The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties

The low density lipoprotein receptor-related protein (LRP) is a multifuncti onal endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domain s, generally referred to as clusters I, II, III, and IV. In this study, sol uble recombinant receptor fragments, representing each of the four individu al clusters, were used to map the binding sites of a set of structurally an d functionally distinct ligands. Using surface plasmon resonance, we studie d the binding of these fragments to methylamine-activated alpha(2)-macroglo bulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen ac tivator (t-PA), plasminogen activator inhibitor-1, t-PA plasminogen activat or inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue fact or pathway inhibitor, lactoferrin, the light chain of blood coagulation fac tor MII, and the intracellular chaperone receptor-associated protein (EAF). No binding of the cluster I fragment to any of the tested ligands was obse rved. The cluster III fragment only bound to the anti-LRP monoclonal antibo dy alpha(2)MR alpha 3 and weakly to RAP. Except for t-PA, we found that eac h of the ligands tested binds both to cluster II and to cluster IV. The aff inity rate constants of ligand binding to clusters II and IV and to LRP wer e 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 seg ment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, s upporting a model in which RAP binding to LRP induces a conformational chan ge in the receptor that is incompatible with ligand binding.