Limited mutations in full-length tetrameric human α2- macroglobulin abrogate binding of platelet-derived growth factor-BB and transforming growth factor-β1

Abstract

α2-Macroglobulin (α2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1), and carries β-amyloid peptide. α2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant α2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the α2M subunit. However, because intact α2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the α2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length α2M. These mutations did not perturb the tetrameric structure of α2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact α2M. E730R did not alter TGF-β1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-β1 binding associated with α2M conformational change. These studies demonstrate that growth factor binding to intact α2M is specific, involving a defined region of the α2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.