Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor

doi:10.1074/jbc.M809259200

Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor*^♦

^‡Université Pierre et Marie Curie, Univerisité Paris 06, UR4, Aging, Stress and Inflammation and ^§Institut Fédératif de Recherche 83, 75252 Paris, France and the ^¶Université Paris-Sud XI, CNRS Unité Mixte de Recherche 8619, IBBMC, 91400 Orsay, France

1 To whom correspondence should be addressed: Université Pierre et Marie Curie, UR4, Aging, Stress and Inflammation, BP256, 7 Quai St-Bernard, 75005 Paris, France. Tel.: 33-1-44-27-21-36; Fax: 33-1-44-27-21-35; E-mail: zhenlin.li{at}upmc.fr.

Abstract

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A₂/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44–77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120–210). A 25-mer peptide named P46 (aa 46–70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

Footnotes

↵2 The abbreviations used are: PEDF, pigment epithelium-derived factor; AD, activation domain; BD, binding domain; KAP, keratin-associated protein; LR laminin receptor; PNPLA2, PLA2/nutrin/patatin-like phospholipase domain-containing 2; RU, resonance unit; TUNEL, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling; Y2H, yeast two-hybrid; aa, amino acid(s); HuBMEC, human bone marrow endothelial cell; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; FBS, fetal bovine serum; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; DAPI, 4′,6′-diamino-2-phenylindole; SPR, surface plasmon resonance; siRNA, small interfering RNA; HA, hemagglutinin; EC, endothelial cell; X-α-Gal, 5′-bromo-4-chloro-3′-indolyl-α-d-galactopyranoside.
↵* This work was supported by funds from the Association pour la Recherche sur le Cancer (ARC, 3587, to Z. L.) and by fellowships from ARC and Naturalia et Biological (to A. B.), Retina France (to J. G.), and Fondation de Recherche Médicale (to C. A. F.).
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and S2.
↵♦ This article was selected as a Paper of the Week.
- Received December 9, 2008.
- Revision received February 11, 2009.
The American Society for Biochemistry and Molecular Biology, Inc.