NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting

Abstract

Endothelial cells express neuropilin 1 (NRP1), endoglin (ENG) and vascular endothelial growth factor receptor 2 (VEGFR2), which regulate VEGF-A-mediated vascular development and angiogenesis. However, the link between complex formation among these receptors with VEGF-A-induced signaling and biology was unclear. Here, we quantified surface receptor interactions by IgG-mediated immobilization of one receptor, and fluorescence recovery after photobleaching (FRAP) measurements of the mobility of another coexpressed receptor. We observed stable ENG/NRP1, ENG/VEGFR2, and NRP1/VEGFR2 complexes, which were enhanced by VEGF-A. ENG augmented NRP1/VEGFR2 interactions, suggesting formation of tripartite complexes bridged by ENG. Effects on signaling were measured in murine embryonic endothelial cells expressing (MEEC+/+) or lacking (MEEC-/-) ENG, along with NRP1 overexpression or knockdown. Optimal VEGF-A-mediated phosphorylation of VEGFR2 and Erk1/2 required ENG and NRP1. ENG or NRP1 increased VEGF-A-induced sprouting, becoming optimal in cells expressing all three receptors, and both processes were inhibited by a MEK1/2 inhibitor. We propose a model where the maximal potency of VEGF-A involves a tripartite complex where ENG bridges VEGFR2 and NRP1, providing an attractive therapeutic target for modulation of VEGF-A signaling and biological responses.