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 ligand-binding 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 alpha2-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.
Highlights
The 39-kDa receptor-associated protein (RAP)1 is an unique receptor antagonist
Using cell surface binding analyses, we have previously shown that there were approximately [5,6,7] times more RAP-binding sites on hepatocytes when compared with t-PA binding sites
Other reported binding studies of RAP to purified lipoprotein receptor-related protein (LRP) concluded that two RAP-binding sites are present on each LRP molecule (27)
Summary
The 39-kDa receptor-associated protein (RAP) 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 complexity of LRP’s structure, largely due to the extensive intradomain disulfide bonds, presents a challenging task for proper folding during its biosynthesis This process may well be assisted by molecular chaperone(s) within the ER. Differential Functions of RAP Repeats ular disulfide bonds and are retained within the ER with little secretion It is not known at present whether the role of RAP in the receptor’s folding is independent from its function in preventing premature ligand interaction with the receptor. We found that while the carboxyl-terminal repeat of RAP functions to the full-length RAP in terms of interacting with the receptor and in assisting the receptor to fold, inhibition of at least one LRP ligand, ␣2macroglobulin, can only be achieved with the amino-terminal or the central repeats of RAP. These differential functions of the RAP repeats suggest that the functions of RAP in receptor folding and inhibition of ligand interactions are independent
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