Signalling by Eph receptors and ephrins is a two-way process — each can act both as a receptor, which transduces signals, but also as a ligand that can send such signals. More is known about the 'forward' signalling pathway — where Eph receptors, as their name suggests, act as receptors — than the events that occur in the reverse direction. Reporting in Nature, however, Cowan and Henkemeyer now describe some of the proteins involved in reverse signalling following the engagement of B-ephrins.

The authors started with a two-hybrid screen to identify proteins that interact with the cytoplasmic domain of ephrin-B1 in a phosphotyrosine-dependent manner. All the resulting 19 clones encoded an adaptor protein called Grb4 (also known as Nck-2 and Nckβ), which contains one Src-homology-2 (SH2) domain, essential for the interaction with ephrin-B1, and three SH3 domains. The authors used glutathione-S-transferase pull-down experiments and co-immunoprecipitation to verify this interaction in mammalian cells, and then visualized the recruitment of Grb4 to ephrin-B1 in live cells.

What is the effect of this interaction? Cowan and Henkemeyer examined the cytoskeleton after ephrin-B1 reverse signalling, and found a marked loss of polymerized actin. Not only that, but cells rounded up and shrank, as though adhesive contacts had been lost, and the focal adhesion marker paxillin was seen to re-distribute from the plasma membrane to the cytoplasm. Finally, catalytic activity of focal adhesion kinase (FAK) was found to increase.

To find out how reverse signalling leads to such effects, the authors used the three Grb4 SH3 domains as bait in another two-hybrid screen. They pulled out five proteins — dynamin, heterogeneous nuclear ribonucleoprotein K (hnRNPK), Abl-interacting protein-1 (Abi-1), c-Cbl-associated protein (CAP) and axin — and showed that one of them, CAP, redistributes with Grb4 to activated spots of ephrin-B1. The authors therefore propose a model, shown below, in which clustered tyrosine-phosphorylated ephrin-B1 forms a high-affinity binding site for the Grb4 SH2 domain, and that the SH3 domain of Grb4 recruits various proteins to this site.