Identical or overlapping sequences in the primary structure of human α2-macroglobulin are responsible for the binding of nerve growth factor-β, platelet-derived growth factor-BB, and transforming growth factor-β

Abstract

α2-Macroglobulin (α2M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-β (NGF-β) to a linear sequence in the 180-kDa human α2M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-β in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-β binding to immobilized FP3 were 300 ± 40 and 180 ± 30 nM, respectively; these values were comparable with those determined using methylamine-modified α2M, suggesting that higher-order α2M structure is not necessary for PDGF-BB and NGF-β binding. PDGF-BB and NGF-β blocked the binding of transforming growth factor-β1 (TGF-β1) to FP3. Furthermore, murinoglobulin, which is the only known member of the α-macroglobulin family that does not bind TGF-β, also failed to bind PDGF-BB and NGF-β. These results support the hypothesis that either a single linear sequence in human α2M or overlapping sequences are responsible for the binding of TGF-β, PDGF-BB, and NGF-β, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF β receptor was fused to the IgG1 Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.