title: Predimerization of recombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency.
abstract: Platelet-derived growth factor (PDGF) is a dimeric growth factor acting through tyrosine kinase alpha- and beta-receptors. In both receptors, the extracellular parts are composed of five Ig-like domains. Functional mapping of the extracellular part of the receptors have shown that ligand-binding occurs to Ig-like domains 2 and 3 and that Ig-like domain 4 is involved in receptor-receptor interactions. Recombinant GST-fusion proteins of PDGF alpha-receptor Ig-like domains 1-4 and beta-receptor Ig-like domains 1-3 (alphaRD1-4-GST and betaRD1-3-GST) were generated and compared with their cleaved counterparts (alphaRD1-4 and betaRD1-3) with regard to their ability to block PDGF binding to cell surface receptors. In the case of both the alpha- and the beta-receptors, 100-1000-fold lower concentrations of the GST-fusion proteins were required, as compared to the cleaved forms, for inhibition of PDGF binding to cell surface receptors. alphaRD1-4-GST and betaRD1-3-GST, in contrast to alphaRD1-4 and betaRD1-3, were shown to occur as ligand independent dimers. Covalently cross-linked alphaRD1-4 dimers displayed a 50-fold increased potency as compared to alphaRD1-4. We thus conclude that the dimeric nature of alphaRD1-4-GST and betaRD1-3-GST is responsible for the high antagonistic potency of the fusion proteins.
authors.affiliation: Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden.
authors.forename: O
authors.initials: O
authors.lastname: Leppänen
authors.forename: K
authors.initials: K
authors.lastname: Miyazawa
authors.forename: G
authors.initials: G
authors.lastname: Bäckström
authors.forename: K
authors.initials: K
authors.lastname: Pietras
authors.forename: T
authors.initials: T
authors.lastname: Sjöblom
authors.forename: C H
authors.initials: CH
authors.lastname: Heldin
authors.forename: A
authors.initials: A
authors.lastname: Ostman
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D005110:Extracellular Space / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013552:Swine; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014316:Tritium
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D014316:Tritium; D005982:Glutathione Transferase; D020796:Receptor, Platelet-Derived Growth Factor alpha; D013936:Thymidine