|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Platelet-derived growth factor (PDGF) is a dimeric protein that exerts its effects through tyrosine kinase α-and β-receptors. The extracellular part of each receptor is composed of five Ig-like domains.
Recombinant α-receptor domains 1-4 (αRD1-4), 1-3 (αRD1-3), and 1-2 (αRD1-2) were prepared and used to study the assembly of soluble ligand-receptor complexes. With micromolar concentrations of PDGF, both αRD1-3 and αRD1-4 formed complexes of 1: 2 molar composition, i.e. one dimeric PDGF molecule bound two soluble receptors. αRD1-3, in contrast to αRD1-4, formed detectable 1:1 complexes under conditions of ligand excess. αRD1-4 displayed an increased ability to form 1:2 complexes as compared with αRD1-3 under conditions of limiting concentrations of ligand. We thus conclude that Ig-4-mediated receptor-receptor interactions stabilize the complex. Since αRD1-4 and αRD1-3 were equipotent in blocking binding of subnanomolar concentrations of PDGF to cell-surface receptors, we also conclude th
at this effect is predominantly achieved through formation of Ig-like domain 4-independent 1:1 ligand-receptor complexes. Finally, since αRD1-2 bound PDGF-BB with high affinity, whereas PDGF-AA was bound only with low affinity, we conclude that Ig-3 of the PDGF α-receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes reside in Ig-1 and 2.
Recombinant GST-fusion proteins of αRD1-4 andβRD1-3 (αRD1-4-GST and βRD1-3-GST) were generated and analyzed with regard to their ability to block PDGF binding to cell surface receptors. Both in the case of the α- and β-receptors, 100-1,000 fold lower concentration of the GST-fusion protein were required, as compared to the cleaved forms. αRD1-4-GST and βRD1-3-GST, in contrast to αRD1-4 and βRD1-3, were shown to occur as ligand independent dimers. Covalently cross-linked αRD1-4 dimers displayed a 50-fold increased potency as compared to αRD1-4. We thus conclude that the dimeric nature of the soluble receptors is responsible for the high antagonistic potency. Less