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 l25I 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 × 105 sites/Y-79 cell; and K d = 3.2 nM, B max = 1.1 × 103 sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the125I-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 (EC50 = 5 nM) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of ∼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.
Pigment epithelium-derived factor (PEDF)1 was initially identified as a protein secreted by cultured human fetal retinal pigment epithelial cells with potent neuronal differentiating activity on retinoblastoma cells (1). Addition of PEDF at nanomolar concentrations to the media of human retinoblastoma Y-79 and Weri cells induces a neuronal phenotype, which is accompanied by the expression of the neuronal markers neuronal-specific enolase and 200-kDa neurofilament (2, 3). PEDF also exhibits neurotrophic activities on primary cultures of rat cerebellar granule (CG) neurons, such as neuronal survival (4) and protection against death by glutamate neurotoxicity (5) and by apoptosis (6). Recent reports indicate that it has effects on other types of neurons. It promotes the survival and differentiation of developing spinal motor neurons (7), and can protect them against glutamate neurodegeneration (8). PEDF can also protect developing primary hippocampal neurons against glutamate neurotoxicity (9). In addition, it delays the death of photoreceptors in mouse models of inherited retinal degenerations (retinitis pigmentosa) (10). Thus, PEDF is a potential neurotrophic factor of the central nervous system and retina. PEDF is also referred to as early population doubling level cDNA (EPC-1), reflecting its up-regulation during G0 in young but not senescent cultured fibroblasts (11), which suggests a role for the protein in cell maintenance.
PEDF is a glycoprotein with a molecular weight of 50,000 identified extracellularly in the vertebrate eye (12-15). It is associated by ionic interactions with glycosaminoglycans in the interphotoreceptor matrix (16) and can be readily purified from the interphotoreceptor matrix and vitreous (12, 13). 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. The antiserum to PEDF also blocks the neuronal survival effects of PEDF on CG cells (5) and its antiproliferative effects on microglia (17).
The sequence of human, bovine, and mouse PEDF cDNA reveals that PEDF is a member of the serpin superfamily of serine protease inhibitors (2, 11, 18, 19). Previous studies have suggested important roles for certain serine proteases and serpins in the development and/or pathology of the nervous system, e.g. thrombin, urokinase, and their inhibitors protease nexin-1 and plasminogen activator inhibitor (20-23). Given these studies, we had previously attempted to determine the inhibitory capacity of PEDF against serine proteases. First, an inhibitory activity against proteases, among others thrombin, could not be demonstrated for PEDF (24, 25). Second, PEDF, like the noninhibitory serpins ovalbumin, angiotensinogen, and maspin, lacks the serpin S → Rconformational change upon cleavage of its serpin-exposed loop (25, 26). Third, 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). Altogether, these observations indicate that the mechanism of action for the neurotrophic activity of PEDF is independent of protease inhibition.
Therefore, it is of interest to investigate the binding properties of PEDF to cells for mechanistic studies. Because the binding of PEDF to its receptor is presumably the first step in the mediation of its physiological effects, basic physicochemical parameters of such binding were established. We have used cultures of human retinoblastoma cells and rat CG cells because they respond to PEDF stimuli, and we used a biologically active form of 125I-PEDF to define the basic physicochemical parameters of such binding. We have used PEDF purified from bovine eyes (12, 13), recombinant human PEDF (26), synthetic peptides derived from BA, and a polyclonal antiserum to PEDF, Ab-rPEDF (12), to further investigate the specificity of the binding. Finally, PEDF affinity column chromatography was used to isolate a PEDF-binding protein from retinoblastoma and CG cell membranes. 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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