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.
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August 1998
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Research Article|
August 01 1998
Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin
Donna SCHILLING;
Donna SCHILLING
*Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, U.S.A.
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James D. REID, IV;
James D. REID, IV
*Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, U.S.A.
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Andrea HUJER;
Andrea HUJER
*Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, U.S.A.
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Dewey MORGAN;
Dewey MORGAN
*Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, U.S.A.
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Edward DEMOLL;
Edward DEMOLL
†Department of Microbiology and Immunology, University of Kentucky, Chandler Medical Center, Lexington, KY 40536, U.S.A.
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Paul BUMMER;
Paul BUMMER
‡College of Pharmacy, University of Kentucky, Chandler Medical Center, Lexington, KY 40536, U.S.A.
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Robert A. FENSTERMAKER;
Robert A. FENSTERMAKER
4Department of Neurosurgery, Roswell Park Cancer Institute, Buffalo, NY 14273, U.S.A.
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David M. KAETZEL
David M. KAETZEL
1
*Department of Pharmacology, University of Kentucky, Chandler Medical Center, MS-305, Lexington, KY 40536, U.S.A.
1To whom correspondence should be addressed (e-mail dmkaetz@pop.uky.edu).
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Publisher: Portland Press Ltd
Received:
February 13 1998
Revision Received:
April 14 1998
Accepted:
April 23 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (3): 637–644.
Article history
Received:
February 13 1998
Revision Received:
April 14 1998
Accepted:
April 23 1998
Citation
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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