Ternary complex formation of EphA4, FGFR and FRS2α plays an important role in the proliferation of embryonic neural stem/progenitor cells

Abstract

EphA4 belongs to a superfamily of receptor tyrosine kinases and interacts with several molecules including fibroblast growth factor receptors (FGFRs) as we reported earlier. Several receptor tyrosine kinases, FGFRs, Trks, Alk and Ret, are currently known to transduce a signal through a docking protein, fibroblast growth factor receptor substrate 2α (FRS2α). However, nothing has been reported about the interaction of FRS2α with EphA4. Using the yeast two-hybrid system and the in vitro binding and kinase assays, we found that the mid-kinase region of EphA4 directly interacts with the FRS2α PTB domain upon tyrosine phosphorylation of the EphA4 juxtamembrane (JM) domain and EphA4 directly phosphorylates FRS2α. We also found that the FRS2α PTB domain and the amino-terminal region of EphA4 bind to the amino- and carboxy-terminal regions of the FGFR JM domain, respectively, suggesting that FRS2α and EphA4 interact with FGFR simultaneously. Furthermore, a kinase-dead EphA4 mutant that constitutively binds to FGFR functions as a dominant-negative molecule for signaling through both EphA4 and FGFR, and so does the truncated FRS2α lacking multiple tyrosine phosphorylation sites. These dominant-negative mutants similarly inhibit the ligand-dependent proliferation of the mouse embryonic neural stem/progenitor cells. These results suggest the formation of a ternary complex comprising EphA4, FGFR and FRS2α. The signaling complex appears to integrate the input from FGFR and EphA4, and release the output signal through FRS2α.