title: 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 alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen 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 alpha(2)MRalpha3 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 ligand-binding 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.
authors.affiliation: Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. j.g.neels@amc.uva.nl
authors.forename: J G
authors.initials: JG
authors.lastname: Neels
authors.forename: B M
authors.initials: BM
authors.lastname: van Den Berg
authors.forename: A
authors.initials: A
authors.lastname: Lookene
authors.forename: G
authors.initials: G
authors.lastname: Olivecrona
authors.forename: H
authors.initials: H
authors.lastname: Pannekoek
authors.forename: A J
authors.initials: AJ
authors.lastname: van Zonneveld
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D006023:Glycoproteins; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D014568:Urokinase-Type Plasminogen Activator; D003545:Cysteine