Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning

Receptor-like roles for ephrin-B proteins are implicated to control axon pathfinding by repulsion, although it is largely unknown how the reverse signals are coupled to downstream intracellular molecules and induce cytoskeletal reorganization at the axon terminal. Here, we show ephrin-B3 (EB3) functions as a repulsive guidance receptor to mediate stereotyped pruning of murine hippocampal mossy fiber (MF) axons during postnatal development. Targeted intracellular point mutants show that axon pruning requires tyrosine phosporylation-dependent reverse signaling and coupling to the SH2/SH3 adaptor protein Grb4 (also known as Nckβ/Nck2). Furthermore, the second SH3 domain of Grb4 is required and sufficient for axon pruning/retraction by mediating interactions with Dock180 and PAK, to bring about guanine nucleotide exchange and signaling downstream of Rac, respectively. These studies reveal a novel pathway that controls axon pruning and elucidate the biochemical mechanism by which ephrin-B reverse signals regulate actin dynamics to bring about the retraction of growth cones.