Characterization of ephrin-A1 and ephrin-A4 as ligands for the EphA8 receptor protein tyrosine kinase

The Eph receptors are the largest known family of receptor protein tyrosine kinases, which play important roles with their ligands called ephrin in th e neural development, angiogenesis, and vascular network assembly. It was p reviously shown that ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase, respectively. In this study, we have examined if there are other additional ephrin ligands interacting with the EphA8 recep tor tyrosine kinase expressed in NIH3T3 fibroblasts, For this purpose, we h ave constructed chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus, Both ephr in-A1 and ephrin-A4 chimeric ligands efficiently bound to the EphA8 recepto r expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands includ ing ephrin-B1, -B2 and -B3 did not. Additionally me have demonstrated that both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in N IH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These re sults strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent recepto r protein tyrosine kinase.