Recognition of alpha(2)-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions

The low density lipoprotein receptor-related protein (LRP) is a scavenger r eceptor that binds several ligands including the activated form of the pan- proteinase inhibitor alpha (2)-macroglobulin (alpha M-2*) and amyloid precu rsor protein, two ligands genetically linked to Alzheimer's disease. To del ineate the contribution of LRP to this disease, it will be necessary to ide ntify the sites on this receptor which are responsible for recognizing thes e and other ligands to assist in the development of specific inhibitors. St ructurally, LRP contains four clusters of cysteine-rich repeats, yet studie s thus far suggest that only two of these clusters (clusters II and IV) bin d ligands. Identifying binding sites within LRP for certain ligands, such a s alpha M-2*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha M-2* uptake, monoclonal an tibody 8G1 and an amino-terminal fragment of receptor-associated protein (R AP D1D2). Surprisingly, the inhibitors recognized different clusters of lig and binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-recept or containing the repeats from cluster I along with three ligand binding re peats from cluster II was effective in mediating the internalization of I-1 25-labeled alpha M-2*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity b inding site for alpha M-2*, and they suggest a strategy for developing spec ific inhibitors to block alpha M-2* binding to LRP by identifying molecules capable of binding repeats in cluster I.