Unlike the other members of the Roundabout (Robo) transmembrane receptor family that mediate axon repulsion in response to binding to Slit, Robo4 is found primarily in vascular endothelial cells. Genetic ablation of Robo4 in mice leads to increased vascular permeability, and Koch et al. sought to identify binding partners of Robo4 that might account for this phenotype. They found that the Ig2 domain in the extracellular domain of Robo4 bound to the extracellular domain of UNC5B, a transmembrane receptor for Netrin. Addition of a soluble protein encompassing the Ig2 domain of Robo4 (which the authors termed sRobo4) to cells expressing UNC5B triggered the internalization of UNC5B. Furthermore, cells expressing UNC5B were repulsed from cells expressing Robo4, resulting in segregation of UNC5B- and Robo4-expressing cells, an effect that required the cytoplasmic domain of UNC5. This suggested that Robo4 acted as a ligand in trans for UNC5B and activated signaling downstream of UNC5B and, accordingly, UNC5B coimmunoprecipitated with phosphorylated and activated Src after endothelial cells were treated with sRobo4. The authors generated an antibody directed against Robo4 (anti–Robo4-1) that blocked the binding of UNC5B to Robo4. In human umbilical artery endothelial cells or wild-type mouse endothelial cells stimulated with vascular endothelial growth factor (VEGF), the association of activated Src with VEGF receptor 2 (VEGFR2) was increased with incubation with anti–Robo4-1 and decreased with sRobo4 treatment, suggesting that stimulation of UNC5B by Robo4 could sequester activated Src from VEGFR2. In addition, intraperitoneal injection of anti–Robo4-1 increased vascularization in corneas. Robo4–/– mice showed increased vessel permeability, a phenotype that was rescued by injection of sRobo4 (and further enhanced by cotreatment with PP2, a Src family tyrosine kinase inhibitor) and that could be mimicked in wild-type mice by injection of anti–Robo4-1. Thus, the authors propose that the UNC5B-Robo4 interaction opposes VEGF-induced angiogenesis and vascular permeability by preventing the association of Src with VEGFR2.
A. W. Koch, T. Mathivet, B. Larrivée, R. K. Tong, J. Kowalski, L. Pibouin-Fragner, K. Bouvrée, S. Stawicki, K. Nicholes, N. Rathore, S. J. Scales, E. Luis, R. del Toro, C. Freitas, C. Bréant, A. Michaud, P. Corvol, J.-L. Thomas, Y. Wu, F. Peale, R. J. Watts, M. Tessier-Lavigne, A. Bagri, A. Eichmann, Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B. Dev. Cell 20, 33–46 (2011). [PubMed]