IDENTIFICATION OF THE SECOND CLUSTER OF LIGAND-BINDING REPEATS IN MEGALIN AS A SITE FOR RECEPTOR-LIGAND INTERACTIONS

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families , To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprot ein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotei n lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor, RAP is known to inh ibit the binding of all ligands to megalin, We identified a ligand-bin ding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped th e epitope for this mAb, mAb AC10 inhibited the binding of apoE-beta VL DL, lipoprotein Lipase, aprotinin, and lactoferrin to megalin in a con centration-dependent manner. When cDNA fragments encoding the four cys teine-rich ligand-binding repeats in megalin were expressed in a bacul ovirus system and immunoblotted with AC10, it recognized only the seco nd cluster of ligand-binding repeats. The location of the epitope reco gnized by mAb AC10 within this domain was pinpointed to amino acids 11 11-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin i s either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites withi n the second cluster of ligand-binding repeats.