Abstract
Purpose: :
PEDF is a neurotrophic and antiangiogenic protein that acts on the retina. A recently identified gene from the retinal pigment epithelium (RPE) codes for a putative binding partner for PEDF, termed PEDF receptor (PEDF–R). Its amino acid sequence predicts four transmembrane domains, with three intracellular and two extracellular regions, and a phospholipase A2 domain. The aim of this study is to investigate the PEDF–R expression and distribution in the retina, its subcellular localization in mammalian cells, and the functionality of its predicted regions.
Methods: :
Expression of PEDF–R mRNA was determined by RT–PCR. Immunohistochemistry was performed with sections of albino rat eyes. COS–7 and 3T3L1 cells were transfected with epitope–tagged PEDF–R expression vectors. Antibodies were developed against peptides derived from presumed extracellular and intracellular domains. Antibodies specificities were assessed in western blots. PEDF conjugated with fluorescein was used in cell binding assays. Epitope–tagged PEDF–R was synthesized in E. coli cell–free extracts and purified by affinity column chromatography. Protein–protein interactions were analyzed by surface plasmon resonance. Phospholipase A activity was assayed spectrophotometrically.
Results: :
PEDF–R mRNA expression was detected in human RPE and retina tissues, and in ARPE19 and rat neural retina cell lines. PEDF–R immunolabeling was distributed in the rat RPE and in the inner segments of photoreceptors. Recombinant epitope–tagged PEDF–R protein partitioned to both membrane and cytosolic fractions of transfected cells. Antibodies against predicted extracellular and intracellular regions labeled both the cytoplasm and surface of permeabilized ARPE19 and 3T3L1 cells. However, only the antibody against a predicted extracellular PEDF–R region labeled the surface of non–permeabilized cells. Live cells exposed to Fl–PEDF showed fluorescence on their cell–surfaces in a dose–response fashion, which decreased by ligand competition with excess of unlabeled PEDF. Kinetic analysis demonstrated that purified recombinant epitope–tagged PEDF–R interacted specifically with high affinity with immobilized PEDF. PEDF–R exhibited a potent phospholipase A activity that liberated fatty acids, which was stimulated by PEDF.
Conclusions: :
These results provide evidence for a phospholipase–linked plasma membrane protein with binding affinity for PEDF that is present in the RPE and photoreceptors. They suggest a molecular pathway by which ligand/receptor interactions on cell–surface could generate a cellular signal.
Keywords: receptors • retina • photoreceptors