Ca2+ and Receptor-associated Protein Are Independently Required for Proper Folding and Disulfide Bond Formation of the Low Density Lipoprotein Receptor-related Protein

doi:10.1074/jbc.273.35.22374

Ca²⁺ and Receptor-associated Protein Are Independently Required for Proper Folding and Disulfide Bond Formation of the Low Density Lipoprotein Receptor-related Protein*

From the Departments of ^‡Pediatrics,^§Molecular Biology and Pharmacology, and ^¶Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Abstract

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.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL59150, American Cancer Society Research Project Grant RPG-97-010-01-CB, and Alzheimer Association Faculty Scholar Award FSA95–051.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Dept. of Pediatrics, Washington University School of Medicine, CB 8116, One Children’s Place, St. Louis, MO 63110. Tel.: 314-454-2726; Fax: 314-454-2685; E-mail: bu{at}kids.wustl.edu.
Abbreviations:

LDL

low density lipoprotein

LRP

LDL receptor-related protein

sLRP

soluble LRP minireceptor

mLRP

membrane-containing LRP minireceptor

RAP

receptor-associated protein

ER

endoplasmic reticulum

NEM

N-ethylmaleimide

MEF

mouse embryonic fibroblast

PAGE

polyacrylamide gel electrophoresis.
- Received May 4, 1998.
- Revision received June 24, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.