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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 apolipoprotein E (apoE)-β very low density lipoprotein (βVLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-βVLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111–1210. From these studies we conclude that the binding of apoE-βVLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

gp330 is a large glycoprotein located in clathrin-coated pits at the surface of the glomerular and proximal tubule epithelia in the rat kidney. It was originally identified as the target of autoimmune antibodies in Heymann nephritis (HN) and has since been shown to be a member of the low density lipoprotein receptor gene family and to form a stable association with receptor-associated protein (RAP), which together constitute the HN antigen complex (HNAC). Progress in defining the normal functions of gp330 as well as the molecular mechanisms of HN has been hampered by the lack of an available kidney cell line that expresses this protein. We here report the identification of a rat yolk sac carcinoma cell line (L2) that synthesizes HNAC and expresses it in coated pits at the cell surface. gp330 and RAP from L2 cells are immunologically identical to their kidney counterparts, and peptide maps of gp330 yielded identical peptide fragments. Characterization of the cell line revealed that there are 3.3 x 10(4) gp330 molecules per L2 cell and that the cells produce a soluble form of gp330 that is released into the medium. Heparin ligand blot analysis demonstrated that RAP but not gp330 binds heparin. By heparin affinity chromatography, gp330 and RAP copurify, indicating that the glycosaminoglycan binding site within RAP is accessible when the subunit is complexed with gp330. These results indicate that the L2 cell line provides a valid and useful model for studies on the function of HNAC and the pathogenesis of HN.

Using antibodies isolated from glomeruli of nephritic rats we have previously identified a 330-kDa cell surface glycoprotein (gp330) as a major pathogenic antigen of Heymann nephritis (HN), an experimental model of human membranous glomerulonephritis. Recently, we have isolated a cDNA clone, C14, encoding a polypeptide that contains a pathogenic epitope of HN responsible for the initiation of the disease. Subsequently, another protein, alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP), which is a subunit of the receptor for human alpha 2-macroglobulin/low density lipoprotein receptor-related protein (LRP), was shown to possess a high degree of sequence homology to the C14 protein (C14p). In this report, we have investigated the relationship between gp330, C14p, and alpha 2-MRAP. Immunoprecipitation studies demonstrate that gp330 forms a heterodimeric association with a 44-kDa polypeptide that is stable to detergent extraction and long-term centrifugation. Further, immunoblotting analysis on the purified complex indicates that the 44-kDa associated protein shares immunological identity to C14p and alpha 2-MRAP. In addition, antibodies eluted from glomeruli of HN rats and antibodies to a C14 fusion protein immunoprecipitated gp330 and the 44-kDa protein, demonstrating that the epitopes responsible for the initial events of HN are accessible within the complex. Based on these data, three models are proposed to explain how pathogenic epitopes in the gp330-44-kDa, HN antigenic complex may be presented at the cell surface and initiate the onset of HN.