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We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time. We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.

The objectives of the present study were to 1) examine mechanisms involved in endothelium-dependent responses of coronary arteries from normal mice and 2) determine whether vascular responses of coronary arteries are altered in two genetic models of hypercholesterolemia [apolipoprotein E (apoE)-deficient mice (apoE -/-) and combined apoE and low-density lipoprotein receptor (LDLR)-deficient mice (apoE + LDLR -/-)]. Plasma cholesterol levels were higher in both apoE -/- and apoE + LDLR -/- compared with normal mice on normal and high-cholesterol diets (normal chow: normal 110 +/- 5 mg/dl, apoE -/- 680 +/- 40 mg/dl, apoE + LDLR -/- 810 +/- 40 mg/dl; high-cholesterol chow: normal 280 +/- 60 mg/dl, apoE -/- 2,490 +/- 310 mg/dl, apoE + LDLR -/- 3,660 +/- 290 mg/dl). Coronary arteries from normal (C57BL/6J), apoE -/-, and apoE + LDLR -/- mice were isolated and cannulated, and diameters were measured using videomicroscopy. In normal mice, vasodilation in response to ACh and serotonin was markedly reduced by 10 microM Nomega-nitro-L-arginine (an inhibitor of nitric oxide synthase) or 20 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of soluble guanylate cyclase). Vasodilation to nitroprusside, but not papaverine, was also inhibited by ODQ. Dilation of arteries from apoE -/- and apoE + LDLR -/- mice on normal diet in response to ACh was similar to that observed in normal mice. In contrast, dilation of arteries in response to serotonin from apoE -/- and apoE + LDLR -/- mice was impaired compared with normal. In arteries from both apoE -/- and apoE + LDLR -/- mice on high-cholesterol diet, dilation to ACh was decreased. In apoE + LDLR -/- mice on high-cholesterol diet, dilation of coronary arteries to nitroprusside was increased. These findings suggest that dilation of coronary arteries from normal mice in response to ACh and serotonin is dependent on production of nitric oxide and activation of soluble guanylate cyclase. Hypercholesterolemia selectively impairs dilator responses of mouse coronary arteries to serotonin. In the absence of both apoE and the LDL receptor, high levels of cholesterol result in a greater impairment in coronary endothelial function.

Screening for structural alterations of the low density lipoprotein (LDL) receptor gene by Southern blot analysis revealed an abnormal band pattern in one subject with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The molecular defect was further characterized by polymerase chain reaction and cDNA sequencing. These analyses identified a 4.8 kb in-frame deletion of exons 2 and 3, where exon 1 was spliced to exon 4. This deletion is expected to produce a receptor that has lost the two first cysteine-rich repeats of the ligand-binding domain. Previously published data of in vitro site-directed mutagenesis has shown that binding of LDL to such a receptor is reduced to 70% of normal. A mild phenotype in our FH homozygote is consistent with that observation. In contrast, heterozygotes carrying this deletion have a relatively more severe phenotype that is comparable to that of heterozygotes carrying a null-allele. A severe phenotype was also found in a compound heterozygote carrying this deletion. Possible mechanisms for this phenotypic variability are discussed.-Rødningen, O. K., S. Tonstad, J. D. Medh, D. A. Chappell, L. Ose, and T. P. Leren. Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene.

We examined the vascular structure and endothelium-dependent relaxation in two genetic models of hypercholesterolemia: apolipoprotein E (apoE)-knockout mice and combined apoE/LDL receptor-double-knockout mice. Intimal area was increased markedly in proximal segments of thoracic aortas from apoE/LDL receptor-knockout mice [0.13 +/- 0.03 (mean +/- SE) mm2] compared with normal (C57BL/6J) mice (0.002 +/- 0.002 mm2, P < .05). Despite intimal thickening, the vascular lumen was not smaller in the aortas of apoE/LDL receptor-knockout mice (0.52 +/- 0.03 mm2) than in normal mice (0.50 +/- 0.03 mm2). In apoE-deficient mice, intimal thickening was minimal or absent, even though the concentration of plasma cholesterol was only modestly less than that in the double-knockout mouse (14.9 +/- 1.1 vs 18.0 +/- 1.2 mmol/L, respectively, P < .05). Relaxation of the aorta was examined in vitro in vascular rings precontracted with U46619. In normal mice, acetylcholine produced relaxation, which was markedly attenuated by the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM). Relaxation to acetylcholine and the calcium ionophore A23187 was normal in apoE-deficient mice (in which lesions were minimal) but greatly impaired in the proximal segments of thoracic aortas of apoE/LDL receptor-deficient mice, which contained atherosclerotic lesions. Vasorelaxation to nitroprusside was similar in normal and apoE-knockout mice, with modest but statistically significant impairment in atherosclerotic segments of apoE/LDL receptor-knockout mice. In distal segments of the thoracic aorta of apoE/LDL receptor-deficient mice, atherosclerotic lesions were minimal or absent, and the endothelium-dependent relaxation to acetylcholine and calcium ionophore was normal. Thus, in apoE/LDL receptor-knockout mice (a genetic model of hyperlipidemia), there is vascular remodeling with preservation of the aortic lumen despite marked intimal thickening, with impairment of endothelium-dependent relaxation to receptor- and nonreceptor-mediated agonists. Atherosclerosis may be accelerated in the apoE/LDL receptor-double-knockout mouse compared with the apoE-knockout strain alone. We speculate that other factors, such as the absence of LDL receptors, may contribute to the differences in the extent of atherosclerosis in these two models of hyperlipidemia.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of plasma triglycerides, promotes binding and catabolism of triglyceride-rich lipoproteins by various cultured cells. Recent studies demonstrate that LPL binds to three members of the low density lipoprotein (LDL) receptor family, including the LDL receptor-related protein (LRP), GP330/LRP-2, and very low density lipoprotein (VLDL) receptors and induces receptor-mediated lipoprotein catabolism. We show here that LDL receptors also bind LPL and mediate LPL-dependent catabolism of large VLDL with Sf 100-400. Up-regulation of LDL receptors by lovastatin treatment of normal human foreskin fibroblasts (FSF cells) resulted in an increase in LPL-induced VLDL binding and catabolism to a level that was 10-15-fold greater than in LDL receptor-negative fibroblasts, despite similar LRP activity in both cell lines. This indicates that the contribution of LRP to LPL-dependent degradation of VLDL is small when LDL receptors are maximally up-regulated. Furthermore studies in LRP-deficient murine embryonic fibroblasts showed that the level of LPL-dependent degradation of VLDL was similar to that in normal murine embryonic fibroblasts. LPL also promoted the internalization of protein-free triglyceride emulsions; lovastatin-treatment resulted in 2-fold higher uptake in FSF cells, indicating that LPL itself could bind to LDL receptors. However, the lower induction of emulsion catabolism as compared with native VLDL suggests that LPL-induced catabolism via LDL receptors is only partially dependent on receptor binding by LPL and instead is primarily due to activation of apolipoproteins such as apoE. A fusion protein between glutathione S-transferase and the catalytically inactive carboxyl-terminal domain of LPL (GST-LPLC) also induced binding and catabolism of VLDL. However GST-LPLC was not as active as native LPL, indicating that lipolysis is required for a maximal LPL effect. Mutations of critical tryptophan residues in GST-LPLC that abolished binding to VLDL converted the protein to an inhibitor of lipoprotein binding to LDL receptors. In solid-phase assays using immobilized receptors, LDL receptors bound to LPL in a dose-dependent manner. Both LPL and GST-LPLC promoted binding of VLDL to LDL receptor-coated wells. These results indicate that LPL binds to LDL receptors and suggest that the carboxyl-terminal domain of LPL contributes to this interaction.

The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPA.PAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPA.PAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of 125I-labeled LpL and uPA.PAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPA.PAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.

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 125I-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 125I-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.

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic lipase (HL) and lipoprotein lipase (LpL) are structurally related lipolytic enzymes that have distinct functions in lipoprotein catabolism. In addition to its lipolytic activity, LpL binds to very low density lipoproteins and promotes their interaction with the low density lipoprotein receptor-related protein (LRP) (Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). In vitro binding assays revealed that HL also binds to purified LRP with a KD of 52 nM. Its binding to LRP is inhibited by the 39-kDa receptor-associated protein (RAP), a known LRP antagonist, and by heparin. 125I-Labeled HL is rapidly internalized and degraded by HepG2 cell lines, and approximately 70% of the cellular internalization and degradation is blocked by either exogenously added RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly diminished capacity to internalize and degrade HL when compared to control fibroblasts. These data indicate that LRP-mediated cellular uptake of HL accounts for a substantial portion of the internalization of this molecule. Proteoglycans have been shown to participate in the clearance of LpL, and consequently a role for proteoglycans in HL clearance pathway was also investigated. Chinese hamster ovary cell lines that are deficient in proteoglycan biosynthesis were unable to internalize or degrade 125I-HL despite the fact that these cells express LRP. Thus, the initial binding of HL to cell surface proteoglycans is an obligatory step for the delivery of the enzyme to LRP for endocytosis. A small, but significant, amount of 125I-HL was internalized in LRP deficient cells indicating that an LRP-independent pathway for HL internalization does exist. This pathway could involve cell surface proteoglycans, the LDL receptor, or some other unidentified surface protein.

The 39-kDa receptor-associated protein (RAP) is cosynthesized 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 Sf 20-60 or 100-400. RAP inhibited 125I-labeled LDL and Sf 100-400 lipoprotein binding at 4 degrees C with KI values of 60 and 45 nM, respectively. The effective concentrations for 50% inhibition (EC50) of cellular degradation of 2.0 nM 125I-labeled LDL, 4.7 nM 125I-labeled Sf 20-60, and 3.6 nM 125I-labeled Sf 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 (KD) 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 125I-labeled LDL and Sf 100-400 VLDL binding to LDL receptors in solid-phase assays with KI 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.

A 39-kDa receptor associated protein (RAP) binds and inhibits ligand binding by two members of the low density lipoprotein (LDL) receptor family, gp330 and low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. To determine if additional members of the LDL receptor family may interact with RAP, Chinese hamster ovary cells were transfected with plasmids directing expression of the very low density lipoprotein (VLDL) receptor cDNA or the LDL receptor cDNA. Detergent-soluble extracts from these and normal Chinese hamster ovary cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which the proteins were transferred to nitrocellulose membranes and incubated with RAP. When detergent extracts from normal cells were incubated with RAP, several polypeptides, including a 130-kDa protein, were observed to bind RAP. In cells transfected with the VLDL receptor cDNA, a substantial increase in RAP binding to the 130-kDa polypeptide was noted. This protein was identified as the VLDL receptor by immunoblotting. The VLDL receptor present in detergent extracts from transfected cells bound to RAP-Sepharose, and a KD of 0.7 nM for the interaction between RAP and the purified VLDL receptor was determined using enzyme-linked immunosorbent assay. The purified VLDL receptor bound 125I-labeled VLDL, but not 125I-labeled LDL, and the binding of 125I-labeled VLDL was completely inhibited by RAP. Further, RAP inhibited the uptake and degradation of 125I-VLDL by cells overexpressing the VLDL receptor. Thus the VLDL receptor represents the third member of the LDL receptor family whose ligand binding properties are antagonized by RAP. This suggests a common functional role for RAP in modulating ligand binding by members of the LDL receptor family.

Lipoprotein lipase (LPL) binds to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and induces catabolism of normal human very low density lipoproteins (VLDL) via LRP in vitro. Recent studies showed that the C-terminal domain of LPL can bind LRP in solid phase assays and inhibit cellular catabolism of two LRP ligands, activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein (Williams, S.E., Inoue, I., Tran, H., Fry, G. L., Pladet, M.W., Iverius, P.-H., Lalouel, J.-M., Chappell, D.A., and Strickland, D.K. (1994) J. Biol. Chem. 269, 8653-8658). The current study investigated the potential for this region of LPL to promote cellular catabolism of VLDL via LRP. A fragment comprising the C-terminal domain of LPL (designated LPLC) was expressed in bacteria and found to promote cellular binding, uptake, and degradation of normal human VLDL in a dose-dependent manner. These effects were present whether LPLC was added simultaneously with 125I-VLDL or was prebound to cell surfaces prior to the assay. Mutations involving Lys407, Trp393, Trp394, or deletion of the C-terminal 14 residues reduced the effects of LPLC. Three LRP-binding proteins, the receptor-associated protein, lactoferrin, and a polyclonal antibody against LRP, competed for 125I-VLDL degradation induced by LPLC. Heparin or heparinase treatment of cells prevented LPLC-induced 125I-VLDL catabolism. Thus, cell-surface proteoglycans play an important role in this pathway. Interestingly, either LPLC or LPL when added in excess could block LPL-induced 125I-VLDL degradation presumably by interacting directly with LRP. However, unlabeled VLDL could not prevent catabolism of 125I-labeled LPLC or LPL. These data show that cellular fates for VLDL versus LPLC or LPL are divergent. This is probably due to independent catabolism of the latter via cell-surface proteoglycans. In summary, these in vitro studies indicate that a fragment of LPL corresponding to the C-terminal domain mimics the native enzyme with respect to induction of VLDL catabolism via LRP. Because LPLC lacks the catalytic site of native LPL, these studies establish that lipase activity is not required for LRP-mediated lipoprotein catabolism.

Very low density lipoproteins (VLDL) are heterogeneous, triglyceride-rich particles that are precursors of low density lipoproteins (LDL). Before conversion to LDL, the majority of VLDL are irreversibly cleared from plasma by uncertain mechanisms. To investigate one potential mechanism for VLDL clearance, we studied the ability of LDL receptors to mediate VLDL uptake in vitro. Small, intermediate, and large VLDL from normolipidemic humans were found to bind and undergo catabolism via LDL receptors on normal human fibroblasts. Binding to cell surfaces was up-regulated by lovastatin, an inducer of LDL receptors. Both LDL and a monoclonal antibody against the LDL receptor (IgG-C7) prevented binding of 125I-VLDL. Also, VLDL binding to mutant fibroblasts lacking LDL receptors was low. Thus, LDL receptors mediated VLDL interactions with cells. Binding affinity decreased near saturation, and the apparent number of high affinity sites decreased with increasing VLDL particle size. Because LDL receptors are small (M(r) 115,000) relative to VLDL (M(r) 9-24 x 10(6)) and are clustered in clathrin-coated pits, these findings suggest that steric hindrance becomes an important binding determinant near saturation and are consistent with a lattice model for LDL receptor-ligand interactions. The capacity for cellular catabolism of VLDL decreased with increasing particle size, consistent with a lattice model. The lattice model was also supported by differences between 125I-VLDL binding to cell surfaces and binding to partially purified LDL receptors in solid-phase assays in which steric constraints resulting from clustering in clathrin-coated pits are not present. In both cell-surface and solid-phase assays, VLDL bound via apoE, not apoB-100. Our studies establish that normal VLDL interact with LDL receptors and that steric hindrance due to crowding of particles on clustered LDL receptors is an important determinant of their binding and catabolism. These findings suggest that LDL receptors may participate in normal VLDL clearance in vivo.

Glycoprotein 330 (gp330), a cell-surface protein that is localized in clathrin-coated pits, is structurally related to both the low density lipoprotein receptor (LDLR) and the LDLR-related protein/alpha 2-macroglobulin receptor (LRP). We recently demonstrated that gp330 and LRP may be functionally related as well; both bind the 39-kDa polypeptide referred to as receptor-associated protein (Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166). In this report, we tested several other LRP ligands for their ability to interact with human and rat gp330 in vitro. Gp330 did not exhibit detectable binding to the LRP ligands, alpha 2-macroglobulin protease complex or Pseudomonas aeruginosa exotoxin A. However, we found that gp330 (purified from human or rat) bound the lipolytic enzyme lipoprotein lipase (LPL) with high affinity (Kd = 6.1 and 2.7 nM, respectively). The binding was saturable, divalent cation dependent, and inhibited by heparin or receptor-associated protein. Because LRP has also been shown to bind LPL, the present findings further extend the functional similarities between gp330 and LRP. By analogy to the postulated role of the LRP-LPL interaction in facilitating hepatic clearance of LPL-associated lipoproteins from the blood (Beisiegel, U., Weber, W., and Bengtsson-Olivercrona, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8342-8346; Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767), we speculate that the gp330-LPL interaction described herein may contribute to the uptake of LPL-associated lipoproteins in tissues expressing gp330. Consistent with this possibility, we found that LPL promoted in vitro binding of 125I-lipoproteins to gp330.

Lipoprotein lipase (LPL), the major lipolytic enzyme involved in the conversion of triglyceride-rich lipoproteins to remnants, was found to compete with binding of activated alpha 2-macroglobulin (alpha 2M*) to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. Bovine milk LPL displaced both 125I-labeled alpha 2M* and 39-kDa alpha 2M receptor-associated protein (RAP) from the surface of cultured mutant fibroblasts lacking LDL receptors with apparent KI values at 4 degrees C of 6.8 and 30 nM, respectively. Furthermore, LPL inhibited the cellular degradation of 125I-alpha 2M* at 37 degrees C. Because both alpha 2M* and RAP interact with LRP, these data suggest that LPL binds specifically to this receptor. This was further supported by observing that an immunoaffinity-isolated polyclonal antibody against LRP blocked cellular degradation of 125I-LPL in a dose-dependent manner. In addition, 125I-LPL bound to highly purified LRP in a solid-phase assay with a KD of 18 nM, and this binding could be partially displaced with alpha 2M* (KI = 7 nM) and RAP (KI = 3 nM). Taken together, these data establish that LPL binds with high affinity to LRP and undergoes LRP-mediated cellular uptake. The implication of these findings for lipoprotein catabolism in vivo may be important if LRP binding is preserved when LPL is attached to lipoproteins. If so, LPL might facilitate LRP-mediated clearance of lipoproteins.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.

Low density lipoproteins (LDL) are large (Mr = 2.5 x 10(6)) in comparison to LDL receptors (Mr = 115,000). Since most LDL receptors are clustered in coated pits, we tested the hypothesis that crowding of receptor-bound LDL particles would cause steric effects. The apparent affinity of LDL for receptors on cultured fibroblasts decreased near saturation causing concave-upward Scatchard plots. Both the higher and lower affinity components of binding were up-regulated by the cholesterol synthesis inhibitor, lovastatin, indicating that the entire binding curve was sterol-responsive. In contrast, neither component of LDL binding was present on lovastatin-treated or untreated null fibroblasts which are incapable of expressing LDL receptors. Therefore, the concave-upward Scatchard plots were entirely due to binding to LDL receptors. These results are consistent with a lattice model in which receptor-bound LDL are large enough to decrease binding to adjacent receptors. A lattice model implies that large LDL should produce steric effects at a lower receptor occupancy than should small LDL. This was tested using seven LDL fractions that differed in diameter from 20 to 27 nm. Fewer large than small LDL were bound to the cell surface at 4 degrees C and 37 degrees C, and fewer were internalized and degraded at 37 degrees C. Since large LDL bound via both apolipoprotein (apo) E and apoB100, receptor cross-linking could have caused fewer large LDL to be bound at saturation. However, when the potential for cross-linking was prevented by an apo-E-specific monoclonal antibody (1D7), the difference in binding by large versus small LDL was not eliminated; instead, it was exaggerated. Taken together, these results support a lattice model for LDL binding and indicate that steric hindrance associated with crowding of LDL particles on receptor lattices is a major determinant for catabolism by the LDL receptor pathway in vitro.

Familial dysbetalipoproteinemia (or type III hyperlipoproteinemia) is characterized by the presence of abnormal, cholesteryl ester-rich beta-very low density lipoproteins (beta-VLDL) in the plasma. Subjects with typical dysbetalipoproteinemia are homozygous for an amino acid substitution in apolipoprotein (apo-) E at residue 158 and have defective apo-E-mediated binding of both pre-beta-VLDL and beta-VLDL to apo-B,E(LDL) (or LDL) receptors (1988. Chappell, D.A., J. Clin. Invest. 82:628-639). To understand the effect of substitutions in apo-E at sites other than residue 158, nine dysbetalipoproteinemic (dys-beta) subjects who were either homozygous or heterozygous for substitutions in apo-E at atypical sites were studied. These substitutions occurred at residue 142 (n = 6), 145 (n = 2), or 146 (n = 1) and are known to cause less defective binding than does the 158 substitution. The chemical composition and electrophoretic mobility of pre-beta-VLDL and beta-VLDL from atypical and typical dys-beta subjects were indistinguishable. However, lipoproteins from atypical and typical dys-beta subjects differed in their affinity for the apo-B,E(LDL) receptor on cultured human fibroblasts. The pre-beta-VLDL and beta-VLDL from atypical dys-beta subjects had 640- or 17-fold higher affinity, respectively, than did corresponding lipoproteins from typical dys-beta subjects. The higher binding affinity of lipoproteins from atypical dys-beta subjects was associated with a higher ratio of apo-E to total apo-C. Since higher binding affinity should cause more rapid receptor-mediated clearance of beta-VLDL in atypical than in typical dys-beta subjects in vivo, the mechanism of beta-VLDL accumulation may differ in these two groups.