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 ...

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. , contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.The 39-kDa receptor-associated protein (RAP) 1 is a 323-amino acid ER chaperone for members of the LDL receptor gene family, which are cysteine-rich endocytic receptors (1, 2). Two unique features differentiate RAP from other general ER chaperones. First, whereas most other ER chaperones function primarily in substrate folding, RAP functions both in receptor folding (3, 4) and subsequent trafficking (5, 6). Second, RAP is a specialized chaperone that functions primarily with members of the LDL receptor gene family, whereas other ER chaperones interact with a variety of structurally and functionally divergent proteins that are synthesized and folded in the ER. Although the chaperone function of RAP was defined primarily with the LDL receptor-related protein (LRP) (3, 5-7), evidence accumulated to date suggests that RAP is likely to function as a chaperone for other members of the LDL receptor gene family (8 -10).The function of RAP during folding may be primarily to inhibit indiscriminate disulfide bond formation, in particular inter-molecularly between different LRP molecules during and after their translation (3). The function of RAP during the trafficking of receptors within the early secretory pathway is to prevent premature ligand interaction with the receptors (2,5,6). This function of RAP is consistent with the fact that RAP universally antagonizes ligand interaction with all members of the LDL receptor gene family. In this respect, it resembles the function of the invariant chain in regulating the peptide binding activity of major histocompatibility complex class II molecules within the secretory pathway (11). Largely because of its ability to inhibit the binding of ligands, recombinant RAP has been used extensively in the study of the biological properties and functions of members of the LDL receptor gene family (2).Several groups of investigators have studied the structure of RAP. In those works, the primary structure of RAP has been shown to comprise of a sequence of about 100 amino acids, r...

The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH + KDEL) or HNEL (GH + HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH + HNEL but also GH + KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-containing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.