Fig. 9: Schematic model for the interactions of ENG with VEGFR2 and NRP1 and their effects on VEGF-A signaling and sprouting.

The receptors, which form homodimers, are depicted as monomers for simplicity. Complex formation of VEGFR2 with ENG is required for signaling, which bridges between VEGFR2 and NRP1 to enhance signaling and sprouting. The receptors expressed by each cell line under specific conditions are shown at the top (MEEC^-/- cells treated with siNRP1 express mainly VEGFR2, MEEC^-/- cells express VEGFR2 and NRP1, MEEC^+/+ cells transfected by siNRP1 express VEGFR2 and ENG, while MEEC^+/+ express all three receptors, with overexpression of NRP1 in MEEC^+/+ transfected with NRP1). As shown by the two cell lines at the left, VEGFR2 alone (Figs. 6 and 8) or together with NRP1 (Figs. 5 and 7) does not induce significant signaling or sprouting upon VEGF-A binding. Expression of ENG with VEGFR2 (MEEC^+/+ + siNRP1) results in a mild response to VEGF-A in both biochemical signaling (Fig. 6) and sprouting (Fig. 8). These responses are enhanced by expression of NRP1 along with ENG and VEGFR2 (MEEC^+/+ cells; Figs. 5 and 7, or by transfection of MEEC^-/- cells with both ENG and NRP1 – Supplementary Fig. 7), and overexpression of NRP1 further increases the signaling and biological response (Figs. 5 and 7, and Supplementary Fig. 7). Together with our finding that ENG binds VEGFR2 and NRP1 at non-overlapping sites (Fig. 4 and Supplementary Fig. 4), we propose that the tripartite complex formed ENG bridging between VEGFR2 and NRP1 regulates the intensity of the VEGF-A-induced signaling and sprouting of ECs.