Abstract
Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Here, we investigated the presence of PEDF receptors on retinoblastoma Y-79 and CG cells. PEDF radiolabeled with (l25)I remained biologically active and was used for radioligand binding analysis. The binding was saturable and specific to a single class of receptors on both cells and with similar affinities (K(d) = 1.7-3.6 nM, B(max) = 0.5-2.7 x 10(5) sites/Y-79 cell; and K(d) = 3.2 nM, B(max) = 1.1 x 10(3) sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the (125)I-PEDF binding. We designed two peptides from a region previously shown to confer the neurotrophic property to human PEDF, synthetic peptides 34-mer (positions 44-77) and 44-mer (positions 78-121). Only peptide 44-mer competed for the binding to Y-79 cell receptors (EC(50) = 5 nM) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of approximately 80 kDa. These results are the first demonstration of a PEDF-binding protein with characteristics of a PEDF receptor and suggest that the region comprising amino acid positions 78-121 of PEDF might be involved in ligand-receptor interactions.
Highlights
From the Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892
We have previously shown that a polyclonal antiserum to human Pigment epithelium-derived factor (PEDF) blocks the neuronal differentiating activity of the purified protein and of the interphotoreceptor matrix extracts on Y-79 cells [12], indicating that PEDF is the sole component of the interphotoreceptor matrix with such activity
We found that 125I-PEDF, prepared from either native bovine or recombinant human PEDF protein, induced the typical morphological differentiation on retinoblastoma Y-79 cells as previously demonstrated for their unlabeled counterparts [12, 13, 24, 26]
Summary
From the Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892. Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Pigment epithelium-derived factor (PEDF) was initially identified as a protein secreted by cultured human fetal retinal pigment epithelial cells with potent neuronal differentiating activity on retinoblastoma cells [1]. We have previously shown that a polyclonal antiserum to human PEDF blocks the neuronal differentiating activity of the purified protein and of the interphotoreceptor matrix extracts on Y-79 cells [12], indicating that PEDF is the sole component of the interphotoreceptor matrix with such activity. Structure-function studies demonstrated that the serpin reactive loop located toward the carboxyl end of the polypeptide is dispensable, whereas a region toward the amino end (BA, amino acid positions 44 –121) confers the neurotrophic activity to the PEDF polypeptide [26]. We describe here that PEDF exhibits a saturable and specific binding to target cells for neurotrophic activity and demonstrate for the first time a PEDF-binding protein with characteristics of a PEDF receptor
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