The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties

Abstract

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated ␣ 2 -macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA⅐plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody ␣ 2 MR␣3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligandbinding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.The low density lipoprotein receptor-related protein (LRP) 1 is a 600-kDa membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors (reviewed in Refs. 1 and 2). LRP can bind and internalize a diverse spectrum of structurally unrelated ligands in a calcium-dependent manner including apolipoproteins, lipases, proteinases, proteinase-inhibitor complexes, Kunitz-type inhibitors, matrix proteins, and other proteins such as lactoferrin, Pseudomonas exotoxin A, and malaria circumsporozoite protein (1, 2). In addition, the blood coagulation factor VIII was recently identified as a ligand of LRP (3). The broad range of ligands suggests a role for the receptor in distinct physiological and pathophysiological processes, ranging from lipoprotein metabolism, cell growth and cell migration, fibrinolysis, and thrombosis to atherosclerosis and Alzheimer's disease.LRP is synthesized as a single polypeptide chain and is cleaved in the trans-Golgi network by the endopeptidase furin into two subunits, resulting in a 515-kDa fragment that contains the ligand binding domains and an 85-kDa fragment comprising the transmembrane and cytoplasmic domains. The subunits rema...

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

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“…The goal of this study was to characterize the interactome of LRP1 in myelin. We designed fusion proteins corresponding to the second and fourth clusters of complement-like repeats (CCRII and CCRIV), two major extracellular ligand-binding domains in LRP1 (15). CCRII or CCRIV fusion proteins were incubated with myelin protein extracts in the presence or absence of RAP, an LRP1 ligand binding inhib-itor (16), and associated partners were identified by tandem mass spectrometry.…”

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