Grabbing onto Ephrin-B

Abstract

Ephrins and ephrin receptors are transmembrane proteins that are expressed on neighboring cells. Upon interaction, they stimulate signaling cascades in both the ephrin-expressing and the receptor-expressing cell. The ephrin-B family has a conserved short cytoplasmic domain with several tyrosine residues; however, it lacks any catalytic domain. Cowan and Henkemeyer used a modified yeast two-hybrid screen to identify Grb4 as a protein that interacted with the tyrosine-phosphorylated ephrin-B1 cytoplasmic domain. Grb4 has one SH2 domain (which recognizes phosphotyrosines) and three SH3 domains (which recognize proline-rich regions in target proteins). The interaction between Grb4 and ephrin-B1 was confirmed with glutathione S -transferase pull-down experiments and coimmunoprecipitation experiments and was specific for Grb4 and did not occur with the related protein Nck. Analysis of cells transfected to express ephrin-B1 showed that ephrin-B1 and Grb4 colocalized and that activation of ephrin-B1 led to the loss of stress fibers, the redistribution of paxillin, and an increase in the phosphorylation of focal adhesion kinase on a specific tyrosine. Finally, screens to identify binding partners for the SH3 domain of Grb4 identified four candidates: Cbl-associated protein CAP (known to have a role in focal adhesions), axin, Abl-interacting protein-1, dynamin, heterogeneous nuclear ribonucleoprotein K, and the kinase Pak1. Many of these putative partners have roles in regulating the actin cytoskeleton. Confirmation of the relevance of the CAP interaction came from experiments showing that activated ephrin-B1 coimmunoprecipitated with CAP and that CAP redistributed to sites of active ephrin-B1 signaling in transfected cells and in native cells. The cellular and biochemical effects of activated ephrin-B1 were inhibited by coexpression of the SH2 domain of Grb4, confirming a role for Grb4 as an adaptor from the phosphorylated ephrin-B1 cytosolic domain to the signaling pathways that control the cytoskeleton. C. A. Cowan, M. Henkemeyer, The SH2/SH3 adaptor Grb4 transduces B-ephrin signals. Nature 413 , 174-179 (2001). [Online Journal]