Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF Receptor (PEDF-R): identification of a functional ligand binding site

• Published in: The Journal of biological chemistry 2013-08, Vol.288 (33), p.23928-23942
• Main Authors: Subramanian, Preeti, Locatelli-Hoops, Silvia, Kenealey, Jason, DesJardin, Jacqueline, Notari, Luigi, Becerra, S Patricia
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Cell Death - drug effects; Cell Line; Cell Survival - drug effects; Eye Proteins - metabolism; Eye Proteins - pharmacology; Humans; Ligands; Models, Biological; Mutant Proteins - metabolism; Nerve Growth Factors - metabolism; Nerve Growth Factors - pharmacology; Neurobiology; Peptides - metabolism; Peptides - pharmacology; Phospholipases - metabolism; Protein Binding; Protein Structure, Tertiary; Rats; Receptors, Neuropeptide - chemistry; Receptors, Neuropeptide - metabolism; Recombinant Fusion Proteins - metabolism; Retina - cytology; Serpins - metabolism; Serpins - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.487884

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 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) 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.