cataract. EphA2 has attracted much attention over the years owing to its dysregulation in many diseases. Previous studies have revealed the unique molecular organizations of Eph receptors, and particularly EphA2, into large clusters of receptor-ligand complexes. One unique feature of Eph receptors is a C-terminal sterile alpha motif (SAM) domain, which has been proposed to alter dimerization and kinase activity in EphA2. However, the precise role of the SAM domain in regulating the function and oligomerization state of EphA2 has not been reported. Here we apply a time-resolve fluorescence spectroscopy, PIE-FCCS, to characterize the oligomerization state of EphA2 in live cells and determine the role of the SAM domain. We deleted the SAM domain in the context of full length EphA2 and an intracellular domain (ICD) construct to assess the effect of the SAM domain on oligomerization state, kinase activity, and cellular behavior. Overall, we find that the SAM domain inhibits ligand-independent clustering and kinase activity in both full-length EphA2 and the isolated ICD construct at the cell membrane. These results are consistent with the autoinhibitory features of the C-terminal tail of EGFR and may help resolve the allosteric regulation of other RTKs.