Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF α-and β-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK → EEE), whereas an RKK → SSS mutant also exhibited a modest loss in affinity for β-receptors. Replacements with serine at either Arg160 (RKK → SKK) or at all three positions (RKK → SSS) had little effect on binding to α-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK → EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29–32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK → EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK → EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of β-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties.
Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin
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Donna SCHILLING, James D. REID, Andrea HUJER, Dewey MORGAN, Edward DEMOLL, Paul BUMMER, Robert A. FENSTERMAKER, David M. KAETZEL; Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin. Biochem J 1 August 1998; 333 (3): 637–644. doi: https://doi.org/10.1042/bj3330637
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