VEGFR N-glycosylation

Vascular endothelial growth factor receptor-2 (VEGFR2) is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. VEGFR2 post-translational modifications (PTMs) and their roles in receptor function and signaling have been extensively studied. However, until recently, co- and post-translational N-glycosylation of RTKs has been regarded as decorative rather than functional. N-glycosylation is a non-template based process that generates heterogeneously modified proteins. Emerging evidence suggests that the tumor microenvironment modulates expression of glycosyltransferases involved in the trimming and remodeling of nascent N-linked glycans into their heterogeneous mature forms, and that these changes alter the functions of modified proteins and thereby impact cellular signaling and adhesion. We sought to understand the consequences of altered glycosylation on VEGFR2 signaling. We used multiple strategies, including: enzymatic removal of N-linked glycans from the receptor, site-directed mutagenesis of specific N-glycosylation sites near the VEGF ligand binding site of VEGFR2, mass spectrometry of VEGFR2 glycopeptides, and concurrent measurement of receptor activation, signaling, and dimerization to interrogate how N-glycosylation modulates VEGFR2 ligand-dependent pro-angiogenic signaling. We demonstrate that VEGFR2 glycosylation at site N247 regulates ligand-dependent receptor activation and signaling. Complex N-glycans with α2-6 linked sialic acid residues at site N247 hinder receptor activation, while asialo-glycans favor VEGFR2 ligand-mediated activation and signaling.