Limited Mutations in Full-length Tetrameric Human α2-Macroglobulin Abrogate Binding of Platelet-derived Growth Factor-BB and Transforming Growth Factor-β1

Abstract

␣ 2 -Macroglobulin (␣ 2 M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-␤1 (TGF-␤1), and carries ␤-amyloid peptide. ␣ 2 M 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 ␣ 2 M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the ␣ 2 M subunit. However, because intact ␣ 2 M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the ␣ 2 M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length ␣ 2 M. These mutations did not perturb the tetrameric structure of ␣ 2 M, 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 ␣ 2 M. E730R did not alter TGF-␤1 binding; however, this mutation in combination with mutations at Glu 714 and Asp 719 eliminated the increase in TGF-␤1 binding associated with ␣ 2 M conformational change. These studies demonstrate that growth factor binding to intact ␣ 2 M is specific, involving a defined region of the ␣ 2 M 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.2 is a 718-kDa homotetrameric glycoprotein that functions as a protease inhibitor and as a carrier of growth factors in the blood and in other extracellular spaces (1). Proteases react with ␣ 2 M by cleaving any of a number of target peptide bonds near the center of the ␣ 2 M subunit, in the "bait region" (2). Cleavage in the bait region induces a major conformational change in ␣ 2 M that entraps the reacting protease within the hollow core of the cylindrical ␣ 2 M structure (2-4). ␣ 2 M conformational change reveals the recognition site for the ␣ 2 M receptor/low density lipoprotein receptor-related protein (LRP-1), which is cryptic in the native ␣ 2 M conformation (5, 6). For this reason, conformationally transformed ␣ 2 M is referred to as "activated." ␣ 2 M thiol ester bonds, which are formed by the side chains of Cys 949 and Glu 952 , also become exposed during ␣ 2

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

“…These include: FP3 (amino acids 591-774), FP4 (amino acids 775-1059), and FP6 (amino acids 1242-1451). FP3 includes the characterized binding site for growth factors (20,21,25). FP6 contains the LRP-1 recognition sequence (22).…”

“…FP6 contains amino acids 1242-1451 and the LRP-1-binding site, in which a single ␣-helix that includes Lys 1370 and Lys 1374 plays a central role (23,24). Assignment of ␣ 2 M activities to specific fusion proteins, such as FP3 and FP6, has been validated by mutagenesis of full-length recombinant ␣ 2 M (25,26). Thus, the ␣ 2 M fusion proteins provide an opportunity to assess activities assigned to different ␣ 2 M domains independently.…”

A derivative of the plasma protease inhibitor α2‐macroglobulin regulates the response to peripheral nerve injury

“…Murinoglobulin lacks the binding site, called PID‐1, which is responsible, for non‐covalent interactions with TGF‐β1 and platelet‐derived growth factor‐BB (Webb et al. 2000b; Arandjelovic et al. 2006).…”

“…1994; Gonias et al. 1994), reflecting the activity of at least one major protein‐interaction site, which has been identified in the structure of the intact protein (Arandjelovic et al. 2006).…”

Dry Eye and Designer Ophthalmics

“…Alpha-2-macroglobulin (A2M) is a broad-acting protease inhibitor. It also binds tear proteins B2M, cathepsin B (CTSB; lysosomal cysteine protease) and IL10; and the growth factors nerve growth factor beta polypeptide, platelet-derived growth factor betal polypeptide and transforming growth factor beta 1 78. This latter property, via interaction of A2M with its receptor LPR1, is thought to be involved in inhibiting proliferation of retinal glial cells 79.…”