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J Biol Chem, Vol. 274, Issue 36, 25877-25882, September 3, 1999


The Carboxyl-terminal Domain of Receptor-associated Protein Facilitates Proper Folding and Trafficking of the Very Low Density Lipoprotein Receptor by Interaction with the Three Amino-terminal Ligand-binding Repeats of the Receptor*

Roger SavonenDagger §, Lynn M. Obermoeller, Julie S. Trausch-Azar, Alan L. SchwartzDagger , and Guojun Buparallel

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The low density lipoprotein (LDL)1 receptor gene family is a growing family of endocytic receptors that bind and internalize various ligands, which include apolipoprotein E (apoE)/lipoproteins and several proteins that are involved in coagulation and hemostasis (for reviews, see Refs. 1 and 2). Common structural features of these receptors include 1) cysteine-rich ligand-binding repeats (LBR), 2) epidermal growth factor precursor homology repeats, 3) spacer regions that include YWTD repeats, 4) a single transmembrane domain, and 5) a carboxyl-terminal cytoplasmic tail with one or two copies of the NPXY motifs, which serve as potential endocytosis signals. The most striking difference among these receptors is the number of LBR. The LDL receptor and the very low density lipoprotein (VLDL) receptor, both with an approximate molecular mass of 130 kDa, contain seven and eight LBR, respectively, whereas the much larger 600-kDa LDL receptor-related protein (LRP) has 31 LBR arranged in four distinct clusters (3-7). Each LBR consists of approximately 40 amino acids that include six cysteine residues, which form three disulfide bonds (8). As shown with LBR 1, 2, and 5 of the LDL receptor, these disulfide bonds are formed between cysteines I and III, II and V, and IV and VI (9-11). These disulfide bonds, along with calcium ions (10), appear to play an important role in stabilizing the structure of the receptor. Whether identical structural arrangements are present within the LBR of other members of the LDL receptor gene family remains to be elucidated but appears to be highly likely due to their sequence homology and functional similarities.

Studies with LRP and its minireceptors indicate that intramolecular disulfide bond formation is of crucial importance for the proper folding and trafficking of the receptor within the early secretory pathway (12-14). Within the endoplasmic reticulum (ER), a 39-kDa receptor-associated protein (RAP) associates with LRP upon receptor synthesis and assists the folding and trafficking of the receptor (12). As the receptors reach the medial Golgi compartments, where post-translational oligosaccharide modifications become endoglucosidase H (Endo H)-resistant, RAP dissociates from the receptor as a result of decreased pH. RAP is then retrieved back to the ER, while the receptor is transported to the cell surface (15). In the absence of RAP co-expression, high molecular weight aggregates of misfolded LRP are seen when analyzed under nonreducing SDS-PAGE. These aggregates are probably due to the formation of intermolecular disulfide bonds during misfolding, since these aggregates are reduced to monomers when analyzed under reducing conditions (12, 13).

In addition to promoting folding, RAP also regulates ligand binding to LRP as a means of preventing premature ligand interactions with the receptor within the early secretory pathway (12, 16). In vitro studies have shown that RAP binds strongly to and prevents ligand binding of all known ligands to LRP, megalin/LRP2, and the VLDL receptor (3, 17-20). In contrast, binding of RAP to the LDL receptor is much weaker, and inhibition of ligand binding to this receptor requires higher concentrations of RAP (21). While recombinant RAP has been used experimentally as a receptor antagonist, this protein is not present extracellularly under normal physiological conditions (22) due to the presence of an ER retention signal at its carboxyl terminus (12, 15). Structurally, RAP consists of relatively homologous triplicate repeats of about 100 amino acid residues (12). The carboxyl-terminal repeat of RAP is the only repeat that promotes the folding and trafficking of LRP, while both the amino-terminal and the central repeats are able to inhibit the binding of certain ligands (14). While the LDL receptor and LRP are highly expressed in the liver, the VLDL receptor is virtually absent therein (6, 23, 24). The main sites of VLDL receptor expression in mammals include the heart, skeletal muscle, and adipose tissue (6, 23, 25, 26). High levels of VLDL receptor expression have also been reported in the ovary (23), brain, and kidney (26). Interestingly, the expression pattern of the VLDL receptor resembles that of lipoprotein lipase, the major enzyme catalyzing the hydrolysis of triacylglycerides in triglyceride-rich lipoproteins (for a review, see Ref. 27). Thus, it was hypothesized that the VLDL receptor was involved in lipoprotein metabolism (6, 28).

Studies in RAP knockout mice have indicated that RAP may play a role in the biogenesis of the VLDL receptor, since VLDL receptor transport to the cell surface was completely blocked in heart muscle cells of RAP-/- mice (16). The objective of the current study was to investigate whether RAP serves as a molecular chaperone during the folding and trafficking of the VLDL receptor, and if so to identify the structural basis for RAP-VLDL receptor interaction. We found that interaction between the carboxyl-terminal repeat of RAP and the three amino-terminal repeats of the VDLD receptor plays important role in the folding and trafficking of this receptor.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of cDNAs-- Construction of cDNAs for sLRPs using polymerase chain reaction has been described previously (13). The same strategies were used for generating constructs for the human VLDL receptor and for the following constructs of truncated human VLDL receptor: LBR 1-8 (i.e. full-length VLDL receptor), LBR1-3, LBR 1-5, LBR 6-8. All of these constructs contained a HA tag inserted after the cleavage site. The cDNA for the full-length human VLDL receptor was kindly provided by Dr. Tokuo Yamamoto (Tohoku University, Japan). A construct of RAP and the properties of the RAP repeats have been described previously (14). The cDNA construct for RAP lacking the HNEL ER retention sequence was described before (12).

Cell Culture and Transfection-- Human glioblastoma U87 cells, human hepatoma HepG2 cells, mouse embryonic fibroblasts MEF-4 (LDLR-/-, LRP-/-) and MEF-7 (RAP-/-) cells were cultured as described before (14, 29). MEF-4 and MEF-7 cells were obtained from Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX). For transient transfection, cells were transfected with various plasmids (total 45 µg of cDNA/100-mm plate in a total volume of 16.5 ml) at 20-30% confluence using the calcium phosphate precipitation method (30). Sixteen hours after the start of transfection, cells were washed with medium and cultured for an additional 24 h before the experiment. For generating stable cell lines, CHO-LRP(-/-) cells (31), were transfected with the respective pcDNA construct and selected for resistance to Geneticin (12). Clones were then screened with anti-HA antibody, and positive clones expressing proteins of the correct size were then expanded and used for experiments.

Metabolic Labeling and Chemical Cross-linking-- Metabolic labeling with [35S]cysteine (0.2 mCi/ml) and intracellular cross-linking with dithiobis(succinimidyl propionate) (DSP; 0.5 mM) were essentially performed as described before (12). For pulse-chase experiments, cells were labeled for 30 or 60 min followed by a chase for 1, 2, or 3 h, as described in each experiment. For continuous labeling cells were labeled for 3 or 4 h, with no chase period, as indicated.

Antibodies, Immunoprecipitation, and SDS-Polyacrylamide Gel Electrophoresis-- Polyclonal anti-RAP and anti-LRP and monoclonal anti-HA antibodies have been described previously (12, 14). Rabbit polyclonal anti-VLDL receptor antibody was raised against a recombinant fragment of the VLDL receptor that contains all of the ligand-binding domains. Immunoprecipitation was essentially carried out as described previously (14, 32). Immunoprecipitation in the absence of SDS was carried out as described by Bu et al. (12). Protein-IgG complexes were precipitated with protein A-agarose beads (Repligen Corp., Cambridge, MA), and the immunoprecipitated material was released from the beads by boiling each sample for 5 min in Laemmli sample buffer (33). If immunoprecipitated material was to be analyzed under reducing conditions, 5% beta -mercaptoethanol was included in the Laemmli sample buffer. Samples were then subjected to SDS-polyacrylamide gel electrophoresis, with the percentage of polyacrylamide indicated in each figure legend. Rainbow molecular weight markers from Bio-Rad were used as molecular weight standards. Quantitation of bands on gels was performed using PhosphorImager analysis techniques on a STORM 840 (Molecular Dynamics, Inc., Sunnyvale, CA). Reported values are mean values ± S.D. (n >=  3).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RAP Is Associated with the VLDL Receptor in Vivo-- To test whether RAP is associated with the VLDL receptor intracellularly, we examined if RAP could be cross-linked to the VLDL receptor in intact cells. U87 cells were transiently transfected with cDNAs for the human VLDL receptor and RAP as described under "Experimental Procedures." Transfected cells were metabolically labeled with [35S]cysteine at 37 °C for 4 h. After labeling, cells were incubated at 4 °C for 30 min with phosphate-buffered saline, in the absence or in the presence of 0.5 mM of the membrane-permeable, thiol-cleavable cross-linker DSP (12). Cells were then lysed; immunoprecipitated with anti-VLDL receptor, anti-LRP, or anti-RAP antibodies; and analyzed via SDS-PAGE under nonreducing (Fig. 1A) or reducing conditions (Fig. 1B). In the absence of the cross-linker, the polyclonal anti-VLDL receptor antibody precipitated a single band of ~130 kDa, the expected size of the VLDL receptor (3) (Fig. 1A, lane 1). Polyclonal anti-LRP antibody immunoprecipitated a high molecular weight band corresponding to the size of LRP (lane 2). No cross-reactivity was observed between the VLDL receptor and LRP antibodies under these conditions. In the presence of the intracellular cross-linker DSP, in addition to the monomeric VLDL receptor band, anti-VLDL receptor antibody also immunoprecipitated high molecular weight complexes probably representing the VLDL receptor cross-linked to RAP (lane 4). High molecular weight complexes were also observed after cross-linking and immunoprecipitation with anti-LRP antibody when analyzed under nonreducing conditions (lane 5) (12). However, these high molecular weight complexes disappeared when analyzed under reducing conditions, which reduce the cross-linker and thus dissociate cross-linked proteins (Fig. 1B). As seen in Fig. 1, A and B, lanes 3, in the absence of cross-linker, anti-RAP antibody immunoprecipitated neither the VLDL receptor nor LRP. Due to the lack of cysteine in its sequence, RAP itself is not labeled in these experiments. However, in the presence of cross-linker, anti-RAP antibody immunoprecipitated both the VLDL receptor and LRP, suggesting that these proteins were associated with RAP at the time DSP was added (Fig. 1, A and B, lanes 6). Taken together, these results indicate that within intact cells, RAP is associated not only to LRP but also to the VLDL receptor.


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  		Fig. 1.   Intracellular cross-linking of RAP with the VLDL receptor (VLDLR). U87 cells were transiently co-transfected with cDNAs for the VLDL receptor and RAP. Transfected cells were metabolically labeled with [35S]cysteine for 4 h and incubated in phosphate-buffered saline in the absence or presence of 0.5 mM DSP cross-linker as described under "Experimental Procedures." Cells were then lysed and immunoprecipitated with anti-VLDL receptor, anti-LRP, or anti-RAP antibodies and analyzed via 6% SDS-PAGE, under either nonreducing (A) or reducing (B) conditions. The positions of the VLDL receptor and LRP are indicated by open and solid arrows, respectively. The molecular size standards in this figure and subsequent figures are given in kilodaltons.

To facilitate immunoprecipitation of different variants of the VLDL receptor via a monoclonal anti-HA antibody, a HA epitope was introduced near the amino terminus of each VLDL receptor construct. To ensure that the HA epitope did not affect the receptor's association with RAP, we transiently transfected U87 cells with cDNAs encoding the HA-tagged VLDL receptor and RAP. The experiment described in Fig. 1 was then repeated exactly as described, except for the use of the monoclonal anti-HA antibody in place of the polyclonal anti-VLDL receptor antibody. The results were identical to those obtained and described above, indicating that addition of the HA epitope on the VLDL receptor had no influence on its interaction with RAP (data not shown).

RAP Promotes Proper Folding and Processing of the VLDL Receptor-- To examine the potential role of RAP in the proper folding and trafficking of the VLDL receptor within the early secretory pathway, we analyzed the kinetics of the VLDL receptor trafficking in the absence or presence of RAP co-expression. Human hepatoma HepG2 cells were transiently transfected with cDNA for the VLDL receptor with co-transfection of either pcDNA3 or cDNA for RAP. Transfected cells were then metabolically pulse-labeled with [35S]cysteine for 30 min and chased for 0, 1, or 2 h. After each chase period, cells were lysed and quantitatively immunoprecipitated with either polyclonal anti-VLDL receptor antibody or monoclonal anti-HA antibody and analyzed by SDS-PAGE. Initially, after 30 min of labeling, a single band of labeled protein was observed in the absence of RAP co-expression (Fig. 2A, lane 1). After 2 h of chase, 26% of the labeled proteins appears as a slower migrating band (Fig. 2A, lane 5). Treatment of immunoprecipitated material with 1.0 milliunits of Endo H indicates that the lower band, which is Endo H-sensitive, represents the ER form of the receptor. Co-expression of RAP significantly promotes the conversion of the Endo H-sensitive ER form to the Endo H-resistant Golgi form (Fig. 2B). After 2 h of chase, 44% of labeled VLDL receptor had been processed into the Golgi-form, indicating that processing of the VLDL receptor is facilitated by RAP co-expression. When samples from identical experiments were analyzed under nonreducing conditions, a broader, fuzzier band was observed in the absence when compared with in the presence of RAP. A likely explanation for this observation is that misfolding of the VLDL receptor in the absence of RAP co-expression is due predominantly to mislinkage of disulfide bonds within the receptor molecule, which slightly alters the VLDL receptor molecules' mobility on SDS-PAGE. This result is distinct from that observed with LRP in which the mislinkage of disulfide bonds primarily occurs between LRPs (14). These findings indicate that RAP promotes proper folding and intracellular trafficking of the VLDL receptor.


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  		Fig. 2.   Pulse-chase analysis of VLDL receptor maturation in the absence or presence of RAP co-expression. Human hepatoma HepG2 cells were transiently transfected with cDNA for the VLDL receptor, with co-transfection of either pcDNA3 (A) or RAP (B). Transfected cells were then pulse-labeled with [35S]cysteine for 30 min followed by a chase for 1 or 2 h. After cell lysis and immunoprecipitation with anti-VLDL receptor antibodies, each sample was split into two equal parts, incubated overnight in the absence or presence of 1.0 milliunit of Endo H, and analyzed via 6% SDS-PAGE under reducing conditions. Samples from another identical experiment were subjected to analysis by SDS-PAGE under nonreducing conditions after immunoprecipitation without Endo H treatment (C). Solid and open arrows indicate Endo H-resistant and -sensitive bands, respectively. Quantitation of band intensity was performed by PhosphorImager analysis.

We have also noted that the conversion of the ER to the Golgi form of the VLDL receptor appears to differ between different cell lines. Pulse-chase analysis with U87 cells (12, 14), instead of HepG2 cells as described above, consistently resulted in a major ER band and a much weaker, sometimes absent, Golgi band of the VLDL receptor. This suggests that various cell types may process the VLDL receptor differently during the post-translational processing and trafficking. However, cell surface iodination studies suggest that only the Golgi form, and not the ER form, traffics to the cell surface (data not shown).

The Carboxyl-terminal Repeat of RAP Is Required for Proper Folding and Secretion of a Soluble VLDL Receptor-- In previous studies with sLRP minireceptors (encoding each of the four ligand binding domains but lacking the transmembrane domain), it was shown that co-expression of RAP is required for proper folding and secretion of sLRP2, sLRP3, and sLRP4 (13). To examine whether secretion of a soluble VLDL receptor was dependent on RAP co-expression, we transiently transfected U87 cells with cDNA for a soluble VLDL receptor encoding the eight ligand binding repeats and the first two epidermal growth factor precursor repeats, with co-transfection of cDNA for the vector, pcDNA3, or RAP. The transfected cells were then pulse-labeled with [35S]cysteine for 1 h and chased for 3 h (Fig. 3A). Both culture media and cell lysates were immunoprecipitated with anti-HA antibody and analyzed by SDS-PAGE under reducing or nonreducing conditions and quantified by PhosphorImager analysis. As seen in Fig. 3, A and C, less than 3% of the soluble VLDL receptor is secreted to the media in the absence of RAP co-expression (lane 2), compared with about 50% when RAP is co-expressed (lane 4). This indicates that RAP significantly promotes proper folding and secretion of the soluble VLDL receptor. Similar to sLRP (13), when analyzed under nonreducing conditions, high molecular weight aggregates, corresponding to dimer, trimer, and multimers, were observed in the absence of RAP co-expression (data not shown).


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  		Fig. 3.   Co-expression of full-length RAP or the carboxyl-terminal repeat of RAP promotes folding and secretion of soluble VLDL receptor. U87 cells were transiently transfected with cDNA for a soluble VLDL receptor with co-transfection of pcDNA3, cDNA for the 323-amino acid full length RAP (A), or cDNAs representing each one of the three internal triplicate repeats of RAP (B). Transfected cells were then pulse-labeled with [35S]cysteine for 1 h followed by a 3-h chase. Cell lysates (C) and harvested media (M) were subjected to immunoprecipitation with anti-HA antibody and analyzed via 7.5% SDS-PAGE under reducing conditions. The intensity of bands was quantitated by PhosphorImager analysis. The diagram in C shows the mean values, in percentage of total label, from three separate experiments (±S.D.). Open and solid bars represent cell lysate (C) and culture media (M), respectively.

The three internal triplicate repeats (about 100 amino acids each) of the 323-amino acid RAP share high sequence homology (14). To investigate which RAP repeat(s) facilitate VLDL receptor folding and secretion, we transiently transfected U87 cells with cDNA for the soluble VLDL receptor together with cDNA for the amino-terminal domain (amino acids 1-110), the central domain (amino acids 91-210), or the carboxyl-terminal domain (amino acids 191-323). When the amino-terminal or the central domain of RAP was co-expressed, 6.6 ± 0.1 and 16 ± 3% of the soluble VLDL receptor was secreted, respectively, compared with 63 ± 8% when the carboxyl-terminal domain of RAP was co-expressed (Fig. 3B, lanes 2, 4, and 6). The amount of labeled receptors retained intracellularly decreased accordingly when the carboxyl-terminal domain of RAP was co-expressed (Fig. 3B, compare lane 5 with lanes 1 and 3). These results indicate that the carboxyl-terminal domain, but not the amino-terminal and the central domain, of RAP is sufficient to promote the proper folding and subsequent secretion of soluble VLDL receptor. To exclude the possibility that the carboxyl-terminal ER retention signal (15), which is present in the full-length and the carboxyl-terminal domain but not in the amino-terminal or central domain constructs of RAP, affected the chaperone function of RAP, we compared the secretion of soluble VLDL receptor with co-expression of RAP or RAPDelta HNEL (12). No difference in the amount of secreted soluble VLDL receptor was observed (data not shown), suggesting that the presence or the absence of the ER retention signal does not affect the chaperone function of RAP during the folding and secretion of the soluble VLDL receptor. Thus, the function of RAP as a folding chaperone resides within its carboxyl-terminal domain.

RAP Interacts with LBR 1-3 of the VLDL Receptor-- To determine which LBR of the VLDL receptor interacts with RAP and thereby facilitate its proper folding and intracellular processing, we generated VLDL receptor constructs containing the first three, the first five, or the last three of its eight LBR (see Fig. 4). Cells were transiently transfected with cDNAs for each one of these constructs with co-transfection of cDNA for the empty vector or RAP. Transfected cells were then metabolically labeled for 4 h. Cell lysates were then divided into two equal parts. One half was subjected to immunoprecipitation with anti-HA antibody, and the other half was subjected to co-immunoprecipitation with anti-RAP antibody under mild conditions (no SDS). The immunoprecipitated material was then analyzed by SDS-PAGE. With MEF-4 cells, which express neither the LDLR nor LRP,2 immunoprecipitation with anti-HA antibody resulted in the same amount of each VLDL receptor construct independent of the presence of RAP co-expression (Fig. 5A). However, co-immunoprecipitation under mild conditions with anti-RAP antibody resulted in detection of abundant VLDL receptor LBR 1-8, 1-3, and 1-5 but not VLDL receptor LBR 6-8 (Fig. 5B). The weaker bands in the absence of RAP co-transfection are probably due to expression of endogenous RAP that co-immunoprecipitated VLDL receptor constructs. To overcome this potential effect of this endogenous RAP, we repeated the same experiment with MEF-7 (RAP-/-) cells (16). Under these conditions, immunoprecipitation with anti-HA antibody also indicated that all the constructs were expressed to the same extent (data not shown). Furthermore, co-immunoprecipitation with anti-RAP antibody resulted in abundant VLDL receptor constructs LBR 1-8, 1-3, and 1-5, but not LBR 6-8, when RAP was co-expressed (data not shown). Furthermore, similar results were found in parallel experiments in CHO LRP-/- cells stably transfected with VLDL receptor constructs (data not shown). Taken together, these results indicate that the RAP binding site resides within the first three LBR of the VLDL receptor.


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  		Fig. 4.   Schematic representation of VLDL receptor constructs. To study which LBR of the VLDL receptor are involved in interactions with RAP, we generated VLDL receptor constructs that contain LBR 1-8 (the full-length VLDL receptor), LBR 1-3, LBR 1-5, and LBR 6-8. For quantitative immunodetection of each VLDL receptor construct, a HA epitope was added near the amino termini of all the constructs.


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  		Fig. 5.   RAP interacts with three amino-terminal ligand binding repeats of the VLDL receptor. MEF-4 cells (LDL receptor-/-; LRP-/-) were transiently transfected with cDNA for the respective VLDL receptor construct containing different groups of LBR (as shown in Fig. 4) with co-transfection of either pcDNA3 or cDNA for RAP. Transfected cells were continuously labeled with [35S]cysteine for 4 h. After cell lysis, half of the lysate was subjected to immunoprecipitation with anti-HA antibody and analyzed via 7.5% SDS-PAGE under reducing conditions (A). The other half of the lysate was immunoprecipitated with anti-RAP antibody under mild conditions (i.e. no SDS) (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous observations that demonstrated that RAP associates with newly synthesized LRP and assists in the proper folding and trafficking of the receptor established that RAP is an intracellular chaperone for LRP within the early secretory pathway (reviewed by Bu and Schwartz (34)). Since RAP also functions as an antagonist of ligand binding to other members of the LDL receptor gene family, it was hypothesized that RAP also serves as a molecular chaperone for these other receptors. The focus of the current investigation was to examine the potential role of RAP as a molecular chaperone for the VLDL receptor. Our results demonstrate that RAP indeed is a molecular chaperone for the VLDL receptor via interaction of its carboxyl-terminal domain with the first three LBR of the VLDL receptor.

One common feature of RAP as a chaperone for LRP and the VLDL receptor is that RAP prevents the formation of mislinked disulfide bonds, which result in misfolding and retention of the receptors. In the absence of RAP co-expression, LRP appears to form high molecular weight aggregates as a result of intermolecular disulfide bonds. However, our results indicate that the major misfolding event for the VLDL receptor is primarily intramolecular. The importance of RAP as an intracellular trafficking chaperone for the VLDL receptor is further supported by the markedly increased intracellular accumulation of soluble VLDL receptor in the absence of RAP co-expression. Studies of RAP knockout mice by Willnow et al. (16) showed that functional LRP was reduced by 75%. In addition, only the ER form of the VLDL receptor was detected by Western blotting, supporting the notion that in these animals RAP is required for intracellular trafficking. Since our immunoprecipitation and cell surface iodination studies demonstrate that only the Golgi form, and not the ER form, traffics to the cell surface, it is likely that a significant portion of the VLDL receptor was misfolded and retained in the early secretory pathway in the RAP knockout mice.

Previous studies have shown that RAP interacts with the LDL receptor with lower affinity when compared with LRP and the VLDL receptor (3, 21, 35-38). Thus, RAP is less likely required for the proper folding and trafficking of the LDL receptor. Consistent with this, the expression of the LDL receptor in RAP-/- mice is normal (16). In transfection studies not shown here, we found that proper folding of a soluble form of the LDL receptor is not dependent on RAP; i.e. similar amounts of soluble LDL receptor are detected in transiently transfected cells independent of RAP co-expression.3 Since the VLDL receptor and the LDL receptor share a high degree of structure homology, this is an interesting observation and suggests that it is the first LBR of the VLDL receptor that may account for the difference in RAP-dependent folding and trafficking between the VLDL and the LDL receptor. However, recent results from Rettenberger et al. (39) suggest that the absence of the third LBR of the VLDL receptor significantly reduces RAP binding. In addition, our data (Fig. 5) indicate that RAP interacts with one or more of the three amino-terminal LBR of the VLDL receptor, while the last three LBR do not bind RAP. Additional studies will be necessary to further identify which of the three LBR are most important for RAP and/or ligand binding and to explain the differences in ligand binding specificity between the LDL receptor and the VLDL receptor.

The VLDL receptor is a multiligand receptor. The receptor probably plays a role in lipoprotein metabolism, since it binds apoE-enriched lipoproteins, such as beta -VLDL (6) and chylomicron remnants (40), and lipoprotein lipase in vitro (41, 42). Furthermore, hypercholesterolemia can be reversed by adenovirus-mediated gene transfer of the VLDL receptor (43, 44) into LDL receptor -/- mice. However, the importance of the receptor in lipoprotein metabolism under normal physiological conditions might be limited, since VLDL receptor knockout mice appear to be essentially normal, except for a slight decrease in body weight, body mass index, and adipose tissue (45). Additional VLDL receptor ligands, tissue plasminogen activator, and urokinase type plasminogen activator complexed with PAI-I (42, 46) are involved in coagulation and hemostasis and may be of physiological importance. Along these lines, it is not currently known whether the sites of interaction of these ligands coincide with RAP binding sites of the VLDL receptor. A recent report by Webb et al. (47) suggests that the VLDL receptor regulates cell surface expression of the urokinase type plasminogen activator receptor and thereby modulates cellular motility. Hence, the VLDL receptor may play a role in tumor invasion and metastasis. In addition, a recent study has shown that the VLDL receptor and apoE receptor 2 are required for neuronal migration (48). In that process, the VLDL receptor and apoE receptor 2 may bridge an extracellular matrix protein Reelin with a cytosolic adaptor protein mammalian Disabled (mDab 1), which activates intracellular kinase pathways. Thus, it will be of interest to examine whether differential RAP expression affects the biogenesis of the VLDL receptor under physiological and pathophysiological conditions.

In summary, the current study demonstrates that RAP functions as a molecular chaperone to facilitate the proper folding and trafficking of the VLDL receptor within the early secretory pathway. Although likely, further studies are required to determine whether RAP is a general chaperone for other members of the LDL receptor gene family.

    ACKNOWLEDGEMENTS

We thank Joachim Herz for providing MEF cell lines, David FitzGerald for providing CHO LRP-/- cells, and Tokuo Yamamoto for providing VLDL receptor cDNA.

    FOOTNOTES

* This work was supported by American Cancer Society Research Project Grant RPG-97-010-01-GB (to G. B.) and National Institutes of Health Grant HL-59150 (to G. B.).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.

§ Supported in part by a grant from STINT, The Swedish Institute, and by a fellowship from SANOFI Association for Thrombosis Research.

parallel 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@kids.wustl.edu.

2 J. Herz, unpublished observations.

3 R. Savonen, L. M. Obermoeller, A. L. Schwartz, and G. Bu, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LDL, low density lipoprotein; DSP, dithiobis(succinimidyl propionate); ER, endoplasmic reticulum; HA, hemagglutinin; LBR, ligand-binding repeat(s); LRP, low density lipoprotein receptor-related protein; MEF, mouse embryonic fibroblast; PAGE, polyacrylamide gel electrophoresis; RAP, receptor-associated protein; VLDL, very low density lipoprotein; Endo H, endoglucosidase H; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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