RAP,a specialized chaperone,prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) forms a complex with a receptor-associated protein (RAP) within the secretory pathway. RAP inhibits ligand binding to LRP and is required for normal functional expression of LRP in vivo, suggesting a physiological function as a specialized chaperone. We have used RAP-deficient mice, generated by gene targeting, to investigate the role of RAP in the biosynthesis and biological activity of LRP and other members of the LDL receptor gene family in various organs and in embryonic fibroblasts. Our results demonstrate that RAP is required for the proper folding and export of the receptors from the endoplasmic reticulum (ER) by preventing the premature binding of co-expressed ligands. Overexpression of apolipoprotein E (apoE), a high affinity ligand for LRP, results in dramatically reduced cellular LRP expression, an effect that is prevented by co-expression of RAP. RAP thus defines a novel class of molecular chaperones that selectively protect endocytic receptors by binding to newly synthesized receptor polypeptides, thereby preventing ligand-induced aggregation and subsequent degradation in the ER.     

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.     

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.     

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.     

Functional mimicry of the LDL receptor-associated protein through multimerization of a minimized third domain

The LDL receptor-associated protein (RAP) is a ligand for the LDL receptor-related protein (LRP1). The first and third domains of RAP can each bind to one of many sequence-related pairs of complement-type repeats (CR) found within the LRP1 ectodomain. Multiple sites of interaction between the multivalent RAP ligand and the multivalent LRP1 receptor yield strong binding avidity for the complex. The third domain of RAP can be significantly truncated, with material retention of monovalent CR pair-binding affinity, provided that the minimized sequence is stabilized with an intramolecular disulfide bond. We demonstrate that the avidity of full-length RAP for LRP1 in vitro can be partially reconstituted by assembly of truncated, disulfide-linked RAP peptides on tetravalent streptavidin or bivalent immunoglobulin scaffolds. The peptide complex with streptavidin shows pronounced hepatotropism in vivo, replicating the biodistribution of full-length RAP.     

A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.     

Dissection of RAP-LRP interactions: binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP

The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7-CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7-CR8, with the higher affinity site (K(d) approximately 1microM) located in the C-terminal two-thirds and the weaker site (K(d) approximately 5microM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5-CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding.     

The low-density lipoprotein receptor-related protein associates with calnexin, calreticulin, and protein disulfide isomerase in receptor-associated-protein-deficient fibroblasts

The low-density lipoprotein receptor-related protein (LRP) is a large (>600 kDa) multi-ligand-binding cell surface receptor that is now known to participate in a diverse range of cellular events. To accomplish this diverse role, LRP is composed of repetitive amino acid motifs consisting of complement-type and EGF precursor-type repeats. Within these repeats are six conserved cysteine residues that form the core disulfide bond structure of each repeat. To accommodate the intricate folding that such a complex structure dictates, a specialized chaperone is present in the endoplasmic reticulum (ER) called the receptor-associated protein (RAP) that binds to LRP immediately following its biosynthesis and assists in its exocytic transport. Interestingly, RAP -/- mice show reduced LRP expression in certain cell types, but not a more global affect on LRP expression that was expected. Such a tissue-restricted effect by RAP prompted an investigation if other ER chaperones associate with LRP to assist in its complex folding requirements and compensate for the absence of RAP in RAP -/- cells. Fibroblasts obtained from RAP -/- mice demonstrate similar LRP expression levels and subcellular distribution as RAP +/+ fibroblasts. Moreover, RAP -/- cells show an identical exocytic trafficking rate for LRP as RAP +/+ cells and comparable cell surface internalization kinetics. In RAP -/- cells, three well-known ER chaperones, calnexin, calreticulin, and protein disulfide isomerase (PDI), associate with LRP and likely compensate for the absence of RAP.     

Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.     

Interaction of lipoprotein lipase and receptor-associated protein

Receptor-associated protein (RAP) is a recognized chaperone/escort protein for members of the low density lipoprotein receptor family. In this report, we show that RAP binds to lipoprotein lipase (LPL) and may play a role in the maturation of LPL. Binding of highly purified RAP to LPL was demonstrated in vitro by solid phase assays, surface plasmon resonance, and rate zonal centrifugation. The dissociation constant for this interaction measured by the first two techniques ranged between 2.4 and 13 nM, values similar to those reported for the binding of RAP to LRP or gp330. The specificity of the interaction was demonstrated by competition with a panel of LPL monoclonal antibodies. Rate zonal centrifugation demonstrated the presence of a stable complex with an apparent Mr consistent with the formation of a complex between monomeric LPL and RAP. RAP x LPL complexes were co-immunoprecipitated in adipocyte lysates or from solutions of purified LPL and RAP. The interaction was also demonstrated in whole cells by cross-linking experiments. RAP-deficient adipocytes secreted LPL with a specific activity 2.5-fold lower than the lipase secreted by control cells. Heparin addition to cultured RAP-deficient adipocytes failed to stimulate LPL secretion in the medium, suggesting defective binding of the lipase to the plasma membrane. These studies demonstrate that RAP binds to LPL with high affinity both in purified systems and cell extracts and that RAP-deficient adipocytes secrete poorly assembled LPL. A function of RAP may be to prevent premature interaction of LPL with binding partners in the secretory pathway, namely LRP and heparan sulfate proteoglycan.     

Structural organization of the receptor associated protein

The receptor associated protein (RAP) is a 38 kDa ER-resident protein that binds tightly to the low density lipoprotein receptor-related protein (LRP), and other members of the LDL receptor family of receptors, and competes with all known LRP ligands for binding to LRP. To better understand the domain structure and organization of RAP, we have expressed RAP subfragments and examined them by two-dimensional HSQC NMR and fluorescence spectroscopies, by differential scanning calorimetry, and by both equilibrium and velocity sedimentation measurements. We found that the protein is organized into three domains located in the first third (1D), middle third (2D), and last third (3D) of the protein. All three domains adopt stable tertiary structure as isolated domains and are monomers. Whereas domains 1D and 2D do not interact with one another, 3D interacts with 2D, both in a 2D-3D construct and in intact RAP. Sedimentation measurements also indicated that intact RAP, although monomeric, is significantly elongated.     

The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Recognition of alpha 2-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.     

Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts

Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.     

An extrahepatic receptor-associated protein-sensitive mechanism is involved in the metabolism of triglyceride-rich lipoproteins

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)-independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor-associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre(+)LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre(+)LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation.     

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs

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. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), 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 low density lipoprotein receptor-related protein modulates levels of matrix metalloproteinase 9 (MMP-9) by mediating its cellular catabolism

Members of the matrix metalloproteinase (MMP) family of enzymes participate in matrix remodeling and share a number of structural and functional features. The activity of this family of proteinases is carefully regulated at the level of zymogen activation and by a family of specific inhibitors termed tissue inhibitors of metalloproteinases (TIMP). It is now becoming clear that levels of certain MMPs are modulated by their association with cellular receptors that mediate their rapid internalization and degradation. In the current investigation we report that the amount of MMP-9 in conditioned cell culture medium is significantly increased when mouse embryonic fibroblasts are grown in the presence of the 39-kDa receptor-associated protein (RAP), an antagonist of ligand binding to low density lipoprotein receptor-related protein (LRP). In vitro assays reveal that the MMP-9.TIMP-1 complex binds to LRP with high affinity and that the binding determinant for LRP appears to reside on MMP-9. Cell lines expressing LRP mediate the internalization of 125I-labeled MMP-9.TIMP-1 complexes, whereas cell lines genetically deficient in LRP show a diminished capacity to mediate the cellular catabolism of MMP-9.TIMP-1 complexes. The results demonstrate that LRP is a functional receptor for MMP-9 and suggest a major role for LRP in modulating remodeling of the extracellular matrix by regulating extracellular proteinase activity.     

Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large receptor that contains extensive glycosylation sites and disulfide bonds. Here we analyzed how N-linked glycosylation and molecular chaperones function during LRP folding. Treatment of cells with a glycosylation inhibitor tunicamycin significantly impaired LRP folding, although binding to receptor-associated protein (RAP), a specialized chaperone for LRP, was not affected. The effects of tunicamycin on LRP folding were not due to an inhibition of RAP glycosylation since a mutant RAP that harbors a mutation at its sole glycosylation site was still capable of promoting LRP folding. The roles of N-linked glycosylation and the lectin chaperone, calnexin, in LRP folding were further dissected using LRP minireceptors that carry mutations at individual glycosylation sites. Interestingly, we found that RAP interacts with oxidoreductase ERp57 and mediates its interaction with LRP. Since previous studies have shown that N-glycan-bound calnexin/calreticulin are also capable of recruiting ERp57, our results suggest that N-linked glycosylation and RAP can independently and cooperatively recruit oxidoreductases to facilitate protein folding and proper disulfide bond formation.