The binding of receptor-recognized α2-macroglobulin to the low density lipoprotein receptor-related protein and the α2M signaling receptor is decoupled by oxidation

Abstract

Receptor-recognized forms of α2-macroglobulin (α2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by α2M. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of α2M using 200 μM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (K(d) - 71 ± 12 pM). Surprisingly, oxidation of native α2- macroglobulin (α2M) with 125μM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (K(d) - 0.7 ± 0.15 μM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of α2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding K(d), supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native α2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (K(d) - 71 pM) are the α2M signaling receptor.