RECEPTOR-ASSOCIATED PROTEIN IS A FOLDING CHAPERONE FOR LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN

The 39-kDa receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to th e low density lipoprotein receptor gene family. Our previous studies h ave demonstrated that RAP interacts with the low density Lipoprotein r eceptor-related protein (LRP) within the endoplasmic reticulum and pre vents premature interaction of ligands with the receptor. To analyze w hether RAP is also involved in the folding of LRP during receptor bios ynthesis, we generated anchor-free, soluble minireceptors that represe nt each of the four putative ligand-binding domains of LRP (SLRP1, -2, -3, and -4, corresponding to the clusters with 2, 8, 10, and 11 cyste ine-rich complement-type repeats, respectively). When these SLRPs were overexpressed by cell transfection, only SLRP1 was secreted. Little o r no secretion was observed for SLR92, -3, and -4. However, when RAP c DNA was cotransfected with SLRP2, -3, and -4 cDNAs, each of these SLRP 2 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 ol igomers of the SLRPs likely resulted from formation of intermolecular disulfide bonds since they were reduced to monomers when analyzed unde r reducing conditions. The oligomers were formed not only among molecu les 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 aggregate d SLRP oligomers upon RAP coexpression. A similar role of RAP in preve nting 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 inter actions may be important for proper folding of LRP by ensuring the for mation of proper intradomain, but not intermolecular or interdomain, d isulfide bonds. Thus, our results strongly suggest that, in addition t o the prevention of premature binding of ligands to LRP, RAP also play s an important role in receptor folding.