Abstract 2047: N-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling
Authors: Chandler Kevin B. , Leon Deborah R. , Kuang Jenevieve , Meyer Rosana D. , Rahimi Nader , Costello Catherine E. 
Published: August 2018

Abstract
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial receptor tyrosine kinase (RTK), is essential for tumor angiogenesis in tumors of diverse origin. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites. Changes in glycosylation influenced by hypoxia, the shunting of glycolytic intermediates via the hexosamine biosynthesis pathway into glycan synthesis, and pro-inflammatory cytokines have the potential to alter VEGFR-2 signaling in tumors. However, the role of glycosylation in VEGFR-2 signaling and function remain largely unknown. The objective of this study is to determine the functional importance of N-glycosylation in VEGFR-2 activation and angiogenic signaling.
Porcine aortic endothelial (PAE) cells with ectopic expression of either wild type VEGFR-2, or one in a series of VEGFR-2 constructs with single glycosylation site mutations in the ligand binding domain, were treated with VEGF-A for 0, 5, 10 or 30 minutes. VEGFR-2 tyrosine phosphorylation was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody to assess activation, and with anti-pTyr-1175-VEGFR-2 and anti-pTyr-1212-VEGFR-2 antibodies. Activation of key molecules involved in downstream signaling of VEGFR-2 was also monitored. To establish the glycosylation status of VEGFR-2 available for ligand-mediated signaling, we isolated plasma membrane localized VEGFR-2, performed enzymatic proteolysis, enriched glycopeptides via hydrophilic interaction liquid chromatography (HILIC), and analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation.
Mutation of one N-glycosylation site near the VEGF binding site increased ligand-dependent phosphorylation (at Tyr-1054, Tyr-1175 and Tyr-1212) within 5 min of ligand addition, and initial observations suggest that this resulted in higher levels of activated c-Src compared to wild type VEGFR-2, which may have implications for endothelial cell proliferation and migration. However, phosphorylation of other downstream signaling targets including Akt and PLCγ1 was not affected. Mutations at other N-glycosylation sites near the VEGF ligand binding site did not appear to impact ligand-mediated activation of VEGFR-2, suggesting that glycans located at certain N-linked sites may influence ligand-mediated activation while others have minimal impact. Targeting VEGFR-2 N-glycosylation may be a valuable strategy to inhibit tumor angiogenesis.
This research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.
Novel aspect: These results demonstrate that site-specific N-glycosylation of VEGFR-2 modulates ligand-mediated phosphorylation of key VEGFR-2 tyrosine residues involved in downstream signaling.
Citation Format: Kevin B. Chandler, Deborah R. Leon, Jenevieve Kuang, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. N-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2047.