A cationic region of the platelet-derived growth factor (PDGF) A-chain (Arg159-Lys160-Lys161) is required for receptor binding and mitogenic activity of the PDGF-AA homodimer.
Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.
Fenstermaker
R A
RA
Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106.
Poptic
E
E
Bonfield
T L
TL
Knauss
T C
TC
Corsillo
L
L
Piskurich
J F
JF
Kaetzel
C S
CS
Jentoft
J E
JE
Gelfand
C
C
DiCorleto
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D004742:Enhancer Elements, Genetic; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D017479:Receptors, Platelet-Derived Growth Factor; D008727:Methotrexate