Together, Neuropilin 1 and Neuropilin 2 direct α5 integrin trafficking through GTPase-activating-protein p120RasGAP in endothelial cells to promote fibronectin fibrillogenesis

Abstract

AbstractIntegrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell’s behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5 integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed traffic remain poorly annotated. Using label-free quantitative mass spectrometry of α5 integrin associations in ECs we identify α5 trafficking pathways that depend on NRP1, NRP2, or both NRPs. We identify a trafficking pathway that depends on both NRPs: one that impinges on the GTPase-activating protein p120RasGAP. This pathway promotes the recycling of α5 integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5 integrin-NRP1-NRP2 complexes to Rab11+recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, results in the accumulation of α5 integrin in early endosomes, a loss of α5 integrin from surface adhesions, and attenuated EC polarisation. Importantly, endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated ourin vitrofindings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors can co-traffic internalised cargoes.