VE-cadherin interaction proteomics identifies ARVCF as a key stabilizer of endothelial adherens junctions

The endothelial cells lining the vascular wall maintain a selective barrier between blood and tissue. Structural connections between these endothelial cells are formed through vascular endothelial (VE)-cadherin-based adherens junctions. The extracellular domain of VE-cadherin forms homotypic bonds between neighboring cells, while its intracellular domain connects to the actin cytoskeleton via a conserved protein complex including α-, ß- and p120-catenins. Whether additional proteins bind to VE-cadherin and contribute to endothelial junction integrity remains unclear. By using mass spectrometry after VE-cadherin immunoprecipitations from human endothelial cells, we have determined the molecular interactions with VE-cadherin. The proteomics identified a core VE-cadherin interactome, consisting of nine proteins, which bind to VE-cadherin even in the absence of tyrosine phosphorylation of its intracellular domain. The core VE-cadherin interactome includes the known catenin proteins as well as four new interactors: ARVCF, ARHGAP23, KEAP1 and NGLY1. Co-immunoprecipitation and co-localization experiments verified that the VE-cadherin-binding protein ARVCF is an important component of endothelial adherens junctions. ARVCF binds to a selective pool of VE-cadherin proteins during junction maturation that is unbound from p120-catenin, through a mechanism involving the C-terminal intrinsically disordered regions of ARVCF. Depletion of ARVCF results in loss of endothelial barrier function and impairs collective cell migration. Accordingly, ARVCF is needed for VE-cadherin-based junction stabilization. Together, our results demonstrate that ARVCF is a key regulator of VE-cadherin to safeguard junctional stability and endothelial integrity.