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1 From the Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, and the 3 Department of Cell and Cancer Biology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
PURPOSE. The neurotrophic activity of pigment epithelium–derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined.
METHODS. Binding reactions were performed with 125I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with 125I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy.
RESULTS. Radioligand-binding assays showed that 125I-PEDF bound in a
specific and saturable fashion to one class of sites on retina
membranes (Kd = 2.5–6.5 nM; maximum
binding [Bmax] = 1–48 x
1010 sites/retina). A peptide of 44 amino acids (44-mer),
identified as the receptor-binding region of PEDF, competed efficiently
for 125I-PEDF binding to retina membranes with kinetics
similar to the full-length PEDF. Ligand blot analysis and
ligand–affinity chromatography revealed a specific and high-affinity
PEDF-binding protein of 
85 kDa in retina plasma membranes. Confocal
microscopy showed that fluorescein-conjugated PEDF stained exclusively
the inner segments of photoreceptors and cells of the ganglion cell
layer in retinal cryosections.
CONCLUSIONS. Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.
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