©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibitory Effect on the Lipolysis-stimulated Receptor of the 39-kDa Receptor-associated Protein (*)

(Received for publication, April 28, 1995)

Armelle A. Troussard , Jamila Khallou , Christopher J. Mann , Patrice André , Dudley K. Strickland (1), Bernard E. Bihain (§) , Frances T. Yen

From the INSERM U391, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes I, 35043 Rennes, France and Biomedical Research and Development, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 µg/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J.(1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations 10 µg/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 µg/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind I-LDL after preincubation with oleate. However, unlike LDL, binding of I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.


INTRODUCTION

Dietary lipids, hydrolyzed and absorbed by the intestine, are repackaged and secreted as chylomicrons. Upon release into the plasma, these particles undergo lipolysis by lipases located on the surface of the endothelium. This process generates chylomicron remnants (CMR)()that are removed from the circulation by the hepatocyte through receptor-mediated endocytosis. It is generally accepted that the low density lipoprotein (LDL) receptor accounts for part of this process and works in synergy with a second genetically distinct receptor(1, 2) . The molecular nature of this second lipoprotein receptor remains disputed(3) . Indeed, two proteins, the LDL receptor-related protein (LRP) and the lipolysis-stimulated receptor (LSR), have been proposed as candidates for this function. The LRP was initially identified by homologous cloning as a member of the LDL receptor gene family (4) and subsequently found to be identical to the -macroglobulin receptor(5) . The LRP is a 600-kDa, Ca-dependent protein that binds -very low density lipoprotein (VLDL) enriched with apoprotein (apo) E(6) , lipoprotein lipase(7) , as well as a series of ligands apparently not related to the lipoprotein system, e.g. activated -macroglobulin (for review, see (8) and (9) ). The biochemical characterization of LSR is far less advanced(10) . Its activity is attributed in human cells to two membrane proteins of apparent molecular masses of 115 and 85 kDa(11) . In rat hepatocytes, the apparent molecular masses of these two proteins are 115 and 90 kDa.()LSR activation by free fatty acids (FFA) induces a conformational shift that unmasks a Ca-independent apoB and apoE binding site that displays the highest affinity for triglyceride-rich lipoproteins(10, 11) . That LSR might significantly contribute to the removal of CMR is supported by the observation of a strong inverse correlation between the level of nonfasting plasma triglycerides and the apparent number of LSR receptors expressed in rat liver.

Significant progress in the understanding of CMR removal stemmed from the study by Willnow et al.(12) using adenovirus vector-mediated gene transfer into mice of a 39-kDa receptor-associated protein (RAP). This protein, which copurifies with the 515-kDa subunit of LRP(5, 13, 14) , binds to LRP in the presence of Ca and inhibits the binding of all LRP ligands thus far identified (15, 16, 17) . RAP overexpression in both wild type and LDL receptor-negative mice increased plasma cholesterol and triglyceride concentrations, primarily through an accumulation of apoB-48 and apoE-containing particles(12) . These data were consistent with RAP's hyperlipidemic effect resulting from the inhibition of the clearance of CMR and pointed toward LRP as being responsible for the removal of these particles. This interpretation assumes that the RAP inhibitory effect is LRP-specific. However, it has been shown that at the concentrations achieved in animals overexpressing RAP, this protein inhibits various other membrane receptors: the LDL receptor(18, 19) , VLDL receptor(20) , and the gp330(21) . The possibility that the RAP hyperlipidemic effect is due to interaction with these receptors was ruled out by the observation of its effect in transgenic homozygous LDL receptor knockout mice and by the fact that neither the gp330 nor the VLDL receptor is expressed in the liver(22, 23, 24) . However, the issue of the specificity of the RAP inhibitory effect is further complicated by the finding that it binds to a yet unidentified 105-kDa protein(20) . This prompted us to reexamine the possibility that RAP interacts with LSR. Previous experiments showed that RAP at concentrations of up to 5 µg/ml had no significant effect on LSR activity, while fully inhibiting the LRP-mediated uptake and degradation of -macroglobulin-methylamine(11) . However, RAP concentrations achieved in the plasma of mice through transfection exceeded this value and ranged between 20 and 200 µg/ml(12) . Here we report that, while ineffective at low concentrations, at the concentrations that in vivo delay remnant clearance, RAP bound LSR candidate proteins and inhibited LSR activity in both intact cells and isolated liver membranes.


EXPERIMENTAL PROCEDURES

Materials

NaI was purchased from Amersham (Les Ulis, France). Oleic acid, bovine serum albumin (A2153) (BSA), CHAPS, Triton X-100, Hepes, leupeptin, benzamidine, and bacitracin were obtained from Sigma (St. Quentin, Fallavier, France). Sodium suramin and glutathione-Sepharose were obtained from FBA Pharmaceutical (West Haven, CT) and Pharmacia (Orsay, France), respectively. Human plasma thrombin was purchased from Calbiochem (Meudon, France), and Dulbecco's modified Eagle's medium (DMEM), trypsin, penicillin-streptomycin, glutamine, and fetal bovine serum were purchased from Life Technologies, Inc. (Eragny, France).

Methods

Preparation and Radiolabeling of LDL

Human LDL (1.025 < density < 1.055 g/ml) was isolated by sequential ultracentrifugation of fresh plasma obtained from the local blood bank(10, 25) . All preparations were used within 2 weeks of their isolation. LDL was radioiodinated as described previously using Bilheimer's modified McFarlane's procedure (26) and used no more than 1 week after radiolabeling. I-LDL was filtered (0.22-µm filter, Gelman, Ann Arbor, MI) immediately prior to use.

Preparation and Radiolabeling of RAP Fusion Protein and RAP

Human RAP was expressed in bacteria as a fusion protein associated with glutathione S-transferase (GST), as described previously(16, 17) . RAP fusion protein was cleaved from its GST moiety by thrombin treatment(17) . For ligand blotting studies, RAP fusion protein was iodinated using IODO-BEADS (Pierce, Asnieres, France), following the manufacturer's instructions.

Cells

Human fibroblasts from a French-Canadian patient homozygous for familial hypercholesterolemia (FH) were kindly provided by Dr. J. Davignon (Montreal). These cells were plated in 36-mm dishes at a density of 1.2 10 cells/dish and grown to confluence (4-5 days) in DMEM containing 20% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mM glutamine. LSR-mediated cell surface binding, uptake, and degradation of lipoproteins were measured as described previously(10) .

Rat Liver Membrane Preparation

Membranes from rat liver were isolated according to the procedure of Belcher et al.(27) . Membrane preparations were stored under N in the dark at 4 °C and used within 1 week of their preparation.

Measurement of LSR Activity in Isolated Rat Liver Membranes

LSR activity was measured in rat liver membranes using a modification of the previously reported procedure(11) . Briefly, membranes were diluted to 1 mg of protein/ml in buffer A (0.1 M phosphate buffer containing 350 mM NaCl and 2 mM EDTA, pH 8) and sonicated (Bioblock Scientific Vibracell, 30 s, 25% pulse, setting 2.5). Aliquots of the membranes (100 µg of protein/tube) were incubated at 37 °C for 30 min in the absence or presence of 800 µM oleate in a total volume of 250 µl/tube adjusted with buffer A. The membranes were then washed by three series of centrifugation (35,000 g, 15 min, 4 °C) and resuspension in buffer A by sonication (5 s, 90% pulse, setting 1). After this, the membranes were incubated at 4 °C for 30 min in the absence or presence of RAP fusion protein or RAP and then incubated at the same temperature for 1 h with I-LDL. To remove unbound ligand, 200 µl of the incubation mixtures were layered over 600 µl of 5% (w/v) BSA in buffer A. The samples were then centrifuged (35,000 g, 25 min, 4 °C), the supernatants were gently aspirated using Pasteur pipettes, and the bottoms of the tubes containing the membrane pellets were cut and counted in a counter (Pharmacia, 1470 Wizard).

Ligand-blotting Studies

Protein was solubilized from rat liver membranes and partially purified LSR fractions were prepared by anion exchange chromatography essentially as described elsewhere. Fractions enriched in LSR activity were separated by SDS-PAGE (4-12% gradient) under non-reducing conditions, transferred to nitrocellulose (0.45 µm, Schleicher and Schuell), and tested for their ability to bind I-LDL or I-RAP fusion protein after incubation with or without oleate.

Protein Determinations

Protein concentrations were determined using Markwell's modified Lowry procedure (28) and BSA as standard.


RESULTS

Experiments were initially undertaken to determine the effect of RAP on the LSR expressed in human FH fibroblasts. FH fibroblasts incubated in the presence of 75 µg/ml RAP fusion protein displayed a consistent reduction of I-LDL binding, uptake, and degradation (Fig. 1, ) induced by 500 µM oleate when compared with the values measured in dishes incubated with the same concentration of oleate but in the absence of RAP (). In contrast, RAP had no detectable effect on the low values of I-LDL binding, uptake, and degradation measured in FH cells incubated in the absence of oleate (data not shown). Since in the presence of RAP, all three parameters were reduced to a similar degree, we hypothesized that its major effect was to inhibit the initial event, i.e. the binding of the lipoprotein particle to the LSR.


Figure 1: Effect of RAP fusion protein on the kinetics of I-LDL binding, uptake, and degradation by LSR in FH fibroblasts. Confluent FH fibroblasts were incubated at 37 °C for 60 min in the absence () or presence () of 75 µg/ml RAP fusion protein, followed by incubation at 37 °C for 90 min in the absence or presence of 500 µM oleate and increasing concentrations of I-LDL (specific activity, 182 cpm/ng) in DMEM containing 0.2% BSA, 2 mM CaCl, and 5 mM Hepes, pH 7.5. After this, cells were washed 3 times in phosphate-buffered saline (PBS) containing 0.2% BSA, pH 7.4, followed by two washes in PBS alone. Cells were then incubated at 4 °C for 1 h with 10 mM suramin in PBS (1 ml/dish). The media were removed and counted for radioactivity; this represented the amount of I-LDL bound (A). Cells were recovered in 0.1 N NaOH containing 0.24 mM EDTA and counted; this represented the amount of I-LDL internalized (B). Degradation products were measured as trichloroacetic acid-soluble products after chloroform extraction (C). Results represent the difference between dishes incubated with and without oleate; each point is the mean of duplicate determinations.



To further explore the mechanism of RAP inhibition of LDL binding to LSR, we used a recently developed rat liver membrane binding assay. With this model, RAP was also found to inhibit LSR activity in a dose-dependent manner (Fig. 2). However, in keeping with our previous observations (11) , low RAP concentrations (5 µg/ml) did not reproducibly nor significantly decrease I-LDL binding to LSR ( Fig. 2and data not shown). The inhibitory effect of RAP fusion protein was due to its RAP rather than GST moiety. Indeed, 81% inhibition was observed in experiments measuring LSR activity in rat liver membranes incubated in the presence of 50 µg/ml RAP cleaved by thrombin treatment from the GST fragment (data not shown). This degree of inhibition was similar to that of 79% achieved with an equivalent concentration of RAP fusion protein (Fig. 2). Thus, there was no added benefit in systematically obtaining cleaved RAP fusion protein.


Figure 2: Effect of increasing concentrations of RAP fusion protein on LSR activity in rat liver membranes. Rat liver membranes were incubated at 37 °C for 30 min with or without 800 µM oleate and then washed 3 times in buffer A. Following this, the membranes were incubated at 4 °C for 30 min with the indicated concentrations of RAP fusion protein, followed by incubation at the same temperature for 1 h with 10 µg/ml I-LDL (specific activity, 140 cpm/ng). Unbound I-LDL was removed by layering 200 µl of the incubation mixture onto a 600-µl 5% (w/v) BSA cushion and centrifugation. The supernatants were removed by aspiration, and the tube bottoms containing the membrane pellets were cut and counted for radioactivity. Results represent the difference between membranes incubated with and without oleate; each point is the mean of duplicate determinations.



In these assays, the incubation medium contained no Ca and was supplemented with 2 mM EDTA. These conditions allow the characterization of LSR activity independently of those of the LDL receptor and LRP that are strictly Ca-dependent(4, 29) . Thus, both LSR activity and RAP inhibitory effect on LSR did not require the presence of divalent cations.

Curve-fitting analysis of the data in Fig. 2yielded an estimate of 20 µg/ml RAP needed to achieve 50% inhibition. To characterize the molecular mechanism responsible for RAP's inhibitory effect on LSR activity, the binding of increasing I-LDL concentrations to LSR was measured in the absence or presence of 20 µg/ml RAP fusion protein. Both direct examination (Fig. 3, panelA) and Lineweaver-Burk transformation of the data (Fig. 3, panelB) showed that the RAP inhibitory effect resulted from a change in maximal binding capacity rather than a change in affinity. This is consistent with RAP not exerting its inhibitory effect through a direct competition with I-LDL for the putative LSR lipoprotein binding domain.


Figure 3: Effect of RAP fusion protein on the binding of I-LDL to LSR in rat liver membranes. Rat liver membranes were incubated at 37 °C for 30 min with or without 800 µM oleate and then washed 3 times in buffer A. Rat liver membranes were then incubated without () or with () 20 µg/ml RAP fusion protein and increasing concentrations of I-LDL (specific activity, 140 cpm/ng) exactly as described in Fig. 2. Results represent the difference between membranes incubated with and without oleate; each point is the mean of duplicate determinations (A). PanelB represents the Lineweaver-Burk transformation of data in panelA.



To test this, solubilized rat liver membrane proteins were separated by anion exchange chromatography; fractions exhibiting LSR activity were pooled, separated on 4-12% gradient SDS-polyacrylamide gels, and transferred to nitrocellulose. The proteins immobilized on the strips were then incubated in the presence or absence of oleate and tested for their ability to bind either I-LDL or I-RAP fusion protein. Preincubation of nitrocellulose strips with oleate induced I-LDL binding to the two LSR candidate membrane proteins of apparent molecular masses 115 and 90 kDa (Fig. 4, lane2). I-RAP fusion protein also bound to two major bands of similar apparent molecular mass (lanes4 and 5). The binding of RAP fusion protein to LSR candidate proteins appeared not to require preincubation with oleate (lane4). In strips incubated with oleate, the pattern of bands revealed by either I-LDL or I-RAP fusion protein was virtually superimposable (lanes2 and 5, respectively). Preincubation of nitrocellulose strips with excess unlabeled RAP prior to incubation with oleate and I-LDL significantly decreased the binding of I-LDL to LSR (lane3). However, the reverse experiment showed that preincubation with unlabeled LDL and 800 µM oleate had little to no inhibitory effect on the subsequent binding of I-RAP (lane6). This latter observation was consistent with the notion that the RAP LSR binding site is distinct from the oleate-induced LDL binding site.


Figure 4: Binding of I-LDL (A) or I-RAP (B) fusion protein in absence or presence of oleate to LSR-enriched solubilized protein fraction separated on 4-12% SDS-PAGE and transferred to nitrocellulose. The LSR-enriched fraction of solubilized liver membrane protein was separated under non-reducing conditions on a 4-12% gradient SDS-PAGE gel, and the separated proteins were transferred to nitrocellulose. After this, the strips were incubated for 30 min in PBS containing 3% (w/v) BSA and washed with PBS. PanelA, the nitrocellulose strips were incubated at 37 °C for 1 h in the absence (lanes1 and 2) or presence (lane3) of 1 mg/ml RAP fusion protein in buffer A. After this, the membranes were washed twice in buffer A and the strips were incubated at 37 °C for 15 min without (lane1) or with (lanes2 and 3) 800 µM oleate and then at 37 °C for 1 h with 20 µg/ml I-LDL (specific activity, 398 cpm/ng). PanelB, the strips were incubated at 37 °C for 15 min without (lane4) or with (lanes5 and 6) 800 µM oleate in buffer A. The strips were then incubated at 37 °C for 1 h in the absence (lanes4 and 5) or presence (lane6) of 1 mg/ml unlabeled LDL, followed by incubation at the same temperature for 1 h with 20 µg/ml I-RAP fusion protein (specific activity, 2696 cpm/ng). All membranes were then washed in PBS containing 0.5% Triton X-100, dried, and exposed for 1 h to a phosphor screen.




DISCUSSION

RAP interaction with LSR protein was confirmed using four different experimental approaches. First, RAP was found to partially block the binding of I-LDL to LSR in human FH fibroblasts, which led to a parallel reduction in their subsequent uptake and proteolytic degradation. Second, RAP inhibited, in a dose-dependent manner, the binding of LDL to LSR in isolated rat liver membranes. Third, RAP was shown by ligand blotting to bind to two main proteins with apparent molecular masses identical to those that are considered as responsible for LSR activity. Fourth, an excess of RAP significantly reduced the binding of I-LDL to partially purified LSR proteins separated by SDS-PAGE and transferred to nitrocellulose.

On the basis of the data currently available, it appears that LSR represents yet another receptor inhibited by RAP. Indeed, LSR apparent molecular mass clearly distinguishes it from the LRP (600 kDa) or the gp330 (600 kDa) but not from the LDL-receptor (120 kDa) or the VLDL receptor (130 kDa). LSR is, however, genetically distinct from the LDL-receptor; LSR is expressed in homozygous FH fibroblasts with null alleles, and its activity is Ca-independent. Circumstantial evidence also suggests that LSR is distinct from the VLDL receptor. In living animals, LSR is expressed to a large extent in the liver, while both in rabbits and mice, VLDL receptor mRNA is at the limit of detectability in this tissue(24, 30) . In addition, retinoic acid appears to increase the expression of the VLDL receptor(30) , while decreasing LSR activity(31) . Also, unlike LSR, the VLDL receptor is inhibited by very low RAP concentrations (half-maximum inhibition at approximately 4 µg/ml; (20) ).

Thus, our observations add one more receptor to the series inhibited by RAP, i.e. the LRP(15, 16, 17) , LDL receptor(18, 19) , VLDL receptor(20) , and gp330(21) . Currently, information regarding the LSR protein primary sequence is not available. Thus, it is yet to be determined whether or not it bears sufficient homology to be considered as a member of the LDL receptor gene family.

Half-maximum LSR inhibition was achieved with RAP fusion protein concentrations of 20 µg/ml (350 nM). Therefore, RAP affinity for LSR is much lower than that for LRP; half-maximum inhibition of -macroglobulin binding to LRP was obtained with RAP concentrations of 0.4 µg/ml (10 nM)(17) . It is not surprising then that at low RAP concentrations, LRP activity in cultured cells is fully inhibited while LSR activity remains unchanged(11) . On the basis of the affinity of RAP for its various currently identified target receptors, two types of interactions can be evidenced. The first is the high affinity group with K ranging between 0.7 and 20 nM; this group includes the LRP, the VLDL receptor, and gp330. The second group, which includes the LDL receptor and LSR, is inhibited only at much higher RAP concentrations.

Analysis of the mechanism of the inhibition of LSR activity by RAP suggested that RAP does not directly compete with I-LDL for binding to LSR. This notion is further strengthened by the observation that unlike LDL, RAP binding to LSR does not require the FFA-induced conformational shift of the receptor. Furthermore, the observation that binding of LDL to LSR did not prevent subsequent binding of RAP to the receptor is consistent with RAP and LDL interacting at different sites on the LSR proteins. The RAP inhibitory effect might therefore result from an interference with the FFA-induced shift in LSR conformation or from another yet unidentified regulatory mechanism.

Previous studies have established that CMR clearance was delayed in both normal and LDL-receptor deficient mice overexpressing RAP(12) . Because gp330 and the VLDL receptor are not expressed to a large extent in the liver(22, 23, 24) , it was therefore concluded that the LRP was responsible for most of CMR removal. The finding of the RAP inhibitory effect on LSR, however, offers an alternative interpretation to Willnow's data. Indeed, RAP inhibition of the LSR could at least partially account for the hyperlipidemic effect of RAP transfection in both LDL receptor knockout and wild type mice. Additional information is needed to ascertain the relative contribution of the LRP and/or the LSR to CMR removal.


FOOTNOTES

*
These studies were supported by grants from the Institut National de la Santé et de la Recherche Médicale, Département d'Ille et Vilaine, Région Bretagne, Fondation pour la Recherche Médicale, District de Rennes, and the Biomed program of the European Commission. 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: INSERM U391, Université de Rennes 1, 2 ave du Prof. Léon Bernard, 35043 Rennes, France. Tel.: 33-99-33-69-40; Fax: 33-99-33-62-08.

The abbreviations used are: CMR, chylomicron remnant(s); apo, apoprotein; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FFA, free fatty acids; FH, familial hypercholesterolemia; gp330, glycoprotein 330; GST, glutathione S-transferase; LDL, low density lipoprotein; LRP, low density lipoprotein receptor-related protein; LSR, lipolysis-stimulated receptor; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RAP, receptor-associated protein; VLDL, very low density lipoprotein; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

Mann, C. J., Khallou, J., Chevreuil, O., Troussard, A. A., Guermani, L. M., Launay, K., Delplanque, B., Yen, F. T., and Bihain, B. E. (1995) Biochemistry, in press.


ACKNOWLEDGEMENTS

We thank Valérie Bordeau, Flora Coulon, and Evan Behre for their excellent technical assistance.


REFERENCES
  1. Brown, M. S., Herz, J., Kowal, R. C., and Goldstein, J. L.(1991)Curr. Opin. Lipidol. 2, 65-72
  2. Soutar, A. K. (1989)Nature 341, 106-107 [Medline] [Order article via Infotrieve]
  3. van Berkel, T. J. C., Ziere, G. J., Bijsterbosch, M. K., and Kuiper, J.(1994) Curr. Opin. Lipidol. 5, 331-338 [Medline] [Order article via Infotrieve]
  4. Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988)EMBO J. 7, 4119-4127 [Abstract]
  5. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S.(1990)J. Biol. Chem. 265, 17401-17404 [Abstract/Free Full Text]
  6. Kowal, R. C., Herz, J., Goldstein, J. L., Esser, V., and Brown, M. S.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5810-5814 [Abstract]
  7. Beisiegel, U., Weber, W., and Bengtsson-Olivecrona, G.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 8342-8346 [Abstract]
  8. Herz, J.(1993) Curr. Opin. Lipidol. 4, 107-113
  9. Krieger, M., and Herz, J. (1994)Annu. Rev. Biochem. 63, 601-637 [CrossRef][Medline] [Order article via Infotrieve]
  10. Bihain, B. E., and Yen, F. T.(1992)Biochemistry 31, 4628-4636 [Medline] [Order article via Infotrieve]
  11. Yen, F. T., Mann, C. J., Guermani, L. M., Hannouche, N. F., Hubert, N., Hornick, C. A., Bordeau, V. N., Agnani, G., and Bihain, B. E.(1994) Biochemistry 33, 1172-1180 [Medline] [Order article via Infotrieve]
  12. Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J.(1994)Science 264, 1471-1474 [Medline] [Order article via Infotrieve]
  13. Ashcom, J. D., Tiller, S. E., Dickerson, K., Cravens, J. L., Argraves, W. S., and Strickland, D. K.(1990)J. Cell Biol. 110, 1041-1048 [Abstract]
  14. Jensen, P. H., Moestrup, S. K., and Gliemann, J.(1989)FEBS Lett. 255, 275-280 [CrossRef][Medline] [Order article via Infotrieve]
  15. Moestrup, S. K., and Gliemann, J.(1991)J. Biol. Chem. 266, 14011-14017 [Abstract/Free Full Text]
  16. Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S.(1991) J. Biol. Chem. 266, 21232-21238 [Abstract/Free Full Text]
  17. Williams, S. E., Ashcom, J. D., Argraves, W. S., and Strickland, D. K.(1992) J. Biol. Chem. 267, 9035-9040 [Abstract/Free Full Text]
  18. Mokuno, H., Brady, S., Kotite, L., Herz, J., and Havel, R. J.(1994)J. Biol. Chem. 269, 13238-13243 [Abstract/Free Full Text]
  19. Medh, J. D., Fry, G. L., Bowen, S. L., Pladet, M. W., Strickland, D. K., and Chappell, D. A. (1995)J. Biol. Chem. 270, 536-540 [Abstract/Free Full Text]
  20. Battey, F. D., Gafvels, M. E., FitzGerald, D. J., Argraves, W. S., Chappell, D. A., Strauss, J. F., III, and Strickland, D. K.(1994)J. Biol. Chem. 269, 23268-23273 [Abstract/Free Full Text]
  21. Kounnas, M. Z., Argraves, W. S., and Strickland, D. K.(1992)J. Biol. Chem. 267, 21162-21166 [Abstract/Free Full Text]
  22. Farquhar, M. G., Kerjaschki, D., Lundstrom, M., and Orlando, R. A.(1994) Ann. N. Y. Acad. Sci. 737, 96-113 [Abstract]
  23. Kounnas, M. Z., Stefansson, S., Louikinova, E., Argraves, K. M., Strickland, D. K., and Argraves, W. S.(1994)Ann. N. Y. Acad. Sci. 737, 114-123 [Medline] [Order article via Infotrieve]
  24. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamoto, T.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9252-9256 [Abstract]
  25. Goldstein, J. L., Basu, S. K., and Brown, M. S.(1983)Methods Enzymol. 98, 241-261 [Medline] [Order article via Infotrieve]
  26. Bilheimer, D. W., Eisenberg, S., and Levy, R. I.(1972)Biochim. Biophys. Acta 260, 212-221 [Medline] [Order article via Infotrieve]
  27. Belcher, J. D., Hamilton, R. L., Brady, S. E., Hornick, C. A., Jaeckle, S., Schneider, W., and Havel, R. J.(1987)Proc. Natl. Acad. Sci. U. S. A. 84, 6785-6789 [Abstract]
  28. Markwell, M. A. K., Haas, S. M., Tolbert, N. E., and Bieber, L. L.(1981) Methods Enzymol. 72, 296-303 [Medline] [Order article via Infotrieve]
  29. Goldstein, J. L., Brown, M. S., and Anderson, M. S. (1977) in International Cell Biology 1976-1977 (Brinkley, B. R., and Porter, K. R., eds) pp. 639-642, Rockefeller University Press, New York
  30. Gafvels, M. E., Paavola, L. G., Boyd, C. O., Nolan, P. M., Wittmaack, F., Chawla, A., Lazar, M. A., Bucan, M., Angelin, B., and Strauss, J. F., III(1994) Endocrinology 135, 387-394 [Abstract]
  31. Bihain, B. E., Yen, F. T., Guermani, L. M., Troussard, A. A., Khallou, J., and Mann, C. J. (1995) in Atherosclerosis X-Proceedings of the Tenth International Symposium on Atherosclerosis, (Woodford, F. P., Davignon, J., and Sniderman, A., eds) pp. 465-470, Elsevier Science Publishers B. V., Amsterdam

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.