Differential Functions of Triplicated Repeats Suggest Two Independent Roles for the Receptor-associated Protein as a Molecular Chaperone

Abstract

The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that binds multiple ligands. The primary function of RAP has been defined as promotion of the correct folding of LRP, and prevention of premature interaction of ligands with LRP within the early secretory pathway. Previous examination of the RAP sequence revealed an internal triplication. However, the functional implication of the triplicated repeats was unknown. In the current study using various RAP and LRP domain constructs, we found that the carboxyl-terminal repeat of RAP possesses high affinities to each of the three ligandbinding domains on LRP, whereas the amino-terminal and central repeats of RAP exhibit only low affinity to the second and the fourth ligand-binding domains of LRP, respectively. Using truncated soluble minireceptors of LRP, we identified five independent RAP-binding sites, two on each of the second and fourth, and one on the third ligand-binding domain of LRP. By coexpressing soluble LRP minireceptors and RAP repeat constructs, we found that only the carboxyl-terminal repeat of RAP was able to promote the folding and subsequent secretion of the soluble LRP minireceptors. However, when the ability of each RAP repeat to inhibit ligand interactions with LRP was examined, differential effects were observed for individual LRP ligands. Most striking, both the amino-terminal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of ␣ 2 -macroglobulin with LRP. These differential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.The 39-kDa receptor-associated protein (RAP) 1 is an unique receptor antagonist. The target receptors for RAP are cysteinerich endocytic receptors that belong to the low density lipoprotein (LDL) receptor family (1). The four representative receptors in this family are the LDL receptor (2), the LDL receptorrelated protein (LRP, Ref.3), glycoprotein gp330/megalin (4), and the VLDL receptor (5). Among these receptors, LRP and gp330/megalin are large multifunctional receptors with multiple ligand-binding domains, which bind several structurally and functionally distinct ligands (for reviews, see Refs. 1 and 6). While RAP exhibits high affinities for LRP, gp330/megalin, and the VLDL receptor, it binds only weakly to the LDL receptor (7). Upon binding to these receptors, RAP inhibits the binding and/or endocytosis of all the ligands by the receptors. This unique feature of RAP has allowed its extensive use in biological studies of these endocytic receptors. Recent evidence has suggested that, under normal physiological conditions, RAP is an endoplasmic reticulum (ER) resident protein and functions within the early secretory pathway (8 -10). Using LRP as the target protein, it was found that RAP retained within the ER functions as a regulator of LRP activity by...

The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties

“…It was demonstrated that both LRP and RAP harbor multiple interaction sites for their mutual interaction (7,8,13). Based on analysis of the primary structure, it was revealed that RAP contains an internal triplication of structural autonomous domains comprising residues 1-100 (D1), 101-200 (D2), and 201-323 (D3) (13,40). When expressed individually, each of the single domains of human RAP maintains its functional integrity and binds to LRP (13,40).…”

“…Indeed, an alternative model for the regulation of ligand binding by RAP proposes a RAP-induced conformational change in the LRP molecule (9,18,39). It was demonstrated that both LRP and RAP harbor multiple interaction sites for their mutual interaction (7,8,13). Based on analysis of the primary structure, it was revealed that RAP contains an internal triplication of structural autonomous domains comprising residues 1-100 (D1), 101-200 (D2), and 201-323 (D3) (13,40).…”

“…Based on analysis of the primary structure, it was revealed that RAP contains an internal triplication of structural autonomous domains comprising residues 1-100 (D1), 101-200 (D2), and 201-323 (D3) (13,40). When expressed individually, each of the single domains of human RAP maintains its functional integrity and binds to LRP (13,40). D1 binds to cluster II, D2 to cluster IV, and D3 to all three RAP binding cluster domains (II, III, and IV) (13).…”

“…When expressed individually, each of the single domains of human RAP maintains its functional integrity and binds to LRP (13,40). D1 binds to cluster II, D2 to cluster IV, and D3 to all three RAP binding cluster domains (II, III, and IV) (13). Based on these reported multiple binding sites on RAP and LRP, one may envision a model in which one molecule of RAP can induce a conformational change in the receptor by interacting simultaneously with multiple receptor domains (Fig.…”

“…Several studies have shown that RAP facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway (8,(11)(12)(13)(14). RAP inhibits the binding of all ligands to the receptor and is thought to prevent premature binding of ligands to the receptor during the trafficking to the cell surface (15)(16)(17).…”