Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R)

Abstract

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. Background: PEDF has neurotrophic activity and interacts with PEDF-R, a membrane-linked lipase. Results: A PEDF-binding region of PEDF-R is required for PEDF-R enzymatic stimulation, and peptides derived from this region block PEDF·PEDF-R-mediated retinal survival activities. Conclusion: A ligand binding domain is identified in PEDF-R, a critical receptor for the survival activity of PEDF. Significance: The findings provide mechanistic insight into the survival activity of PEDF.