Abstract
Purpose :
The vascular glycocalyx constituent and endothelial-specific type I integral membrane glycoprotein, endomucin (EMCN), regulates VEGF165-induced internalization of VEGF receptor (VEGFR) 2 and its downstream activities. The aim of this study is to investigate the molecular mechanism of EMCN's role in VEGFR2 clathrin-mediated endocytosis (CME) and its specificity in cell surface angiogenesis-related receptor tyrosine kinase endocytosis and signaling.
Methods :
Immunoprecipitation (IP) of EMCN in human retinal endothelial cells (HRECs) followed by mass spectrometry was employed to identifiy potential EMCN binding partners. The STRING database was used to analyze protein-protein interactions and functional clustering of these partners. Interactions between EMCN and AP2 α2 and β2 subunits in HRECs were confirmed through IP. EMCN was knocked down using siEMCN, with non-targeting siRNA as a control. A wound healing assay examined endothelial cell migration, while angiogenic receptor internalization (VEGFR2, VEGFR1, and FGFR1) was assessed by biotin labeling, extraction using avidin-conjugated beads, and visualization via western blot.
Results :
From the 416 EMCN-binding proteins identified by mass spectrometry, STING functional clustering revealed 15 proteins involved in endocytosis. Our IP validated EMCN interactions with AP2 subunits AP2α2 and AP2β2. VEGFR2 and AP2 interacted in the presence EMCN but not after EMCN knockdown. EMCN depletion had no effect on PlGF-2-induced HREC migration (1.11 ± 0.0707 vs. 1.38 ± 0.0601, P < 0.05, n=6 or 8) or VEGF165 (10 ng/ml) induced VEGFR1 internalization after 30 min, compared to the control (1.50 ± 0.117 vs. 0.625 ± 0.108, P < 0.001, n=6). FGF-induced HREC migration and FGFR1 internalization was unaffected by EMCN knockdown. EMCN knockdown significantly reduced VEGF165- and VEGF121-induced HREC migration (1.15 ± 0.0235 vs. 1.01 ± 0.0348, P<0.05 and 1.19 ± 0.0352 vs. 1.04 ± 0.0152, P<0.01, respectively, n=10). VEGF121-induced significant VEGFR2 internalization after 60 min (1.00 vs. 0.780 ± 0.00925, P<0.05, n=3). VEGFR2 internalization continued up to 120 min after VEGF121 stimulation but was prevented in EMCN-knockdown cells (0.577 ± 0.0359 vs. 0.787 ± 0.0192, P<0.05, n=3).
Conclusions :
Our data suggest that EMCN regulates VEGFR2 endocytosis through the AP2 complex and that EMCN modulation of ligand-driven VEGFR2 CME and signaling is specific for VEGFR2.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.