The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu¹⁵⁹–Met³²⁵) with affinity similar to the full-length PEDF-R (Met¹–Leu⁵⁰⁴). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile¹⁹³–Leu²³²) and P1 (Thr²¹⁰–Leu²⁴⁹) peptides. Recombinant C-terminal truncated PEDF-R4 (Met¹–Leu²³²) and internally truncated PEDF-R and PEDF-R4 (ΔHis²⁰³–Leu²³²) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His²⁰³–Leu²³² region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

Purpose.: Pigment epithelium-derived factor (PEDF) is a serpin with antiangiogenic properties. Previously, the authors showed that PEDF injected into the subconjunctiva reaches the choroid. Here, they examined the effects of PEDF polypeptide fragments on vessel sprouting and on choroidal neovascularization (CNV) after subconjunctival administration. Methods.: Recombinant human PEDF (rhuPEDF) was cleaved at its serpin-exposed loop by limited chymotrypsin proteolysis. Synthetic PEDF peptides 34-mer (Asp⁴⁴-Asn⁷⁷) and 44-mer (Val⁷⁸-Thr¹²¹) were used. Ex vivo chick aortic vessel sprouting assays were performed. CNV was induced in rats by laser injury of Bruch's membrane. Daily subconjunctival injections (0.01–10 pmol/d protein) were performed for 5 days starting at day of injury or at the seventh day after injury. New vessel volumes were quantified using optical sections of choroid/RPE flat-mounts labeled with isolectin-Ib4. PEDF distribution was evaluated by immunofluorescence of choroid/RPE/retina cross-sections. Results.: Full-length rhuPEDF, cleaved rhuPEDF, or peptide 34-mer exhibited ex vivo antiangiogenic activity, but peptide 44-mer was inefficient. PEDF immunostaining around CNV lesions diminished after laser injury. Subconjunctival administration of rhuPEDF or 34-mer at 0.1 pmol/d decreased CNV lesion volumes by 52% and 47%, respectively, whereas those of 44-mer were similar to vehicle injections. Doses of 0.1 and 1 pmol/d rhuPEDF decreased fully developed CNV complex volumes by 45% and 50%, respectively, compared with vehicle injections. Conclusions.: A functional region for the inhibition of vessel sprouting and CNV resides within the 34-mer region of PEDF. Furthermore, subconjunctival administration of optimal range dosages of rhuPEDF or 34-mer can suppress and regress rat CNV lesions, demonstrating that these agents reach the choroid/RPE complex as functionally active molecules.

Pigment epithelium‐derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a ∼ 60 kDa PEDF‐binding protein in bovine retina with Bos taurus F₁‐ATP synthase β‐subunit, and that F₁F_o‐ATP synthase components have been identified recently as cell‐surface receptors, we examined the direct binding of PEDF to F₁. Size‐exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F₁. Real‐time binding as determined by surface plasmon resonance demonstrated that yeast F₁ interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F₁ and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF‐binding and angiostatin‐binding sites on F₁. Surfaces of endothelial cells exhibited affinity for PEDF‐binding proteins of ∼ 60 kDa. Antibodies to F₁β‐subunit specifically captured PEDF‐binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell‐surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell‐surface F₁, these results show that PEDF is a ligand for endothelial cell‐surface F₁F_o‐ATP synthase. They suggest that PEDF‐mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.Structured digital abstract MINT‐7711286: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F‐ATPase alpha subunit (uniprotkb:P07251), F‐ATPase beta subunit (uniprotkb:P00830), F‐ATPase gamma subunit (uniprotkb:P38077), F‐ATPase delta subunit (uniprotkb:Q12165) and F‐ATPase epsilon subunit (uniprotkb:P21306) by competition binding (MI:0405) MINT‐7711113: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F‐ATPase epsilon subunit (uniprotkb:P21306), F‐ATPase delta subunit (uniprotkb:Q12165), F‐ATPase gamma subunit (uniprotkb:P38077), F‐ATPase beta subunit(uniprotkb:P00830) and F‐ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) MINT‐7711060: F‐ATPase gamma subunit (uniprotkb:P38077), F‐ATPase beta subunit (uniprotkb:P00830), F‐ATPase alpha subunit (uniprotkb:P07251) and PEDF (uniprotkb:P36955) physically interact (MI:0915) by molecular sieving (MI:0071) MINT‐7711313: F‐ATPase epsilon subunit (uniprotkb:P21306), F‐ATPase delta subunit (uniprotkb:Q12165), PEDF (uniprotkb:P36955), F‐ATPase alpha subunit (uniprotkb:P07251), F‐ATPase beta subunit (uniprotkb:P00830) and F‐ATPase gamma subunit(uniprotkb:P38077) physically interact (MI:0915) by molecular sieving (MI:0071) MINT‐7711083: PEDF (uniprotkb:P36955) physically interacts (MI:0915) with F‐ATPase epsilon subunit (uniprotkb:P21306), F‐ATPase delta subunit (uniprotkb:Q12165), F‐ATPase gamma subunit (uniprotkb:P38077), F‐ATPase beta subunit (uniprotkb:P00830) and F‐ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107)