Receptor-associated Protein Is a Folding Chaperone for Low Density Lipoprotein Receptor-related Protein

Abstract

The 39-kDa receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low density lipoprotein receptor gene family. Our previous studies have demonstrated that RAP interacts with the low density lipoprotein receptor-related protein (LRP) within the endoplasmic reticulum and prevents premature interaction of ligands with the receptor. To analyze whether RAP is also involved in the folding of LRP during receptor biosynthesis, we generated anchor-free, soluble minireceptors that represent each of the four putative ligandbinding domains of LRP (SLRP1, -2, -3, and -4, corresponding to the clusters with 2, 8, 10, and 11 cysteine-rich complement-type repeats, respectively). When these SLRPs were overexpressed by cell transfection, only SLRP1 was secreted. Little or no secretion was observed for SLRP2, -3, and -4. However, when RAP cDNA was cotransfected with SLRP2, -3, and -4 cDNAs, each of these SLRPs was secreted. The cellular retention of SLRPs in the absence of RAP coexpression appeared to be a result of the formation of SDS-resistant, oligomeric aggregates observed under nonreducing conditions. Such oligomers of the SLRPs likely resulted from formation of intermolecular disulfide bonds since they were reduced to monomers when analyzed under reducing conditions. The oligomers were formed not only among molecules of a given SLRP, but also between different SLRPs. The role of RAP in the process of LRP folding was shown by the reduction in aggregated SLRP oligomers upon RAP coexpression. A similar role of RAP in preventing the aggregation of newly synthesized receptor was also observed using membrane-containing minireceptor of LRP. Coimmunoprecipitation and ligand binding studies demonstrated that RAP binds avidly to SLRP2, -3, and -4, but not to SLRP1. These results suggest that these interactions may be important for proper folding of LRP by ensuring the formation of proper intradomain, but not intermolecular or interdomain, disulfide bonds. Thus, our results strongly suggest that, in addition to the prevention of premature binding of ligands to LRP, RAP also plays an important role in receptor folding.The 39-kDa receptor-associated protein (RAP) 1 is a potent inhibitor of endocytic receptors that belong to the low density lipoprotein receptor gene family. The four representative receptors in this family are the low density liproprotein receptor (Yamamoto et al., 1984), the low density liproprotein receptorrelated protein (LRP) (Herz et al., 1988), the membrane glycoprotein gp330/megalin (Raychowdhury et al., 1989;Saito et al., 1994), and the very low density lipoprotein receptor (Takakashi et al., 1992). Among these receptors, LRP and gp330/megalin are large multifunctional receptors, each of which can bind and endocytose several structurally and functionally distinct ligands (for reviews, see Krieger and Herz (1994) and Kounnas et al. (1994)). Upon binding to these receptors, RAP inhibits the binding and/or endocytosis of all the ligands by ...

Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis

“…LRP is a member of the low-density lipoprotein receptor gene family of endocytic receptors (26)(27)(28). The 39-kDa RAP can be used as a ligand (23). Internalization of cell-surface bound recombinant RAP is rapid and after dissociation from the receptor in the endosomes, it is transported to the lysosome whereas the LRP recycles back to the plasma membrane (29).…”

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

“…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).…”

“…Evidence is accumulating that the clusters of LDLRA domains constitute the ligand binding domains of the receptor (6 -10). Except for receptor-associated protein (RAP) binding to clusters III and IV (1,7,8), only cluster II was shown to be involved in ligand binding (6 -10). Specifically, it has been demonstrated that ␣ 2 -macroglobulin-light chain, complexes between urokinase-type plasminogen activator (u-PA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1), lactoferrin, an anti-LRP Fab fragment (denoted Fab A8), and RAP bind to cluster II.…”

“…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).…”

The Carboxyl-terminal Domain of Receptor-associated Protein Facilitates Proper Folding and Trafficking of the Very Low Density Lipoprotein Receptor by Interaction with the Three Amino-terminal Ligand-binding Repeats of the Receptor

“…Whether identical structural arrangements are present within the LBR of other members of the LDL receptor gene family remains to be elucidated but appears to be highly likely due to their sequence homology and functional similarities. Studies with LRP and its minireceptors indicate that intramolecular disulfide bond formation is of crucial importance for the proper folding and trafficking of the receptor within the early secretory pathway (12)(13)(14). Within the endoplasmic reticulum (ER), a 39-kDa receptor-associated protein (RAP) associates with LRP upon receptor synthesis and assists the folding and trafficking of the receptor (12).…”