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J. Cai, S. Han, Q. Ruan, L. Wu, M. B. Grant, M. E. Boulton; Identification of a -Secretase-Catalyzed Transmembrane Cleavage Site for VEGFR1 in Retinal Vascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):63.
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© ARVO (1962-2015); The Authors (2016-present)
The antiangiogenic activity of PEDF is associated with the γ-secretase-catalysed cleavage of VEGF receptors. The aim of this study was to identify the VEGFR1 cleavage site and determine how amino acid substitution affects VEGFR1 binding to γ-secretase and the intracellular translocation of VEGFR-1.
The GFP-tagged human VEGFR1 expression vector (pVEGFR1-EGFP) underwent site-directed mutagenesis to substitute valine767 (a putative cleavage site) with alanine (pVEGFR1-EGFP-A167). Porcine aortic endothelial cells (PAECs) lacking VEGF receptors were transfected with either pVEGFR1-EGFP, pVEGFR1-EGFP-A167 or empty vector. Stably transfected cells were selected using kanamycin resistance and exposed to VEGF (100ng/ml) in the presence or absence of PEDF (100ng/ml) for up to 24 hours. VEGFR1 binding to γ-secretase complex was determined by immunoprecipitation of VEGFR1 followed by Western Blot analysis for the components of the γ-secretase complex (presenilin [PS1], nicastrin [NTC], APH-1 and PEN-2). Peptide analysis by LC-MS/MS confirmed the identity of the bound proteins. Intracellular translocation of VEGFR1 was analyzed using confocal microscopy and Western blot following subcellular fractionation.
Neither VEGF nor PEDF affected the overall expression levels of VEGFR1 in PAECs. Both Western blot analysis and LC-MS/MS demonstrated that PEDF+VEGF resulted in NTC binding to VEGFR1 by 5 minutes followed by APH-1 and PS1 at 30min and finally, PEN-2 at 1hr for both wild type and mutant-VEGFR1. Neither VEGF nor PEDF alone promoted binding of VEGFR1 to the γ-secretase complex. Intracellular translocation of VEGFR1-EGFP was followed by confocal microscopy. VEGF alone induced nuclear translocation of VEGFR1 in cells transfected with both wild type and mutant-VEGFR1, whereas addition of PEDF alone did not induce VEGFR1 translocation. However, VEGF+PEDF in combination blocked the VEGF-induced nuclear translocation of VEGFR1 in the cells transfected with wild type-VEGFR1 vector but not mutant, confirming that V767 plays a critical role in translocation. Western blot for the intracellular fragment of VEGFR1 confirmed PEDF-induced cleavage of wild type-VEGFR1 in the presence of VEGF resulting in the appearance of a cytosolic VEGFR1 fragment which was absent in cells with mutant VEGFR1.
V767 is a γ-secretase-catalyzed cleavage site on VEGFR-1 and may offer a novel therapeutic strategy to regulate VEGF activity in conditions associated with pathological angiogensis.
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