April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Identification of a -Secretase-Catalyzed Transmembrane Cleavage Site for VEGFR1 in Retinal Vascular Endothelial Cells
Author Affiliations & Notes
  • J. Cai
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • S. Han
    Surgery,
    University of Florida, Gainesville, Florida
  • Q. Ruan
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • L. Wu
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • M. B. Grant
    Pharmacology and Therapeutics,
    University of Florida, Gainesville, Florida
  • M. E. Boulton
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  J. Cai, None; S. Han, None; Q. Ruan, None; L. Wu, None; M.B. Grant, None; M.E. Boulton, None.
  • Footnotes
    Support  NIH Grant EY018358
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 63. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: vascular endothelial growth factor • receptors • neovascularization 
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