©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycoprotein 330/Low Density Lipoprotein Receptor-related Protein-2 Mediates Endocytosis of Low Density Lipoproteins via Interaction with Apolipoprotein B100 (*)

(Received for publication, May 10, 1995; and in revised form, June 16, 1995)

Steingrimur Stefansson (1) David A. Chappell (2) Kelley M. Argraves (1) Dudley K. Strickland (1) W. Scott Argraves (1)(§)

From the  (1)Biochemistry Department, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855 and the (2)Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The ability of glycoprotein 330/low density lipoprotein receptor-related protein-2 (LRP-2) to function as a lipoprotein receptor was investigated using cultured mouse F9 teratocarcinoma cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which induces F9 cells to differentiate into endoderm-like cells, produced a 50-fold increase in the expression of LRP-2. Levels of the other members of the low density lipoprotein (LDL) receptor (LDLR) family, including LDLR, the very low density lipoprotein receptor, and LRP-1, were reduced. When LDL catabolism was examined in these cells, it was found that the treated cells endocytosed and degraded at 10-fold higher levels than untreated cells. The increased LDL uptake coincided with increased LRP-2 activity of the treated cells, as measured by uptake of both I-labeled monoclonal LRP-2 antibody and the LRP-2 ligand prourokinase. The ability of LDL to bind to LRP-2 was demonstrated by solid-phase binding assays. This binding was inhibitable by LRP-2 antibodies, receptor-associated protein (the antagonist of ligand binding for all members of the LDLR family), or antibodies to apoB100, the major apolipoprotein component of LDL. In cell assays, LRP-2 antibodies blocked the elevated I-LDL internalization and degradation observed in the retinoic acid/dibutyryl cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed that was likely mediated by LDLR since it could not be inhibited by LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated protein, or apoB100 antibody. The results indicate that LRP-2 can function to mediate cellular endocytosis of LDL, leading to its degradation. LRP-2 represents the second member of the LDLR family identified as functioning in the catabolism of LDL.


INTRODUCTION

Lipoprotein receptors comprise a family of proteins that are structurally related to the low density lipoprotein (LDL) (^1)receptor (LDLR). In addition to LDLR, the family includes the very low density lipoprotein receptor (VLDLR) (Takahashi et al., 1992; Gafvels et al., 1993), the LDLR-related protein (LRP-1) (Herz et al., 1988; Jensen et al., 1989; Strickland et al., 1991), and glycoprotein 330/LRP-2 (^2)(Raychowdhury et al., 1989; Saito et al., 1994; Korenberg et al., 1994). The roles of VLDLR, LDLR, and LRP-1 as mediators of lipoprotein endocytosis have been established through numerous studies using cultured cells and animal models (for reference, see Gianturco and Bradley(1987), Yamamoto et al. (1993), and Krieger and Herz(1994)). In contrast, the role of LRP-2 in lipoprotein metabolism has largely been inferred from in vitro binding data showing that it binds apoE-enriched beta-VLDL, lipoprotein lipase-enriched VLDL (Willnow et al., 1992; Kounnas et al., 1993), and apolipoprotein J (Kounnas et al., 1995).

To investigate the cellular function of LRP-2, we have identified several LRP-2-expressing cell lines (Stefansson et al., 1995). One of these cell lines is F9 teratocarcinoma cells, which when treated with retinoic acid (RA) and dibutyryl cyclic AMP (Bt(2)cAMP), differentiate to endoderm-like epithelial cells that express 50-fold higher levels of LRP-2 than untreated cells. The levels of the other members of the LDLR family are reduced by the treatment. During characterization of the expression and activity of LDLR family members in this cell system, we discovered a novel function of LRP-2, namely that it mediates endocytosis of LDL.


MATERIALS AND METHODS

Proteins

LRP-2 was purified from pig kidney by RAP-Sepharose affinity chromatography as described previously (Kounnas et al., 1994a). Human RAP was expressed in bacteria as a fusion protein with glutathione S-transferase and purified free of glutathione S-transferase as described by Williams et al.(1992). Human prourokinase was provided by Dr. Jack Henkin (Abbott Laboratories, Abbott Park, IL). Human alpha(2)-macroglobulin was purified according to Barrett(1981) and complexed to trypsin, and the complex was purified as described by Ashcom et al.(1990). LDL (d = 1.02-1.05 g/ml) and VLDL (S 100-400) were isolated as described from normolipodemic human subjects who had the most common apoE phenotype (E3/3) (Havel et al., 1955; Chappell, 1988). Lipoproteins were labeled with [I]iodine (Amersham Corp.) to specific activities of 1000-2000 cpm/ng by the iodine monochloride method (Bilheimer et al., 1972) and used within 24 h. All other proteins were iodinated using IODO-GEN (Pierce).

Antibodies

IgG was isolated from rabbit polyclonal anti-LRP-2 serum (rb239 or rb784) (Kounnas et al., 1994b) and anti-LRP-1 serum (rb777 and rb810) (Strickland et al., 1991) by affinity chromatography on protein G-Sepharose followed by affinity selection on either LRP-2- or LRP-1-Sepharose (1-2 mg/ml of resin). IgG was isolated from the rabbit polyclonal antiserum raised against a synthetic peptide corresponding to the last 11 residues of the LRP-1 cytoplasmic domain (rb704) (Kounnas et al., 1992) by protein G-Sepharose chromatography. The mouse anti-LRP-2 monoclonal antibody 1H2 was provided by Dr. Robert McCluskey (Harvard/Massachusetts General Hospital, Boston). The mouse anti-LRP-1 monoclonal antibody 5A6, which is directed against the 85-kDa light chain of the receptor, has been previously described (Strickland et al., 1990). IgGs from 1H2 and 5A6 ascitic fluids were purified by protein G-Sepharose chromatography. The monoclonal antibody 4G3 to the ligand-binding domain of apolipoprotein B100 (Milne et al., 1983) and the mouse anti-apoE monoclonal antibody 1D7 (Weisgraber et al., 1983) were provided by Drs. Ross Milne and Yves Marcel (Clinical Research Institute of Montreal). Rabbit anti-LDLR serum was provided by Dr. Joachim Herz (Russell et al., 1984). Rabbit antiserum to a synthetic peptide corresponding to the carboxyl terminus of human and rabbit VLDLRs has been described previously (Battey et al., 1994).

Cells

Mouse embryonic F9 teratocarcinoma cells (ATCC CCL 185) were obtained from American Type Culture Collection and grown on plates (Corning, Corning, NY) coated with 0.1% gelatin in Dulbecco's modified Eagle's media (DMEM; Life Technologies, Inc.) supplemented with 10% bovine calf serum (Hyclone Laboratories, Logan, UT), penicillin, and streptomycin. Subconfluent cultures were treated with 0.1 µM RA (Calbiochem; diluted from a 0.1 M stock in dimethyl sulfoxide) and 0.2 µM Bt(2)cAMP (Sigma; diluted from a 0.1 M stock in dimethyl sulfoxide) for periods of up to 7 days without a change of medium.

Immunoblotting

Detergent extracts of the cells were prepared by solubilizing cells in 1% Triton X-100, 0.5% Tween 20, 0.5 M NaCl, 50 mM Hepes, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA. Equal amounts of protein from the extracts were loaded onto 4-12% polyacrylamide gradient gels in the presence of SDS. Following electrophoresis, the proteins were transferred onto nitrocellulose membranes, blocked with 3% non-fat milk and phosphate-buffered saline, and probed with antibodies to LDLR family members or RAP. Detection of bound antibody was achieved by autoradiography using the Renaissance chemiluminescence kit (DuPont NEN). Autoradiographs were analyzed by densitometry using a Lynx imaging system (Applied Imaging Corp.).

Solid-phase Binding Assays

I-Labeled LDL (0.9 µg/ml) was incubated in microtiter wells coated with LRP-2 or BSA (3 µg/ml coating concentration) in the presence of increasing concentrations of unlabeled competitor (RAP, monoclonal IgG 4G3, or polyclonal IgG to either LRP-1 or LRP-2) as described by Williams et al.(1992). The computer program Ligand (Munson and Rodbard, 1980) was used to analyze the competition data and to determine dissociation constants (K) for receptor-ligand interactions.

Assay of Cellular Internalization and Degradation of Ligands

To evaluate ligand internalization and degradation in F9 cells treated with RA/Bt(2)cAMP, cells from successive days of treatment were released by trypsin/EDTA and reseeded onto gelatin-coated wells (0.5-1.5 10^5 cells/wells, 24-well plates) in serum-containing DMEM with RA and Bt(2)cAMP. Prior to the addition of radiolabeled ligand, the cells were cultured for 18 h at 37 °C and 5% CO(2). Ligand internalization and degradation assays were performed as described previously (Kounnas et al., 1993; Stefansson et al., 1995). Briefly, F9 monolayers were washed twice with serum-free DMEM and incubated in serum-free DMEM containing 1.5% BSA and Nutridoma serum substitute (DMEM/BSA/SS). Antagonists of receptor ligand binding, RAP (800 nM), or polyclonal LRP-2 antibodies (250 µg/ml) in DMEM/BSA/SS were incubated with the cells for 30 min prior to the addition of radiolabeled ligand. Radiolabeled ligands (prourokinase (5 nM); alpha(2)-macroglobulin-trypsin (1 nM); mouse IgG 1H2, 5A6, and control IgG (3 nM); and LDL (2 µg/ml)) in DMEM/BSA/SS were incubated with the cells for 3.5-5 h at 37 °C and 5% CO(2). Determination of the amount of radiolabeled ligand internalized and degraded was done as described previously (Kounnas et al., 1993; Stefansson et al., 1995). The amount of I-LDL specifically internalized and degraded was determined by subtracting the amount of radioactivity internalized or degraded in the presence of a 20-fold molar excess of unlabeled ligand from the amount of radioactivity that was internalized or degraded in the absence of excess unlabeled LDL. Of the total I-LDL internalized after 3.5 h, typically 70% could be blocked by excess unlabeled LDL. Similarly, of the total cell-mediated I-LDL degradation observed after 3.5 h, typically 80% could be blocked by excess unlabeled LDL.


RESULTS

LRP-2 Expression by F9 Cells Is Enhanced by Retinoic Acid and Dibutyryl Cyclic AMP Treatment

We had previously found that F9 cells expressed both LRP-1 and LRP-2 (Stefansson et al., 1995). Knowing that F9 cells can be induced to differentiate into an absorptive endoderm-like cell type by treatment with RA and Bt(2)cAMP (Sherman and Miller, 1978), the effects on the expression of LDLR family members were examined. Immunoblot analysis was performed on detergent extracts of cells from successive days of treatment. As shown in Fig. 1, over the 7-day course of the differentiation, LRP-2 levels increased and after 7 days were 50-fold higher compared with untreated cells. In contrast, LRP-1 levels decreased 10-fold during differentiation. The level of 39-kDa RAP increased 5-fold after 7 days of treatment, which is in agreement with findings reported by Furukawa et al.(1990). The results indicate that LRP-2 and RAP levels increase coordinately during RA/Bt(2)cAMP-induced F9 cell differentiation, while levels of the other members of the LDLR family decrease.


Figure 1: Immunoblot analysis of the expression of members of the LDLR family in extracts of F9 cells treated with RA/Bt(2)cAMP. Labels on the right of each panel indicate the LDLR family member (or RAP) that is immunologically stained. The M(r) values of the stained bands shown are as follows: 600,000 for LRP-2, 85,000 for the LRP-1 light chain, 110,000 for LDLR, 130,000 and 120,000 for the two forms of VLDLR, and 39,000 for RAP. In each panel, lane0 corresponds to detergent extracts made from untreated F9 cells. Lanes 1-7 correspond to extracts made from F9 cells on successive days of RA/Bt(2)cAMP treatment.



Functional Activity of Individual LDLR Family Members in Differentiated F9 Cells

To determine whether changes in receptor levels observed during the course of differentiation correlated with changes in receptor activity displayed by the cells, endocytosis of receptor-specific antibodies or ligands was examined. Internalization of radioiodinated monoclonal antibodies to LRP-1 and LRP-2 was used as a means to measure the levels of endocytosis mediated by each receptor. As shown in Fig. 2A, the amount of I-LRP-2 antibody (mAb 1H2) internalized by cells increased over the course of the RA/Bt(2)cAMP-induced differentiation. The pattern of increase in the uptake of I-LRP-2 antibody over the course of treatment paralleled the increase in LRP-2 protein levels as shown in Fig. 1. In contrast, the level of I-LRP-1 antibody (mAb 5A6) that was internalized by the cells over the course of treatment was not significantly different from that of a control IgG of the same isotype. The lack of I-LRP-1 antibody (mAb 5A6) internalization by F9 cells was not due to failure of the antibody to recognize mouse LRP-1 since a different mouse teratocarcinoma cell line (SCC-PSA1 cells; ATCC CRL 1535) specifically internalized the antibody (data not shown). Despite the fact that the monoclonal LRP-2 antibody was internalized, it was not degraded (Fig. 2B), which is in agreement with previous observations (Stefansson et al., 1995). The basis for this may be that the antibody does not dissociate from the receptor in the low pH environment of the endosomes, but is recycled back to the surface with the receptor, as has been described for antibodies to LRP-1 (Herz et al., 1990). Our results suggest that there is little LRP-1 expressed on the surface of the F9 cells. Consistent with this conclusion was the observation that the F9 cells failed to endocytose or degrade alpha(2)-I-macroglobulin-trypsin complexes, a LRP-1-specific ligand, during the course of RA/Bt(2)cAMP treatment (Fig. 2, C and D). Internalization and degradation of I-prourokinase, a ligand for both LRP-2 (Stefansson et al., 1995) and LRP-1 (Kounnas et al., 1993), increased over the course of treatment (Fig. 2, C and D). Because F9 cells display little LRP-1 activity, this suggests that the increasing level of endocytosis and degradation of I-prourokinase is mediated by LRP-2.


Figure 2: Assay of cellular internalization and degradation of LDLR family member ligands or antibodies by F9 cells treated with RA/Bt(2)cAMP. A and B show the cellular internalization (A) and degradation (B) of I-LRP-2 antibody 1H2 (▪), I-LRP-1 antibody 5A6 (▴), or a control mouse IgG () by F9 cells at the indicated days of treatment with Bt(2)cAMP (DBC). C and D show the cellular internalization (C) and degradation (D) of I-prourokinase (Pro-uPA; ▪), I-prourokinase plus RAP (), and alpha(2)-I-macroglobulin (alpha(2)M)-trypsin (bullet) by F9 cells at the indicated days of treatment. E and F show the cellular internalization (E) and degradation (F) of I-VLDL (bullet) and I-VLDL plus RAP () by F9 cells at the indicated days of treatment. G and H show the cellular internalization (G) and degradation (H) of I-LDL (bullet) and I-LDL plus RAP () by F9 cells at the indicated days of treatment. The data presented are representative of three experiments, each performed in duplicate. Each plotted value represents the average of duplicate determinations with the range indicated by the bars. DBC, Bt(2)cAMP.



The two other LDLR family members that were detected in extracts of F9 cells (untreated and RA/Bt(2)cAMP-treated) are LDLR and VLDLR. To examine the activity of these receptors in the F9 cells, we used the VLDLR- and LDLR-specific lipoprotein ligands, VLDL and LDL, respectively. Although VLDL binds to LDLR, the capacity of LDLR to mediate their catabolism is considerably less than that of LDL (Chappell et al., 1993). I-VLDL was found to be internalized and degraded by F9 cells during the course of RA/Bt(2)cAMP treatment; however, there was no change in the amount taken up and degraded by the cells (Fig. 2, E and F). RAP was found to completely block the internalization and degradation of I-VLDL, as has been reported previously (Battey et al., 1994; Medh et al., 1995). These findings were consistent with immunoblot analysis of detergent extracts of the F9 cells (Fig. 1C), which indicated that the level of VLDLR did not increase throughout the course of treatment. Furthermore, VLDL has been shown not to interact with LRP-2 unless it is enriched with lipoprotein lipase (Kounnas et al., 1993). Therefore, the increased levels of LRP-2 in the RA/Bt(2)cAMP-treated cells would not be expected to promote an increase in VLDL uptake.

In contrast to the observation that LDLR levels did not increase in response to RA/Bt(2)cAMP treatment, there was a 10-fold increase in the amount I-LDL that was internalized and degraded by the cells over the course of RA/Bt(2)cAMP treatment (Fig. 2, G and H). The similarity between the pattern of increase in I-LDL uptake over the course of treatment and that of the uptake of I-LRP-2 antibody and the LRP-2 ligand I-prourokinase suggests that LRP-2 might be mediating the endocytosis of LDL.

LRP-2 Binds to LDL in Solid-phase Binding Assays

To evaluate the ability of LRP-2 to bind directly to LDL, in vitro binding assays were performed using purified LRP-2. As shown in Fig. 3A, I-LRP-2 bound to microtiter wells coated with LDL, but not to BSA-coated wells. This binding could be inhibited by unlabeled LRP-2. Based on a fit of these data, a dissociation constant (K) of 45 nM (n = 4) was derived. In a like manner, I-LDL bound to microtiter wells coated with LRP-2, and binding could be inhibited by increasing doses of unlabeled LDL (Fig. 3B). By fitting these data, assuming a molecular mass of 0.513 10^6 Da for LDL (Chappell et al., 1993), a K of 50 nM (n = 2) was determined. RAP, the antagonist of LRP-2 ligand binding, inhibited the binding of I-LDL to LRP-2 (Fig. 3C), with an inhibition constant (K) of 3.3 nM (n = 3). The binding of I-LDL to LRP-2 could also be blocked by polyclonal LRP-2 antibodies (Fig. 3D), but not by polyclonal LRP-1 antibodies (data not shown). The results indicate that LRP-2 can bind LDL particles. By comparison to the LDLR-LDL interaction, the LDL-LRP-2 affinity is 10-fold lower (Medh et al., 1995).


Figure 3: Binding of LRP-2 and LDL in solid-phase binding assays. In A, I-LRP-2 (0.1 nM) and various concentrations of unlabeled LRP-2 were incubated with wells coated with LDL (bullet) or BSA (). In B, I-LDL (1.6 nM) and various concentrations of unlabeled LDL were incubated with wells coated with LRP-2 (▪) or BSA (). In C, I-LDL and various concentrations of RAP were incubated with wells coated with LRP-2 (▪) or BSA (). In D, I-LDL and various concentrations of polyclonal LRP-2 antibody (rb239) were incubated with wells coated with LRP-2 (▪) or BSA (). In E, I-LDL and various concentrations of either apoB100 antibody (mAb 4G3) (▪) or apoE antibody (mAb 1D7) () were incubated with wells coated with LRP-2 (▪) or BSA (). The curves represent the best fit of the data to a single class of sites. The data presented in A-E are representative of two, three, five, three, and two experiments, respectively, with each performed in duplicate. Each plotted value represents the average of duplicate determinations with the range indicated by the bars.



The major structural component of LDL is apoB100. This apolipoprotein is known to mediate the binding of LDL to LDLR (Milne et al., 1983). To evaluate the role of apoB100 in LDL binding to LRP-2, a monoclonal anti-apoB100 IgG (mAb 4G3) was used as a blocking agent in the solid-phase I-LDL-LRP-2 binding assays. The antibody 4G3 binds to the receptor-binding region of apoB100 and can block interaction with LDLR (Milne and Marcel, 1982; Milne et al., 1983). As shown in Fig. 3E, mAb 4G3 inhibited the binding of I-LDL to LRP-2 coated on microtiter wells. Neither a control IgG of the same isotype as mAb 4G3 nor the apoE antibody 1D7 (known to block apoE-mediated binding to LDLR (Weisgraber et al., 1983)) had inhibitory effects on the binding. The results indicate that apoB100 serves as the ligand that mediates interaction of LDL with LRP-2.

LRP-2 Mediates Endocytosis of LDL in F9 Cells

To demonstrate that LRP-2 expressed by F9 cells functions to mediate LDL endocytosis, we measured the endocytosis and degradation of I-LDL in the presence of polyclonal antibodies that had previously been shown to block LRP-2 function (Stefansson et al., 1995). As shown in Fig. 4, LRP-2 antibodies blocked 75% of the I-LDL internalized by the cells treated with RA/Bt(2)cAMP. The LRP-2 antibody was not able to block I-LDL internalization to the same extent as did excess unlabeled LDL. However, RAP was able to block I-LDL uptake to a similar extent compared with excess unlabeled LDL. The results indicate that while LRP-2 accounts for the increase in LDL uptake observed during RA/Bt(2)cAMP treatment, there is a basal level of I-LDL uptake that does not involve LRP-2. The fact that this basal level of LDL uptake is RAP-sensitive suggests that some LDLR family member, most likely LDLR, is involved.


Figure 4: LRP-2 antibody inhibits the increased cellular uptake of LDL that occurs in F9 cells treated with RA/Bt(2)cAMP. Shown are the amounts of I-LDL internalized by F9 cells on successive days of treatment with RA/Bt(2)cAMP (DBC) in the presence of LRP-2 antibody (250 µg/ml; ▪), control IgG (250 µg/ml; ), RAP (800 nM; ), or LDL (40 µg/ml; ) or in the absence of competitor (bullet). Each plotted value represents the average of duplicate determinations with the range indicated by the bars. The data presented are representative of two experiments, each performed in duplicate.



The effect of monoclonal anti-apoB100 IgG (mAb 4G3) on cellular uptake and degradation of LDL was also examined. As shown in Fig. 5, mAb 4G3 blocked the endocytosis and degradation of I-LDL to a similar extent compared with excess LDL and RAP. The apoE antibody 1D7 had little or no effect on these processes. The results indicate that LDL uptake and degradation by F9 cells are apoB100-dependent. This, along with the observed inhibitory effects of apoB100 antibody on in vitro LDL-LRP-2 binding (Fig. 3) and the fact that LRP-2 antibodies block the increased I-LDL uptake and degradation in the treated cells (Fig. 4), indicates that LRP-2 interaction with apoB100 mediates the increased LDL clearance exhibited by the treated cells.


Figure 5: Monoclonal apoB100 antibody inhibits the increased cellular uptake and degradation of LDL that occurs in F9 cells treated with RA/Bt(2)cAMP. Shown are the amounts of I-LDL internalized (A) and degraded (B) by normal F9 cells (open bars) and by F9 cells treated with RA/Bt(2)cAMP (DBC) for 7 days (filled bars) in the presence of RAP, LRP-2 antibody, LRP-1 antibody, and apoB100 antibody (mAb 4G3). All values depicted have been corrected by subtraction of nonspecifically internalized or degraded LDL as described under ``Materials and Methods.'' The data presented are representative of five experiments, each performed in duplicate. Each plotted value represents the average of duplicate determinations with the range indicated by the bars.




DISCUSSION

This study establishes for the first time that LRP-2 is a LDL receptor capable of mediating LDL endocytosis and lysosomal degradation. This conclusion is supported by in vitro binding assays showing high affinity binding of LRP-2 and LDL and cell assays showing that the uptake and degradation of radiolabeled LDL in F9 cells are inhibited by LRP-2 antibodies. In addition, the LRP-2 interaction with LDL was inhibitable with apoB100 antibody in both solid-phase and cellular assays, thereby indicating that apoB100 is the component of LDL recognized by LRP-2. RAP was shown to be a potent inhibitor of LDL binding to LRP-2, having a lower K(3.3 nM) than that reported for inhibition of LDL binding to LDLR (140 nM (Medh et al., 1995)).

The major question raised by these findings is the in vivo relevance of LRP-2-mediated uptake of LDL. The fact that LRP-2 is apparently expressed only in extravascular sites (Kounnas et al., 1994b) seems to preclude its role in the clearance of LDL directly from blood in the adult. However, LRP-2 is expressed by embryonic trophectoderm and on parietal and visceral endoderm (Buc-Caron et al., 1987; Gueth-Hallonet et al., 1994). During early placental formation, trophectoderm differentiates into trophoblast giant cells, which surround the conceptus and make contact with decidual tissue and maternal blood (Cross et al., 1994). Parietal endoderm forms a layer underlying the trophoblast giant cells and mediates nutrient exchange from the trophoblast giant cells to the yolk. RA/Bt(2)cAMP-differentiated F9 cells have been shown to have characteristics consistent with parietal endoderm (Damjanov et al., 1994). The observed LRP-2-mediated uptake of LDL by cultured RA/Bt(2)cAMP-differentiated F9 cells may represent an experimental model for the uptake of maternal LDL by embryonic trophoblast and endodermal cells of the yolk sac placenta. In addition to cells of the placenta, LRP-2 has been found to be expressed by a number of other specialized epithelial cells including those of choroid plexus, lung alveoli, and kidney proximal tubules (Kounnas et al., 1994b; Zheng et al., 1994; Assmann et al., 1986). Each of these epithelia is in contact with extravascular fluids with LRP-2 localized on the apical surface of the cells, exposed to the fluids. However, with the exception of cerebrospinal fluid of the adult human, which contains low levels (0.77 mg/liter) of apoB (Carlsson et al., 1991), little or no information exists as to the LDL content in these fluids.

It is conceivable that LRP-2 acts as part of a back-up system to LDLR for the uptake of cholesterol-rich LDL. In animals genetically deficient for LDLR, developmental abnormalities are not evident (Goldstein and Brown, 1983). It has been speculated that increased de novo synthesis of cholesterol may compensate for the absence of cholesterol derived via LDLR-mediated uptake of LDL (Dietschy et al., 1983). However, de novo synthesis of cholesterol cannot compensate for cellular requirements for lipid-soluble vitamins, such as vitamin E, that are associated with LDL. In humans and mice that are genetically deficient for apoB and hence deficient in apoB-containing lipoproteins, neurological abnormalities are apparent (Homanics et al., 1993; Kane and Havel, 1989). Vitamin deficiency has been speculated to be a contributing factor in the abnormal neurological phenotype associated with genetic deficiency of apoB (Homanics et al., 1993; Farese et al., 1995). LRP-2-mediated uptake of LDL may therefore serve as a mechanism to acquire lipid-soluble vitamins.

The identification of the apoB component of LDL as a ligand for LRP-2 adds to a growing list of ligands that bind to this receptor. In addition to LDL, the current list of LRP-2 ligands includes lipoprotein lipase and the lipoprotein lipase-VLDL complex (Willnow et al., 1992; Kounnas et al., 1993), apoE-enriched beta-VLDL (Willnow et al., 1992), apolipoprotein J (Kounnas et al., 1995), prourokinase (Stefansson et al., 1995), plasminogen activator inhibitor-1 (Stefansson et al., 1995), complexes of tissue-type plasminogen activator or urokinase with plasminogen activator inhibitor-1 (Willnow et al., 1992; Moestrup et al., 1993; Stefansson et al., 1995), thrombospondin-1 (Godyna et al., 1995), lactoferrin (Willnow et al., 1992), and RAP (Kounnas et al., 1992; Orlando et al., 1992; Christensen et al., 1992). It is not obvious whether there is a functional linkage among these ligands that accounts for their having a common receptor. It seems that LRP-2, and also LRP-1, can function in two major physiological arenas, lipoprotein metabolism and proteinase regulation. It remains to be determined whether there exists a link between these apparently distinct physiological processes that could explain the evolution of a single class of receptors.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK45598 (to W. S. A.), HL49264 (to D. A. C.), and GM42581 and HL50787 (to D. K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biochemistry Dept., J. H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0725; Fax: 301-738-0794; argraves{at}hlsun.red-cross.org.

(^1)
The abbreviations used are: LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; VLDL, very low density lipoprotein; VLDLR, very low density lipoprotein receptor; LRP-1, alpha(2)-macroglobulin receptor low density lipoprotein receptor-related protein; LRP-2, glycoprotein 330/low density lipoprotein receptor-related protein-2; RA, retinoic acid; Bt(2)cAMP, dibutyryl cyclic AMP; RAP, receptor-associated protein; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; mAb, monoclonal antibody.

(^2)
Glycoprotein 330 is synonymous with gp330, brushin, megalin, gp600, and LRP-2.


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