Receptor-associated Protein and Members of the Low Density Lipoprotein Receptor Family Share a Common Epitope

Abstract

Heymann nephritis is an experimental rat model for human membranous glomerulonephritis. Two target an-tigens have been identified in the proximal tubule brush border of rat kidneys. One of them is megalin, a 600-kDa membrane protein that belongs to the family of low density lipoprotein receptor (LDLR)-related proteins. The other one is receptor-associated protein (RAP), a polypeptide of 40 kDa that associates with members of the LDLR family. Here we show that antibodies produced against recombinant human RAP strongly cross-react with the chicken oocyte receptor for very low density lipoprotein and vitellogenin (LR8), and with two other members of the LDLR family, LDLR-related protein and megalin. The interaction of this antibody with LR8 showed binding characteristics exactly as those demonstrated for the physiological ligands of this receptor , in that binding of the antibody: (i) is Ca 2-dependent ; (ii) is abolished by unfolding of the cysteine-rich binding domain by reduction; and (iii) interferes with the binding of very low density lipoprotein and vitel-logenin. Immunopurification of the LR8-specific sub-population of the polyclonal antiserum yielded an IgG fraction strongly reacting with LR8 as well as with RAP. Using recombinant fragments of RAP and peptide mapping , the cross-reacting epitope(s) could be narrowed down to three short sequences (5-7 residues) in the COOH-terminal part of the protein. After immunization with RAP, anti-LR8 antibodies and anti-RAP antibodies arise simultaneously, indicating that the receptor-specific activity is not due to anti-idiotypic antibodies. These findings suggest the existence of a common epitope(s) on RAP and members of the LDL receptor family. Based on these results, we present an extended molecular model for the development of passive Hey-mann nephritis. Receptor-associated-protein (RAP) 1 was initially described as a 39-40-kDa protein copurifying with LDL receptor (LDLR)-related protein (LRP) (1, 2). At the same time, a puta-tive target antigen in Heymann nephritis (HN), an experimental rat model of human membranous nephropathy, was identified and cloned (3). On molecular characterization of RAP, it became evident that the protein identified as the target antigen in HN was identical to RAP and not, as previously assumed, a fragment of glycoprotein 330/megalin (4). RAP subsequently was shown to bind not only to LRP but also to other members of the LDLR family, including megalin (5), the VLDLR (6), the chicken oocyte receptor for VLDL and vitellogenin (LR8) (7, 8) and, to a lesser extent, to the LDLR itself (8, 9). The ability to interfere with ligand binding to these receptors has made RAP a perfect tool to study ligand-receptor interaction both in vitro (8, 10-14) and in vivo (15). The latter experiments especially have established the role of LRP as a chylomicron remnant receptor in the liver. Despite the successful use of RAP to study the physiological function(s) of members of the LDLR family and of LRP in particular, clues about the function of RAP in vivo emerged only recently. The tetrapeptide sequence HNEL at the carboxyl terminus of RAP was shown to be necessary for the retention of RAP in the endoplasmic reticulum; thus, RAP associates in vivo with LRP early in the secretory pathway and dissociates from the receptor before reaching the cell surface (16). These results suggested a specialized role of RAP as a chaperon for LRP, possibly regulating the interaction of the receptor with ligands along the secretory pathway. This was confirmed by elegant studies by Herz and colleagues in RAP knockout mice (17) and in cultured cells in experiments relating the biosynthesis and functional expression of LRP and megalin with that of RAP (18). These experiments show that at least one of the physiological functions of RAP can be defined as that of a specialized escort protein, which protects certain receptors from ligand-induced aggregation along their intracellular itinerary. We have previously shown that LR8 can serve as a model system to study ligand binding and structural aspects of LDLR family members (8, 19, 20). Here we have used LR8 to demonstrate that all receptors that strongly bind RAP share a common immunological epitope with the escort protein. Based on this observation, we propose a model for the development of passive Heymann nephritis induced by anti-RAP antibodies, in which megalin present in the brush borders of the proximal kidney tubule in rats constitutes an additional target antigen. EXPERIMENTAL PROCEDURES Preparation and Radiolabeling of Ligands-VLDL was prepared from plasma of estrogen-treated roosters by sequential ultracentrifuga-tion according to the method of George et al. (21). VLDL was labeled with 125 I to a specific activity of 250-400 cpm/ng using the iodine 1 The abbreviations used are: RAP, receptor-associated protein; LDLR, low density lipoprotein receptor; LRP, LDLR-related protein; VLDLR, very low density lipoprotein receptor; LR8, LDLR relative with eight ligand binding repeats; HN, Heymann nephritis; GST, glutathi-one S-transferase; HRP, horseradish peroxidase.