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P. Subramanian, S. Locatelli-Hoops, S. Choxi, G. M. Seigel, S. P. Becerra; Mechanistic Insight into the Interaction of Pigment-epithelium Derived Factor and Its Receptor PEDF-R for Retinal Cell Survival. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2336.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment epithelium-derived factor (PEDF), a neurotrophic factor, has high affinity receptors on the surface of target cells. PEDF-R is a transmembrane protein present in the retina. It exhibits phospholipase A2 (PLA2) activity and high affinity for PEDF. Recently we have identified a PEDF-binding region in PEDF-R. In this study, we investigate the biological significance of PEDF: PEDF-R interactions for survival of R28 rat retinal cells.
Plasma membrane proteins were isolated from rat retina R28 cells with 0.1% NP-40. PEDF-R protein was detected by immunoblotting. PLA2 activity was determined by spectrophotometric assays. Cell survival and death was monitored using real-time microelectronic cell sensor system (ACEA) and CellTiter-Glo luminescent assay. Peptides were chemically synthesized and recombinant PEDF protein was purified from conditioned media of BHK cells harboring expression vectors for full-length human PEDF.
PEDF-R was immunodetected as an 80-kDa protein in the plasma membrane fractions of R28 cells. The immunodetected protein band was blocked by incubating the antibody with a specific peptide antigen. R28 plasma membrane fraction exhibited PLA2 activity, which was further stimulated by the addition of PEDF in a concentration-dependent manner. Serum starvation of R28 cells induced cell death. Exogenous addition of nanomolar concentrations of PEDF promoted R28 cell survival in a dose-dependent fashion. Peptides from the longest extracellular region of PEDF-R were generated and tested for PEDF binding. Preincubation of PEDF with PEDF-R-derived peptides containing PEDF affinity blocked the survival effect of PEDF on R28 cells. No effect on cell viability was observed with peptide alone or with peptides lacking PEDF affinity.
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