Structure‐function relationships of pigment epithelium‐derived factor receptor (PEDF‐R): Identification of a PEDF binding region

Abstract

PEDF‐R/ATGL is found in the retina and has high affinity for pigment epithelium‐derived factor (PEDF), a retina cell survival protein. PEDF‐R exhibits phospholipase A2 (PLA) activity, which is stimulated by PEDF. We aim at identifying the structural determinant for PEDF binding in PEDF‐R that relates to retina survival. Recombinant C‐terminal truncated PEDF‐R (M1‐L232) retained PLA activity, which increased with PEDF additions, as did full‐length PEDF‐R (M1‐L504). However, a single D166A alteration in PEDF‐R caused loss of PLA activity. Surface plasmon resonance (SPR) revealed that recombinant PEDF‐R, PEDF‐R[D166A] and L4 (L159‐M325) had similar PEDF affinity. Ligand blot, SPR and affinity chromatography demonstrated that synthetic peptides E5b (I193–L232), P1 (T210‐L249) and E5d (T210‐L232) specifically bound PEDF. Their effect on PEDF‐mediated survival activity was examined in retina R28 cells. PEDF‐R protein was detected in R28 membrane fractions and its PLA activity was stimulated by PEDF additions. PEDF mediated R28 cell survival, which was attenuated by E5b or P1 additions. Thus, the C‐terminal half of PEDF‐R is dispensable for PLA activity and PEDF binding. D166 is crucial for PLA activity. E5b contains structural determinants for binding PEDF. This novel site may form part of the heterodimer interface of PEDF‐R with the ligand, which is required for retina cell survival. Supported by NIH‐IRP.