Identification of a Lipase-linked Cell Membrane Receptor for Pigment Epithelium-derived Factor

Abstract

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDFbinding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDFbinding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A 2 (PLA 2 ) and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA 2 /nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A 2 activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A 2 activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.

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

“…The structure of the exons of PNPLA2 reflects the proposed domain structure of the protein with three endodomains (L1, L3, and L5) and two ectodomains (L2 and L4) (35). Immunoreactivity of non-permeabilized cells, FACS, antibody capture experiments using antibodies to peptides from intracellular L3 and extracellular L4 domains, and cell surface biotinylation experiments agree with the predicted PEDF-R topology and show that PEDF-R is one of the proteins labeled at the surface of ARPE-19 cells (26). The amino acid sequence reveals a phospholipase A 2 (PLA 2 ) domain toward its amino end.…”

“…The amino acid sequence reveals a phospholipase A 2 (PLA 2 ) domain toward its amino end. Indeed, PEDF-R exhibits PLA 2 , triglyceride lipase, and acylglycerol transacylase activities (26,34). Moreover, we have shown that PEDF stimulates the PLA 2 activity of the PEDF-R enzyme, resulting in the release of fatty acids from phospholipids (26,35).…”

“…The PEDF-R protein specifically binds PEDF with high affinity (K D ϭ 3 nM) and does not have affinity for other serpins like maspin and ovalbumin (26). It is detected in the inner segments of the photoreceptors, at lower levels in the inner retina and retinal ganglion cell layer of the native retina, and also in the retinal pigment epithelium.…”

“…The molecular mechanism of PEDF multifunctionality could be explained by responses to interactions with distinct cell surface receptors. We have identified the novel gene PNPLA2 in the retina that encodes a lipase-linked cell membrane protein with high affinity for PEDF and termed it PEDF-R (26). Later, other PEDF-binding proteins were reported in endothelial and tumor cells (37/67-kDa non-integrin laminin receptor (27) and cell surface F 1 F 0 -ATP synthase (28,29)) and * This work was supported, in whole or in part, by the National Institutes of on ARPE-19 cells (LRP6, a Wnt co-receptor (30)).…”

“…Interestingly, the distribution of PEDF-R in the retina matches that of PEDF binding sites (25), implying that these cells contain PEDF-R molecules available to interact with PEDF. Most tissues express PNPLA2, including retina and brain (26,(31)(32)(33). PEDF-R is a member of the PNPLA2 (patatin-like phospholipase domain-containing 2) family and is also known as adipocyte triglyceride lipase, transport secretion protein-2.2, Ca 2ϩ independent phospholipase A2, and desnutrin (32)(33)(34).…”