Specific Binding of α-Macroglobulin to Complement-Type Repeat CR4 of the Low-Density Lipoprotein Receptor-Related Protein

Abstract

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (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-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface 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 complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized 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.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

“…These receptors are transported through the ER-Golgi secretory pathway bound to the receptor-associated protein RAP (LRPAP1 or 2-macroglobulin receptor-associated protein) that acts as a chaperone and presumably protect receptors from early binding to ligands until dissociation occurs at the lower pH in later Golgi compartments (39). Studies with E. coli produced LA modules without glycans established that RAP binds to multiple LA modules through electrostatic interaction between three calcium-coordinating acidic residues in each LA and lysine residues (9, [40][41]. In LRP1 the most juxtamembrane cluster of LA modules LA3-6 that contains linker O-glycans ( Fig.…”

Hypoxia Induces Metalloproteinase-9 Activation and Human Vascular Smooth Muscle Cell Migration Through Low-Density Lipoprotein Receptor–Related Protein 1–Mediated Pyk2 Phosphorylation

“…Thus, LRP1 may contribute to the sustained increase in intracellular calcium required for Pyk2 phosphorylation, probably because of the capacity of LRP1 to retain calcium in its fifth and sixth CR-domain (CR56). 45 Moreover, our results showed that hypoxia-induced LRP1 overexpression is linked to Pyk2 phosphorylation and MMP-9 activation in hVSMC. In accordance, LRP1 deficiency prevented hypoxia-induced MMP-9 overexpression, and that both LRP1 and MMP-9 silencing delayed hypoxiainduced hVSMC migration.…”

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

“…With Golgi being the site for initiation of GalNAc-type O-glycosylation it is possible that O-glycosylation plays a role in the interaction with RAP. RAP binding to LDLR and its related receptors occurs via the LDLR class A repeats overlapping several of the O-glycan sequence motifs described here (9,10). Curiously, RAP itself is also O-glycosylated at several sites (19), two of which are in the RAP-D3 domain, the major domain participating in LDLR binding (57).…”