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

Abstract

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 putative 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 PROCEDURESPreparation and Radiolabeling of Ligands-VLDL was prepared from plasma of estrogen-treated roosters by sequential ultracentrifugation 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

Receptor-associated Protein in an Oviparous Species Is Correlated with the Expression of a Receptor Variant

“…8), in which we have also obtained supporting evidence for our previous observation that RAP and certain members of the LDLR gene family share common epitope(s) (27). Here, the antibody was prepared against a synthetic peptide corresponding to residues 101-116 of chicken RAP, suggesting that receptor cross-reactive epitopes may not be limited to the region(s) of RAP previously identified in the mammalian system (27). Shared epitope recognition is, at least in part, responsible for the development in rats of the autoimmune disease, passive Heymann nephritis (27).…”

“…The high level of RAP in these cells has already allowed us to use crude cell extracts in a novel reverse ligand blotting procedure termed EXLBlot (Fig. 8), in which we have also obtained supporting evidence for our previous observation that RAP and certain members of the LDLR gene family share common epitope(s) (27). Here, the antibody was prepared against a synthetic peptide corresponding to residues 101-116 of chicken RAP, suggesting that receptor cross-reactive epitopes may not be limited to the region(s) of RAP previously identified in the mammalian system (27).…”

“…Incubation with extracts of control cells (lane 2) reveal a much weaker 95-kDa signal, which is possibly due to the cross-reactivity of anti-RAP with endogeneous receptor(s) (Ref. 27; cf. Fig.…”

“…Under these conditions, endogenous simian RAP did not cross-react with our anti-peptide antibody. However, the antibody recognized a COS-7 cell membrane protein doublet of about 100 -105 kDa, which may represent endogeneous simian LDLR homologues (27).…”

“…In ligand blots, both forms were shown to react with mammalian RAP (18). Recently, we have obtained preliminary evidence for expression of RAP or a RAP-like protein in the chicken (27). To investigate the relevance of RAP expression in correlation to that of the variant receptor forms and in regard to the information on RAP in nonmammalian species, we have initiated studies to identify and molecularly and functionally characterize the first nonamniotic RAP.…”