Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.
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 alpha-and beta-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 beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-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 beta-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.
Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, USA.
D
D
Schilling
J D
JD
Reid IV
A
A
Hujer
D
D
Morgan
E
E
Demoll
P
P
Bummer
R A
RA
Fenstermaker
D M
DM
Kaetzel
D000818:Animals; D001254:Astrocytoma / Q000378:metabolism; D000077214:Becaplermin; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D006224:Cricetinae; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; C075278:platelet-derived growth factor A; D000077214:Becaplermin; D006493:Heparin; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor