FUNCTIONAL IMPORTANCE OF PLATELET-DERIVED GROWTH-FACTOR (PDGF) RECEPTOR EXTRACELLULAR IMMUNOGLOBULIN-LIKE DOMAINS - IDENTIFICATION OF PDGF BINDING-SITE AND NEUTRALIZING MONOCLONAL-ANTIBODIES

The biological effects of platelet-derived growth factor (PDGF) are me diated by alpha- and beta-PDGF receptors (PDGFR), which have an intrac ellular tyrosine kinase domain and an extracellular region comprising five immunoglobulin-like domains (D1-D5). Using deletion mutagenesis w e mapped the PDGF binding site in each PDGFR to the D2-D3 region, In t he case of alpha-PDGFR, I-125-PDGF AA and I-125-PDGF BB bound to the f ull-length extracellular domain, D1-D5, and D2-D3 with equal affinity (K-d = 0.21-0.42 nM). Identical results were obtained for I-125-PDGF B B binding to beta-PDGFR mutants D1-D5 and D2-D3, establishing that D1, D4, and D5 do not contribute to PDGF binding. Monoclonal antibodies ( mAb) directed against individual PDGFR Ig-like domains were used to ex tend these observations. The anti-D1 mAb 1E10E2 and anti-D5 mAb 2D4G10 had no effect on alpha- or beta-PDGFR function, respectively. In cont rast, mAb 2H7C5 and 2A1E2 directed against D2 of the alpha- and beta-r eceptor, respectively, blocked PDGF binding, receptor autophosphorylat ion and mitogenic signaling with IC50 values of 0.1-3.0 nM. An anti-D4 mAb 1C7D5 blocked beta-receptor autophosphorylation and signaling wit hout inhibiting PDGF binding consistent with the observation that D4 i s essential for PDGFR dimerization (Omura, T., Heldin, C.-H., and Ostm an, A. (1997) J. Biol. Chem. 272, 12676-12682), mAbs identified here a ct as potent PDGFR antagonists that can be used as research tools and potentially as therapeutic agents for the treatment of diseases involv ing unwanted PDGFR signaling.