Pigment epithelium-derived factor receptor (PEDF-R): a plasma membrane-linked phospholipase with PEDF binding affinity

Abstract

Pigment epithelium-derived factor (PEDF), a multifunctional protein, acts in retinal differentiation, survival and maintenance by interacting with high affinity receptors on the surface of target cells. We have recently identified PEDF-R, a new member of the patatin-like phospholipase domain-containing 2 (PNPLA2) family with characteristics of a PEDF receptor. The PEDF-R sequence reveals a patatin-like phospholipase domain toward its amino-end, and four transmembrane domains interrupted by two extracellular loops and three intracellular regions along its polypeptide sequence. This newly identified protein is present on the surface of retina and RPE cells, and has the expected transmembrane topology. It has specific and high binding affinity for PEDF, and exhibits a potent phospholipase A(2) activity that liberates fatty acids. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In summary, PEDF-R is a novel component of the retina that is a phospholipase-linked membrane protein with high affinity for PEDF. The results suggest a molecular pathway by which PEDF ligand/receptor interactions on the cell surface could generate a cellular signal. These conclusions enhance our understanding of the role of PEDF as a neurotrophic survival factor.

Fig. 4.1

In silico information of gene, transcript and protein structure of PEDF-R. (Top) Transcript summary information for human PEDF-R obtained from http://www.ensembl.org for ENST00000336615 (Hubbard et al. 2005). PEDF-R gene has 10 exons (top line), the transcript length is 2,071 bps (exons are illustrated by boxes; coding regions are black and grey; introns are lines flanking boxes); and the translation length is 504 amino acid residues. (Bottom) Computer programs predict a transmembrane topology for PEDF-R. Exons are mapped on the illustration and are coded as in Top Panel. Features illustrated are: the coding region corresponding to the yeast-2 hybrid clone p12c; the locations of peptide antigens for antibodies Ab-R^A and Ab-R^C, and the Ser⁴⁷-Asp¹⁶⁶ catalytic dyad

Fig. 4.2

Subcellular localization of PEDF-R in retinal cells. Cell lysates (Lys) were fractionated by high speed centrifugation (80,000×g) into cytosolic (cyt) and membrane fractions (Mb). Western blots of fractions were immunostained with anti-PEDF-R^A antibody. Total protein loaded in lanes were as follows: (ARPE-19, Lys) 19.8 μg; (ARPE-19, Cyt) 12 μg; (ARPE-19, Mb) 6.5 μg; (RPE, Cyt) 67 μg; (RPE, Mb) 10 μg; (Retina, Cyt) 71 μg; (Retina, Mb) 10 μg; (RGC-5, Mb) 9.5 μg; and (R29, Mb) 5.7 μg respectively. The asterisks point to the migration position of PEDF-R

Fig. 4.3

PLA activity of PEDF-R. Detergent-soluble membrane fractions from R28 cells were assayed for PLA activity using (1,2-dilinoleoyl)-phosphatidylcholine as substrate and lipoxygenase as the coupling enzyme in reaction buffer (50 mM Tris-HCl, pH 7.5, 3 mM deoxycholate) as described (Jimenez-Atienzar et al. 2003). Formation of the product was measured spectrophotometrically by increase in the absorbance at 234 nm per min (dA/min; y-axis). (a) Dose response of PLA activity of R28-derived PEDF-R. Proteins from R28 cell membranes were solubilized with phosphate buffered saline pH 6.5 containing 0.1% NP-40, and the PLA activity was determined for increasing amounts of detergent-soluble protein fractions, (b) Effects of PEDF on the R28-derived PEDF-R activity. Extracts were preincubated with PEDF for 10 min and then assayed for PLA activity. Concentrations of PEDF used in each assay are indicated in the x-axis. (c) PEDF-R proteins were solubilized from ARPE-19 membrane protein precipitate with phosphate buffered saline pH 6.5 containing 0.5% CHAPS in the absence (grey boxes) or presence of 0.8 μM PEDF (black boxes). PLA activity was measured as in Panel B in the absence (none) or presence of 10 nM PEDF