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

alpha 2-Macroglobulin (alpha M-2) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta 1), and carries beta-amyloid peptide. alpha M-2 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 alpha M-2 fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor- binding site near the center of the alpha M-2 subunit. However, because intact alpha M-2 forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha M-2 core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha M-2. These mutations did not perturb the tetrameric structure of alpha M-2, 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 alpha M-2. E730R did not alter TGF-alpha 1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-alpha 1 binding associated with alpha M-2 conformational change. These studies demonstrate that growth factor binding to intact alpha M-2 is specific, involving a defined region of the alpha M-2 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.