©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The 39-kDa Receptor-associated Protein Modulates Lipoprotein Catabolism by Binding to LDL Receptors (*)

(Received for publication, June 7, 1994; and in revised form, September 26, 1994)

Jheem D. Medh (1) Glenna L. Fry (1) Susan L. Bowen (1) Marc W. Pladet (1) Dudley K. Strickland (2) David A. Chappell (1)(§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 39-kDa receptor-associated protein (RAP) is co-synthesized and co-purifies with the low density lipoprotein receptor-related protein (LRP)/alpha(2)-macroglobulin receptor and is thought to modulate ligand binding to LRP. In addition to binding LRP, RAP binds two other members of the low density lipoprotein (LDL) receptor family, gp330 and very low density lipoprotein (VLDL) receptors. Here, we show that RAP binds to LDL receptors as well. In normal human foreskin fibroblasts, RAP inhibited LDL receptor-mediated binding and catabolism of LDL and VLDL with S 20-60 or 100-400. RAP inhibited I-labeled LDL and S100-400 lipoprotein binding at 4 °C with Kvalues of 60 and 45 nM, respectively. The effective concentrations for 50% inhibition (EC) of cellular degradation of 2.0 nMI-labeled LDL, 4.7 nMI-labeled S 20-60, and 3.6 nMI-labeled S 100-400 particles were 40, 70, and 51 nM, respectively. Treatment of cells with lovastatin to induce LDL receptors increased cellular binding, internalization, and degradation of RAP by 2.3-, 1.7-, and 2.6-fold, respectively. In solid-phase assays, RAP bound to partially purified LDL receptors in a dose-dependent manner. The dissociation constant (K) of RAP binding to LDL receptors in the solid-phase assay was 250 nM, which is higher than that for LRP, gp330, or VLDL receptors in similar assays by a factor of 14 to 350. Also, RAP inhibited I-labeled LDL and S100-400 VLDL binding to LDL receptors in solid-phase assays with K values of 140 and 130 nM, respectively. Because LDL bind via apolipoprotein (apo) B100 whereas VLDL bind via apoE, our results show that RAP inhibits LDL receptor interactions with both apoB100 and apoE. These studies establish that RAP is capable of binding to LDL receptors and modulating cellular catabolism of LDL and VLDL by this pathway.


INTRODUCTION

The alpha(2)-macroglobulin receptor-associated protein (RAP) (^1)is a 39-kDa polypeptide that co-purifies with the alpha(2)-macroglobulin (alpha(2)M) receptor/low density lipoprotein receptor-related protein (LRP)(1) . A fraction of intracellular RAP associates with LRP immediately after its synthesis and is transferred to the cell surface in a complex with LRP; the remainder remains intracellular(2, 3) . RAP is not secreted into the extracellular fluid and is not found in plasma. RAP also co-purifies with gp330, another member of the LDL receptor family(4, 5) . Mature human RAP consists of 323 amino acids and has 73% identity with the rat Heymann nephritis antigen and 77% identity with a mouse heparin-binding protein called HBP-44(2) . The carboxyl-terminal domain of RAP also has 26% sequence identity with a region of apolipoprotein (apo) E containing the LDL receptor binding domain(2) .

The physiological role of RAP is not yet clear. It is well established that RAP inhibits the interactions of LRP with all of its known ligands including alpha(2)M-proteinase complexes(6, 7) , apoE-enriched beta-migrating very low density lipoproteins (beta-VLDL)(6) , lipoprotein lipase(8, 9) , complexes of tissue-type and urokinase plasminogen activators with their inhibitor(10, 11, 12, 13, 14) , and Pseudomonas exotoxin A(15) . In addition, fibroblasts genetically engineered to overexpress RAP also overexpress LRP. (^2)Thus, it is believed that RAP modulates LRP-ligand interactions and may function in the intracellular transport of LRP as well. Kounnas et al. (3) have shown that RAP also binds gp330 with a high affinity and may modulate interactions of gp330 with its ligands(3, 16) .

Because RAP binds to LRP (17, 18) and gp330(3, 19) , both members of the LDL receptor family, we speculated that RAP might also bind LDL receptors themselves. Here, we demonstrate that RAP binds to LDL receptors both in normal human foreskin fibroblasts and solid-phase binding assays. Our findings suggest that RAP can modulate LDL receptor-mediated binding and catabolism of normal LDL and VLDL.


EXPERIMENTAL PROCEDURES

Materials

Plasma LDL and the VLDL subclasses S(f) 100-400 and S(f) 20-60 representing large and small VLDL, respectively, were isolated from the blood of fasted normolipidemic human subjects (apoE phenotype E3/3) by ultracentrifugation as previously described(20) . Recombinant human RAP was produced in Escherichia coli as a fusion protein with glutathione-S-transferase as previously described(6, 18) . Thrombin was used to digest the fusion protein then neutralized by the addition of D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone. The cleaved glutathione-S-transferase was removed by absorption to glutathione-agarose(18) . Monoclonal antibodies IgG-C7 and IgG-4A4 directed against the first cysteine-rich repeat and the cytoplasmic 14 amino acids, respectively, of the LDL receptor were obtained as previously described(21) . Activated alpha(2)M (alpha(2)M*), monoclonal anti-RAP antibody 7F1, and polyclonal rabbit anti-LRP antibody Rb-777 were produced as previously described(1, 22, 23) . Lipoproteins were iodinated by the iodine-monochloride method(24) . RAP, alpha(2)M*, and IgG-7F1 were iodinated using IODO-GEN (Pierce) as previously described(23) .

Whole Cell Binding Assays

Human foreskin fibroblasts were cultured as previously described(25, 26) . Prior to assays, LDL receptors were up-regulated by incubation for 48 h with media containing 2 mg/ml lipoprotein-deficient serum and for 24 h with media containing 1 µg/ml of lovastatin. Surface binding to metabolically inactive cells at 4 °C was studied after incubating cells with I-labeled ligands for 3 h as previously described(25, 26) . Steady-state ligand degradation, defined as the trichloroacetic acid-soluble radioactivity in the incubation medium, was measured after incubating cells with I-labeled ligands at 37 °C for 5 h. Cellular protein as determined by the Lowry assay (27) was 45-60 µg/well but varied by less than 15% within each experiment. However, wells treated with lovastatin had 50% of the protein in untreated wells. Thus, when the two treatments were compared, results were corrected for cellular protein. In all other cases, results are shown as ligand present in units of ng/well.

Solid-phase Binding Assays

LDL receptors were partially purified by fractionation of total cell extracts over DE52-cellulose (Whatman) as previously described(28) . Microtiter wells (96-well plates, immulon 2, Dynatech) were coated for 30 min at 37 °C with 3 µg of IgG-4A4 in 100 µl of buffer. When I-labeled RAP was used as the ligand, wells were coated with IgG-C7 because I-labeled RAP had relatively high binding to IgG-4A4. The wells were then blocked with 1% BSA at 37 °C for 1 h. LDL receptors from DE52 eluants were specifically immobilized by incubating IgG-coated wells for 16 h at 4 °C with 0-100 µg DE52 eluant protein in 100 µl of buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM CaCl(2), 0.5% BSA. The wells were washed and incubated for 3 h at 4 °C with I-labeled ligands as specified in the figure legends. After washing to remove unbound ligands, bound ligands were desorbed in 0. 1 N NaOH and quantitated by measuring the radioactivity.

In some experiments, bound ligands were detected by an enzyme-linked immunoabsorbent assay. Wells were coated with 0-10 µg of RAP for 5 h at 4 °C, blocked with 1% BSA for 2 h at 4 °C, washed, and incubated for 16 h at 4 °C with 100 µl of buffer containing 1 µg/µl DE52 eluant. The wells were washed again and incubated for 2 h at 4 °C with 100 µl of buffer containing 30 µg/ml IgG-C7. Bound IgG-C7 was quantitated by incubating the wells for 2 h at 4 °C with 100 µl of a 1:10,000 dilution of alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma). Then, wells were washed and 100 µl of substrate (1 mg/ml p-nitrophenylphosphate, Sigma) was added. After 20 min at 25 °C, absorbance at 405 nm was measured. Standard curves prepared simultaneously by coating wells with known amounts of IgG-C7 were linear (correlation coefficients >0.98).

Data Analysis

All data obtained in cellular and solid-phase assays were measured in duplicate. For steady-state experiments, the EC was determined using ALLFIT as previously described(29) . The equilibrium dissociation and inhibition constants (K(D) and K(I)) and nonsaturable binding were determined using LIGAND(30) . This program uses data from displacement curves to calculate K(I) values using the equation K(I) = EC/{1 + ([L]/K(D))}, where [L] is the concentration of the radiolabeled ligand and K(D) is its dissociation constant. Data in units of protein mass were converted to moles using average molecular weights of 0.513, 1.41, and 1.07 times 10^6 for the protein components of LDL, S(f) 100-400, and 20-60 particles, respectively(31, 32) , as previously described(33) .


RESULTS

RAP Inhibits Lipoprotein Binding and Catabolism by Normal Fibroblasts

Previous studies have shown that in the absence of added apoE or lipoprotein lipase, native LDL and VLDL bind to LDL receptors but not to LRP(18, 25, 26, 33, 34, 35) . LDL receptor-mediated surface binding of I-labeled LDL and S(f) 100-400 particles to fibroblasts at 4 °C was inhibited by increasing concentrations of RAP with K(I) values of 60 and 45 nM, respectively (Fig. 1). Inhibition of binding by unlabeled LDL is shown for comparison. We next investigated the effect of RAP on steady-state catabolism of lipoproteins. Results shown in Fig. 2are representative of three experiments. RAP inhibited binding and uptake (data not shown) and degradation of 2.0 nMI-labeled LDL, 4.6 nM S(f) 20-60, and 3.6 nM S(f) 100-400 particles with EC values of 40, 70, and 51 nM, respectively. As a control, we also tested the ability of RAP to inhibit LRP-mediated alpha(2)M* binding, uptake, and degradation by these cells (data not shown). The EC for degradation of 3.9 nM alpha(2)M* was 5 nM.


Figure 1: RAP inhibits cell-surface binding of I-labeled LDL and S 100-400 to human fibroblasts at 4 °C. Fibroblasts were treated with lipoprotein-deficient serum and lovastatin to up-regulate their LDL receptor number as described under ``Experimental Procedures.'' Cells were then incubated for 3 h at 4 °C in media containing 3 nM of either I-labeled LDL (A) or I-labeled S 100-400 VLDL (B) in the presence of increasing concentrations of RAP (closedcircles) or LDL (opentriangles). After washing as described, bound radioactivity was dissociated by incubating cells for 30 min at 4 °C with buffer containing 10 mg/ml polyphosphate. The moles of bound ligand were calculated from the radioactivity released. The curves represent the best fit of the data to a single class of sites using K values of 2.4 and 2.0 nM for I-labeled LDL and I-labeled S 100-400 particles, respectively.




Figure 2: RAP competes for I lipoprotein degradation by human fibroblasts. Cells were incubated for 5 h at 37 °C with media containing 2.0 nMI-labeled LDL (opencircles), 4.7 nMI-labeled S 20-60 VLDL (closedcircles), or 3.6 nMI-labeled S 100-400 VLDL (opentriangles) in the presence of various concentrations of RAP. Degradation was measured as described under ``Experimental Procedures.''



Lovastatin Up-regulates Catabolism ofI-Labeled RAP by Fibroblasts

To test the possibility that RAP competes for lipoprotein catabolism by binding to LDL receptors, we investigated the regulation of I-labeled RAP catabolism by lovastatin, an inducer of cellular LDL receptor activity (25) (Fig. 3). In three separate experiments, binding and uptake (data not shown) and degradation of I-labeled RAP were consistently higher in lovastatin-treated cells compared with controls by averages of 2.3-, 1.7-, and 2.6-fold, respectively. However, binding, uptake, and degradation of I-labeled LDL were induced 11-, 19-, and 34-fold, respectively, upon up-regulation with lovastatin (data not shown). The lower induction of RAP catabolism by lovastatin as compared with LDL catabolism at least partially reflects the LRP-mediated catabolism of RAP, which is not regulated by lovastatin (data not shown and (36) ). We investigated the LRP-mediated catabolism of I-labeled RAP using the polyclonal anti-LRP antibody Rb-777(8, 23) . In the presence of 30 µg/ml Rb-777, 80% of I-labeled alpha(2)M* degradation was inhibited, whereas only 40% of 13 nMI-labeled RAP degradation was inhibited (data not shown), suggesting that LRP-independent catabolism was significant.


Figure 3: Lovastatin treatment increases I-labeled RAP catabolism by fibroblasts. Cells were treated either with lipoprotein-deficient serum and lovastatin (closedcircles) as described under ``Experimental Procedures'' or maintained in lipoprotein-containing media (opencircles). They were then incubated for 5 h at 37 °C in the presence of various concentrations of I-labeled RAP. Degradation was calculated as described under ``Experimental Procedures.'' The data are corrected for cellular protein content in each well.



Binding of RAP to LDL Receptors in Solid-phase Assays

We further characterized RAP-LDL receptor interactions in solid-phase assays using LDL receptors partially purified by fractionating total cell extracts over DE52-cellulose. These preparations contain significant amounts of LRP and RAP. LDL receptors were selectively immobilized on plastic wells using LDL receptor-specific antibodies IgG-C7 or IgG-4A4. Immunoblots of the immobilized material showed that it contained LDL receptors but not LRP or RAP (data not shown).

Fig. 4A shows that increasing amounts of I-labeled RAP bound to increasing amounts of LDL receptors immobilized to plastic wells coated with IgG-C7. In three separate experiments, specific binding of I-labeled RAP directly correlated with the amount of immobilized receptor. To verify these results, we also measured binding of unlabeled RAP using the I-labeled monoclonal anti-RAP antibody, IgG-7F1 (Fig. 4A). Like I-labeled LDL binding, I-labeled RAP binding to LDL receptors was EDTA sensitive (data not shown). However, 1 mM EDTA was sufficient to totally inhibit I-labeled LDL binding, whereas 10 mM EDTA was required to completely eliminate I-labeled RAP binding.


Figure 4: RAP binding to immobilized LDL receptors in a solid-phase assay. Microtiter wells were first coated with IgG-C7 and blocked with BSA as described under ``Experimental Procedures'' and then incubated with various concentrations of DE52 eluant of the whole cell extract for 16 h at 4 °C. The wells were then incubated for 3 h at 4 °C with buffer containing (A) either 13 nMI-labeled RAP (closedcircles) or 260 nM unlabeled RAP (opentriangles) or (B) 9.8 nMI-labeled LDL (closedcircles) or 3.9 nMI-labeled alpha(2)M* (opentriangles). Unlabeled RAP was detected by incubating with 3.3 nMI-labeled 7F1 for 3 h at 4 °C. Nonspecific binding to BSA-coated wells was subtracted from total binding.



We also measured I-labeled alpha(2)M* binding to immobilized LDL receptors (Fig. 4B). Whereas I-labeled LDL bound in a dose-dependent manner, I-labeled alpha(2)M* failed to bind proteins immobilized by anti-LDL receptor antibodies. These data exclude a significant component of LRP binding in these assays. The ligand binding capacity of partially purified receptors varied among different preparations and decreased with storage at -70 °C. Thus, it is not appropriate to compare the stoichiometry of I-labeled RAP binding in Fig. 4A with that of I-labeled LDL binding in Fig. 4B. DE52 eluants prepared from fibroblasts lacking LDL receptors bound RAP at 50-60% lower levels than did eluants from normal fibroblasts under identical assay conditions (data not shown), indicating that LDL receptors were responsible for a majority of RAP binding.

To further confirm the interaction of RAP with LDL receptors in solid-phase assays, we coated plastic wells with increasing amounts of RAP and allowed LDL receptors in the DE52 eluant to bind (Fig. 5). Bound LDL receptors were detected using IgG-C7, a monoclonal antibody against the LDL receptor. Receptor binding was directly proportional to the amount of RAP immobilized. Negligible amounts of IgG-C7 bound in the absence of DE52 eluant, providing evidence for the specificity of binding.


Figure 5: LDL receptor binding to immobilized RAP in a solid-phase assay. Microtiter wells were coated with various amounts of RAP, blocked, and incubated in the presence (closedcircles) or absence (opentriangles) of DE52 eluant as described. Bound LDL receptors were quantitated using the anti-LDL receptor antibody IgG-C7.



We determined the K(D) for RAP binding to LDL receptors in the solid-phase assay. Immobilized LDL receptors were incubated with increasing concentrations of RAP, and the amount of RAP bound was determined using I-labeled 7F1, an anti-RAP monoclonal antibody. Fig. 6A shows the data obtained in a representative experiment. The best fit of this data to a single-site model predicted a K(D) of 250 nM for RAP binding to LDL receptors. Fig. 6B shows inhibition of I-labeled RAP binding to LDL receptors in the solid-phase assay by unlabeled RAP (K(I) = 290 nM).


Figure 6: Affinity of RAP binding to LDL receptors in a solid-phase assay. Microtiter wells coated with IgG-C7 were used to immobilize LDL receptors from DE52 eluants. The wells were then incubated for 3 h at 4 °C with buffer containing either (A) increasing concentrations of unlabeled RAP followed by another incubation with 3.3 nMI-labeled 7F1 as in Fig. 4A or (B) 13 nMI-labeled RAP in the presence of various concentrations of unlabeled RAP. Curve fitting to a single-site model is shown.



Ligand blotting to DE52 eluants was attempted but required micromolar concentrations of RAP to visualize LDL receptors. Under these conditions, background binding was much increased as was binding to several unidentified proteins in both normal fibroblasts and those lacking LDL receptors (data not shown).

RAP Competes for Lipoprotein Binding to Immobilized LDL Receptors

Fig. 7A shows that RAP competed for I-labeled LDL binding to LDL receptors in solid-phase assays with a K(I) of 140 nM. LDL competed for its own binding with a K(I) of 0.2 nM in this assay (data not shown). RAP also inhibited I-labeled S(f) 100-400 lipoprotein binding to LDL receptors with a K(I) of 130 nM (Fig. 7B). Corresponding K(I) values for LDL and S(f) 100-400 lipoproteins were 1.0 and 1.5 nM, respectively (data not shown). In other experiments not shown, both LDL and S(f) 100-400 particles completely inhibited I-labeled RAP binding to immobilized LDL receptors but only at micromolar concentrations. Control experiments in which wells were coated with increasing amounts of LDL or S(f) 100-400 particles indicated that I-labeled RAP did not bind directly to lipoproteins (data not shown).


Figure 7: RAP competes for I lipoprotein binding to LDL receptors in a solid-phase assay. LDL receptors from DE52 eluants were immobilized onto IgG-4A4-coated microtiter wells. The wells were then incubated with buffer containing (A) 1 nMI-labeled LDL or (B) 2.1 nMI-labeled S100-400 particles in the presence of various concentrations of unlabeled RAP as described under ``Experimental Procedures.'' Curve fitting to a single-site model is shown.




DISCUSSION

These data establish that RAP inhibits both LDL and VLDL binding and catabolism by normal skin fibroblasts. We confirmed the interaction of RAP with LDL receptors by several independent methods using solid-phase assays. RAP bound to LDL receptor-coated wells, and LDL receptors bound to RAP-coated wells. Also, RAP inhibited lipoprotein binding to partially purified LDL receptors and vice versa. It is unlikely that interaction of RAP with other members of the LDL receptor family confounds our findings. Gp330 and VLDL receptors are not expressed in normal fibroblasts to a significant degree, and VLDL receptors do not bind LDL, as was observed in our assays. Although LRP is present in fibroblasts, previously published data show that neither LRP nor gp330 binds normal lipoproteins in the absence of exogenously added lipoprotein lipase or apoE(8, 25, 33, 34, 35, 37) . The absence of alpha(2)M* binding in the solid-phase assay eliminates the possibility of a significant component of LRP-mediated binding. Also, induction of LDL receptors with lovastatin induced catabolism of RAP, which would not be expected with other members of the LDL receptor family. Finally, the affinity of RAP for other members of the LDL receptor family is substantially higher than that reported here.

Because LDL and VLDL bind LDL receptors via apoB100 and apoE, respectively, RAP may share binding sites for these ligands. Studies by Russell et al.(38, 39) and others suggest that repeats 3-7 of the LDL receptor are required for binding apoB100, whereas repeat 5 is sufficient for binding apoE. RAP may prevent lipoprotein binding by inducing a conformational change in the LDL receptor. It is also possible that a single LDL receptor may bind more than one RAP molecule by direct interaction with more than one ligand-binding repeat. A definitive demonstration of the binding stoichiometry has not been done. The affinity of RAP for LDL receptors (250 nM) is less than that predicted from its ability to inhibit lipoprotein binding to LDL receptors on cells (50 nM) and in solid-phase assays (140 nM). We currently have no explanation for these differences. However, they suggest that the interaction of RAP with LDL receptors is stronger in the presence of lipoproteins.

Both LDL receptors and LRP contribute to catabolism of RAP by normal fibroblasts. At a concentration of RAP greater than saturation for LRP but below saturation for LDL receptors, a polyclonal antibody against LRP inhibited 40% of I-labeled RAP degradation by normal fibroblasts. This suggests the existence of an LRP-independent pathway. When LDL receptors were up-regulated with lovastatin, catabolism of I-labeled RAP was increased by 2.5-fold (Fig. 3), despite the fact that catabolism of LDL increased >20-fold. These results suggest that both LDL receptors and LRP contribute to the total cellular catabolism of RAP. Consistent with this idea, RAP inhibited the catabolism of I-labeled lipoproteins, but the reverse is not true (data not shown). Although we cannot completely explain this finding, it partially reflects the fact that native lipoproteins cannot bind LRP and therefore would not inhibit LRP-mediated catabolism of RAP. Another factor to consider is that RAP is a heparin-binding protein (2) and conceivably could interact with cell-surface proteoglycans, an interaction that may not be blocked by lipoproteins.

RAP is the first new ligand for the LDL receptor since the discovery that apoB100 and apoE bind to this receptor(38, 40) . Previous studies establish that RAP binds to two other members of the LDL receptor family, LRP (K(D) = 18 nM) and gp330 (K(D) = 8 nM)(3, 17, 18, 19) . Recently, Battey et al. (41) found that RAP also binds to the VLDL receptor (K(D) = 0.7 nM). The affinity of RAP for LDL receptors in solid-phase assays is lower by a factor of 14-350 than that for LRP, gp330, or the VLDL receptor (41) and, in contrast to these receptors, is too low to permit ligand blotting. The LDL receptor family members share several regions of homology, particularly the ligand binding and growth factor-type repeats(42) . It is known that RAP binds to a cluster of 8 complement-type repeats in LRP(43) , and it is likely that a similar region in the LDL receptor is responsible for RAP binding. Williams et. al(18) found that a single LRP may bind two RAP molecules, supporting the notion that more than one ligand-binding repeat interacts with RAP. RAP contains a region (amino acids 203-321) with 26% sequence homology to apoE (2) that could form an amphipathic helix homologous to the secondary structure of apoE known to be involved in apoE-LDL receptor interactions(44, 45) . This region of RAP has been shown to inhibit the interactions of LRP with its ligand (46) and may be responsible for interactions with other LDL receptor family members.

The physiological significance of an interaction between RAP and LDL receptors is currently unknown. Our results suggest that RAP may modulate lipoprotein catabolism in vivo. In a recent study, Mokuno et al.(47) show that intravenous injection of glutathione-S-transferase-RAP into rats reduces the clearance of human I-labeled LDL from the blood. This is consistent with a regulatory role for RAP in lipoprotein catabolism via LDL receptors. RAP is present both intracellularly and on the cell surface but is not secreted(2) . Conceivably, RAP could act as a chaperone to assist proper LRP and LDL receptor folding and transport to the cell surface. On the other hand, intracellular RAP may promote receptor recycling and uncoupling from endocytosed ligands. RAP exists in specialized intracellular compartments, in which the affinity for LDL receptors may be dependent on its local concentration, proximity to membranes, local pH, and calcium concentration. Clearly, more studies are needed to address these possibilities. However, our results and those of Mokuno et al. (47) demonstrate that RAP may not be an appropriate experimental reagent to discriminate between LDL receptor and LRP-mediated phenomena.


FOOTNOTES

*
This work was supported by grants from the Department of Veteran Affairs Research Fund (to D. A. C.), a grant-in-aid from the American Heart Association, Bristol-Myers Squibb, and National Institutes of Health Grants HL49264 and HL50787. 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 reprint requests should be addressed: Dept. of Internal Medicine, E318 GH, University of Iowa College of Medicine, Iowa City, IA 52242. Tel.: 319-356-8285; Fax: 319-353-6343.

(^1)
The abbreviations used are: RAP, alpha(2)M receptor-associated protein; alpha(2)M, alpha(2) macroglobulin; alpha(2)M*, activated alpha(2)M; LDL, low density lipoproteins; LRP, LDL receptor-related protein; VLDL, very low density lipoproteins; apo, apolipoprotein; BSA, bovine serum albumin.

(^2)
S. E. Williams, K. M. Argraves, F. D. Battey, and D. K. Strickland, unpublished observations.


ACKNOWLEDGEMENTS

-We thank Dr. Arthur Spector for critical review of the manuscript, Mary Lou Booth and Gregory Aylsworth for assistance with tissue culture, and Nicole Bates for excellent technical assistance.


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