Ca²⁺ and receptor-associated protein are independently required for proper folding and disulfide bond formation of the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca²⁺ and its relationship to RAP during LRP folding. We found that depletion of Ca²⁺ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca²⁺ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca²⁺ depletion. The role of Ca²⁺ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca²⁺ depletion, Furthermore, Ca²⁺ depletion- induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca²⁺ and RAP independently participate in LRP folding.