Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large surfa ce receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites a re located in clusters of complement-type repeats (CR), where the general a bsence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to th e LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglo bulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-termin al part of the cluster comprising CR3-CR6, using ligand blotting and surfac e plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the com plex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is char acterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.