Structural coincidence of alpha PDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody.
We have generated two groups of deletion mutants of the alpha PDGFR and one group of chimeras between alpha PDGFR and beta PDGFR to further investigate the structural requirements of the alpha PDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against alpha PDGFR (designated as mAb-alpha R1). The mAb-alpha R1 has recently been reported to block high affinity binding of PDGF-AA to alpha PDGFR. The first group of mutants were carboxyl-terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (alpha R1-216), the first four Ig-like domains (alpha R1-415), or all five Ig-like domains (alpha R1-530) of the alpha PDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated alpha PDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-alpha R1 was able to immunoprecipitate each of these secreted form of alpha PDGFRs, suggesting that the epitope recognized by mAb-alpha R1 is located within Ig-like domains 1 and 2 of the alpha PDGFR. Furthermore, PDGF-AA exhibited detectable binding to alpha R1-415 or alpha R1-530 but failed to interact with alpha R1-216, suggesting that the first two Ig-like domains of the alpha PDGFR are not sufficient for PDGF-AA binding. The second group of alpha PDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (alpha R delta 49-100), Ig-like loop 2 (alpha R delta 150-189), Ig-like loop 3 (alpha R delta 235-290), or part of Ig-like loops 4 and 5 (alpha R delta 375-450). The internal deletion mutants were transfected into 32D cells which lack both alpha PDGFR and beta PDGFR. PDGF-AA bound with high affinity to 32D cells expressing alpha R delta 375-450 but not to 32D cells expressing the other three internal deletion mutants, suggesting that the region required for PDGF-AA binding should be within the first three Ig-like domains of the alpha PDGFR. In addition, mAb-alpha R1 bound to 32D cells expressing alpha R delta 235-290 but failed to bind 32D cells transfected with alpha R delta 49-100 or alpha R delta 150-189.(ABSTRACT TRUNCATED AT 400 WORDS)
Yu
J C
JC
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Mahadevan
D
D
LaRochelle
W J
WJ
Pierce
J H
JH
Heidaran
M A
MA
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007136:Immunoglobulins; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor; C075278:platelet-derived growth factor A; D017479:Receptors, Platelet-Derived Growth Factor