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.
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
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Obermoeller
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Chen
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Schwartz
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D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D017510:Protein Folding; D011971:Receptors, Immunologic / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D004220:Disulfides; D006023:Glycoproteins; D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D002118:Calcium