Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding.
To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.
Heidaran
M A
MA
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Mahadevan
D
D
Larochelle
W J
WJ
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D000077214:Becaplermin; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D051379:Mice; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D020574:Proto-Oncogene Proteins c-sis; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; C498150:IgG D1.3; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins; D000077214:Becaplermin; D013498:Suramin; D017479:Receptors, Platelet-Derived Growth Factor