Structural coincidence of αPDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody

Jin Chen Yu, Daruka Mahadevan, William J. LaRochelle, Jacalyn H. Pierce, Mohammad A. Heidaran

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

We have generated two groups of deletion mutants of the αPDGFR and one group of chimeras between αPDGFR and βPDGFR to further investigate the structural requirements of the αPDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against αPDGFR (designated as mAb-αR1). The mAb-αR1 has recently been reported to block high affinity binding of PDGF-AA to αPDGFR. The first group of mutants were carboxyl- terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (αR1-216), the first four Ig-like domains (αR1-415), or all five Ig-like domains (αR1-530) of the αPDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated αPDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-αR1 was able to immunoprecipitate each of these secreted form of αPDGFRs, suggesting that the epitope recognized by mAb-αR1 is located within Ig-like domains 1 and 2 of the αPDGFR. Furthermore, PDGF-AA exhibited detectable binding to αR1-415 or αR1-530 but failed to interact with αR1-216, suggesting that the first two Ig-like domains of the αPDGFR are not sufficient for PDGF-AA binding. The second group of αPDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (αR(Δ49-100)), Ig-like loop 2 (αR(Δ150-189)), Ig-like loop 3 (αR(Δ235-290)), or part of Ig-like loops 4 and 5 (αR(Δ375-450)). The internal deletion mutants were transfected into 32D cells which lack both αPDGFR and βPDGFR. PDGF-AA bound with high affinity to 32D cells expressing αR(Δ375-450) 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 αPDGFR. In addition, mAb-αR1 bound to 32D cells expressing αR(Δ235-290) but failed to bind 32D cells transfected with αR(Δ49-100) or αR(Δ150-189). Since this antibody could immunoprecipitate αR(Δ49-100), but not αR(Δ150-189) in cell lysates of the 32D transfectants, these results suggest that Ig-like loop 1 is dispensible for mAb-αR1 recognition and deletion of Ig-like loop 1 of αPDGFR significantly inhibits cell surface expression. The two α/β PDGFR chimeras generated were an αPDGFR containing the first Ig-like domain of βPDGFR (β126α127R) and a βPDGFR possessing the first Ig-like domain of αPDGFR (α119β119R). PDGF-AA bound to 32D cells expressing β126α127R, but not to those expressing α119β119R, suggesting that Ig-like domain 1 is not required for conferring PDGF-AA binding specificity. Taken together, we conclude that major high affinity sites for PDGF-AA binding are located within Ig-like domains 2 and 3 and that the epitope recognized by mAb-αR1 is located within Ig-like domain 2 of the αPDGFR.

Original languageEnglish (US)
Pages (from-to)10668-10674
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number14
StatePublished - Apr 8 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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