Vascular endothelial growth factor (VEGF) signals through VEGF receptor 2 (VEGFR2) to regulate arterial morphogenesis. Noting that mice lacking the scaffolding protein synectin show decreased arterial morphogenesis and that synectin–/– arterial endothelial cells (AECs) have an impaired response to VEGF, Lanahan et al. investigated the role of synectin in VEGFR2 signaling. Comparison of wild-type and synectin–/– AECs revealed no difference in the abundance of total or cell-surface VEGFR2, and their stimulation with VEGF-A elicited similar degrees of VEGFR2 tyrosine residue 1054 (Y1054) phosphorylation, indicating similar VEGFR2 activation. However, VEGF-A–dependent phosphorylation of VEGFR2 Y1175, a residue critical for vasculogenesis, was decreased in synectin–/– AECs, as were downstream signaling events [including extracellular signal–regulated kinase 1 and 2 (ERK1/2) phosphorylation]. Full-length synectin rescued phosphorylation of Y1175 and ERK1/2, whereas mutants unable to bind myosin-VI did not; furthermore, myosin-VI–/– AECs showed decreased VEGF-A–dependent Y1175 and ERK1/2 phosphorylation. Although synectin–/– and myosin-VI–/– AECs showed normal VEGF-dependent VEGFR2 internalization, immunofluorescence analysis revealed a delay in its association with EEA1-positive endosomes and live cell imaging revealed decreased movement of VEGFR-containing vesicles. Like synectin–/– mice, mice lacking myosin-VI showed impaired arterial morphogenesis; similarly, myosin-VI knockdown in zebrafish phenocopied arterial defects produced by synectin knockdown and, like synectin knockdown, decreased proliferation of arterial endothelial cells. Knockdown of the protein tyrosine phosphatase PTP1b increased phosphorylation of Y1175 and ERK1/2 in synectin–/– AECs and enhanced their migration so that it was comparable to that of wild-type AECs. Moreover, arterial phenotype was rescued by PTP1b knockdown in myosin-VI knockdown zebrafish embryos or its inhibition in synectin–/– mice. The authors propose that phosphorylation of VEGFR2 Y1175, and thereby the generation of signals crucial to arterial morphogenesis, depends on synectin- and myosin-VI–mediated VEGFR2 trafficking to EEA1-positive endosomes and away from regions where it is exposed to PTP1b.
A. A. Lanahan, K. Hermans, F. Claes, J. S. Kerley-Hamilton, Z. W. Zhuang, F. J. Giordano, P. Carmeliet, M. Simons, VEGF receptor 2 endocytic trafficking regulates arterial morphogenesis. Dev. Cell 18, 713–724 (2010). [PubMed]