Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.     

Boca-dependent maturation of beta-propeller/EGF modules in low-density lipoprotein receptor proteins

The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed beta-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these beta-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the beta-propeller. Subsequently, once the EGF repeat is translated, the beta-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent beta-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand.     

The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.     

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.     

Structural insights into recognition of β2‐glycoprotein I by the lipoprotein receptors

The interactions of β2 glycoprotein I (B2GPI) with the receptors of the low‐density lipoprotein receptor (LDLR) family are implicated in the clearance of negatively charged phospholipids and apoptotic cells and, in the presence of autoimmune anti‐B2GPI antibodies, in cell activation, which might play a role in the pathology of antiphospholipid syndrome (APS). The ligand‐binding domains of the lipoprotein receptors consist of multiple homologous LA modules connected by flexible linkers. In this study, we investigated at the atomic level the features of the LA modules required for binding to B2GPI. To compare the binding interface in B2GPI/LA complex to that observed in the high‐resolution co‐crystal structure of the receptor associated protein (RAP) with a pair of LA modules 3 and 4 from the LDLR, we used LA4 in our studies. Using solution NMR spectroscopy, we found that LA4 interacts with B2GPI and the binding site for B2GPI on the ¹⁵N‐labeled LA4 is formed by the calcium coordinating residues of the LA module. We built a model for the complex between domain V of B2GPI (B2GPI‐DV) and LA4 without introducing any experimentally derived constraints into the docking procedure. Our model, which is in the agreement with the NMR data, suggests that the binding interface of B2GPI for the lipoprotein receptors is centered at three lysine residues of B2GPI‐DV, Lys 308, Lys 282, and Lys317. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Low-density lipoprotein receptor family: endocytosis and signal transduction

The low-density lipoprotein receptor (LDLR) family is composed of a class of single transmembrane glycoproteins, generally recognized as cell surface endocytic receptors, which bind and internalize extracellular ligands for degradation by lysosomes. Structurally, members of the LDLR family share homology within their extracellular domains, which are highlighted by the presence of clusters of ligand-binding repeats. Recently, information regarding the structural and functional elements within their cytoplasmic tails has begun to emerge, which suggests that members of the LDLR family function not only in receptor-mediated endocytosis, but also in transducing signals that are important during embryonic development and the pathogenesis of Alzheimer's disease. This review focuses on recent knowledge of the structural and functional aspects of LDLR family members in endocytosis and signal transduction. The relationship of these functions to the development of the neuronal system and in the pathogenesis of Alzheimer's disease is specifically discussed.     

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.     

Unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes

The receptor-associated protein (RAP) functions as an escort protein for receptors of the low-density lipoprotein receptor (LDLR) family by preventing premature intracellular binding of ligands and assisting with delivery of mature receptors to the cell surface. The modulation of affinity by pH is believed to play an important role in the escort function of RAP, because RAP binds tightly to proteins of the LDLR family at near-neutral pH early in the secretory pathway where its high affinity precludes premature binding of ligands but then dissociates from bound receptors at the lower pH of the Golgi compartment. The third domain of RAP (RAP-D3), which forms a three-helix bundle, is sufficient to reconstitute the escort activity. Here, we test the hypothesis that low-pH induced unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. First, variants of RAP-D3 resistant to low pH-induced unfolding were constructed by replacing interior histidine residues with phenylalanines. In contrast to native RAP-D3, which exhibits an unfolding pKa of 6.3 and a Tm of 42 degrees C, the most hyperstable variant of RAP-D3, in which four histidine residues are replaced with phenylalanine, has an unfolding pKa of 4.8, and a Tm of 58 degrees C. The phenylalanine substitutions in RAP-D3 confer increased stability to pH-induced dissociation of complexes formed between RAP-D3 and a two-repeat fragment of the LDLR (LA3-4). When introduced into full-length RAP, the four mutations that confer hyperstability on RAP-D3 interfere with transport of endogenous LRP-1 to the cell surface in a dominant negative fashion under conditions where expression of normal RAP has no effect on LRP-1 transport. Our studies support a model in which low pH-dependent unfolding of RAP-D3 facilitates dissociation of RAP from the LA repeats of LDLR family proteins in the mildly acidic pH of the Golgi.     

Compensated endocytosis of LDL by hamster cells co-expressing the two distinct mutant LDL receptors defective in endocytosis and ligand binding

The low density lipoprotein receptor (LDLR) regulates the plasma cholesterol level by mediating endocytosis of LDL. We established stable hamster cell lines expressing two LDLRs with distinct functional defects, i.e., endocytosis and ligand binding. In the cell line expressing only I189D h/r (human-rat chimeric) LDLR, defective in LDL binding, very little amount of LDL was internalized, although the receptor was endocytosed efficiently. In the cell line expressing Y807C LDLR solely, very few receptors were located in coated pits or endocytosed, while LDL binding to the receptor was not disrupted. In striking contrast, in the cells co-expressing both receptors, a much larger number of Y807C LDLR were internalized and co-located with I189D h/r LDLR in the perinuclear region. In these cells, LDL was bound exclusively to Y807C LDLR and its uptake was enhanced by 80% as compared to the cell expressing Y807C LDLR solely, whereas LDL binding affinity was not changed. These results suggest that a defect of the essential motif for endocytosis, cysteine 807, could be compensated by co-expression of I189D h/r LDLR, but the LDL binding was not affected.     

Solution structure of the sixth LDL-A module of the LDL receptor

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of plasma cholesterol into cells and serves as a prototype for an entire class of cell surface receptors. The amino-terminal domain of the receptor consists of seven LDL-A modules; the third through the seventh modules all contribute to the binding of low-density lipoproteins (LDLs). Here, we present the NMR solution structure of the sixth LDL-A module (LR6) from the ligand binding domain of the LDLR. This module, which has little recognizable secondary structure, retains the essential structural features observed in the crystal structure of LDL-A module five (LR5) of the LDLR. Three disulfide bonds, a pair of buried residues forming a hydrophobic "mini-core", and a calcium-binding site that serves to organize the C-terminal lobe of the module all occupy positions in LR6 similar to those observed in LR5. The striking presence of a conserved patch of negative surface electrostatic potential among LDL-A modules of known structure suggests that ligand recognition by these repeats is likely to be mediated in part by electrostatic complementarity of receptor and ligand. Two variants of LR6, identified originally as familial hypercholesterolemia (FH) mutations, have been investigated for their ability to form native disulfide bonds under conditions that permit disulfide exchange. The first, E219K, lies near the amino-terminal end of LR6, whereas the second, D245E, alters one of the aspartate side chains that directly coordinate the bound calcium ion. After equilibration at physiologic calcium concentrations, neither E219K nor D245E folds to a unique disulfide isomer, indicating that FH mutations both within and distant from the calcium-binding site give rise to protein-folding defects.     

Structural independence of ligand-binding modules five and six of the LDL receptor

The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.     

Ligand-binding domains in vitellogenin receptors and other LDL-receptor family members share a common ancestral ordering of cysteine-rich repeats

Insect vitellogenin and yolk protein receptors (VgR/YPR) are newly discovered members of the low-density lipoprotein receptor (LDLR) family, which is characterized by a highly conserved arrangement of repetitive modular elements homologous to functionally unrelated proteins. The insect VgR/YPRs are unique in having two clusters of complement-type cysteine-rich (class A) repeats or modules, with five modules in the first cluster and seven in the second cluster, unlike classical LDLRs which have a single seven-module cluster, vertebrate VgRs and very low density lipoprotein receptors (VLDLR) which have a single eight-module cluster, and LDLR-related proteins (LRPs) and megalins which have four clusters of 2–7, 8, 10, and 11 modules. Alignment of clusters across subfamilies by conventional alignment programs is problematic because of the repetitive nature of the component modules which may have undergone rearrangements, duplications, and deletions during evolution. To circumvent this problem, we ``fingerprinted' each class A module in the different clusters by identifying those amino acids that are both relatively conserved and relatively unique within the cluster. Intercluster reciprocal comparisons of fingerprints and aligned sequences allowed us to distinguish four cohorts of modules reflecting shared recent ancestry. All but two of the 57 modules examined could be assigned to one of these four cohorts designated A, B, C, and D. Alignment of clusters based on modular cohorts revealed that all clusters are derived from a single primordial cluster of at least seven modules with a consensus arrangement of CDCADBC. All extant clusters examined are consistent with this consensus, though none matches it perfectly. This analysis also revealed that the eight-module clusters in vertebrate VgRs, insect VgR/YPRs, and LRP/megalins are not directly homologous with one another. Assignment of modules to cohorts permitted us to properly align 32 class A clusters from all four LDLR subfamilies for phylogenetic analysis. The results revealed that smaller one-cluster and two-cluster members of the family did not originate from the breakup of a large two-cluster or four-cluster receptor. Similarly, the LRP/megalins did not arise from the duplication of a two-cluster insect VgR/YPR-like progenitor. Rather, it appears that the multicluster receptors were independently constructed from the same single-cluster ancestor. Received: 16 January 1997 / Accepted: 21 August 1997     

Differential functions of members of the low density lipoprotein receptor family suggested by their distinct endocytosis rates

The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.     

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.     

Functions of lipoprotein receptors in neurons

The LDL receptor (LDLR) family is comprised of several multifunctional cell surface proteins that bind and endocytose ligands with diverse biological functions. One ligand common to all LDLR family members is apolipoprotein E (apoE), a lipid transport protein that also plays a central role in the pathogenesis of neurodegeneration in Alzheimer's disease. This review discusses the role of apoE and its receptors in the central nervous system and, in particular, the signaling mechanisms by which two members of the LDLR gene family, apoE receptor-2 and VLDL receptor, control brain development, normal neuronal positioning, and neurotransmission in the adult brain.     

The structure, dynamics, and binding of the LA45 module pair of the low-density lipoprotein receptor suggest an important role for LA4 in ligand release

The low-density lipoprotein receptor (LDLR), the primary receptor for cholesterol uptake, binds ligands through its seven LDL-A modules (LAs). We present nuclear magnetic resonance (NMR) and ligand binding measurements on the fourth and fifth modules of the LDLR (LA45), the modules critical for ApoE binding, at physiological pH. Unlike LA5 and all other modules in LDLR, LA4 has a very weak calcium affinity, which probably plays a critical role in endosomal ligand release. The NMR solution structure of each module in the LA45 pair only showed minor differences compared to the analogous domains in previously determined crystal structures. The 12-residue linker connecting the modules, though slightly structured through an interaction with LA4, is highly flexible. Although no intermodule nuclear Overhauser effects were detected, chemical shift perturbations and backbone dynamics suggest cross talk between the two modules. The ligand affinity of both modules is enhanced when the two are linked. LA4 is more flexible than LA5 and remains so even in the module pair, which likely is related to its weaker calcium binding affinity.     

Anionic phospholipids inhibit apolipoprotein E--low-density lipoprotein receptor interactions

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity.     

Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors

Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.     

The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism

It has been proposed that clearance of cholesterol-enriched very low density lipoprotein (VLDL) particles occurs through a multistep process beginning with their initial binding to cell-surface heparan sulfate proteoglycans (HSPG), followed by their uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the low density lipoprotein receptor-related protein (LRP). We have further explored the relationship between HSPG binding of VLDL and its subsequent internalization by focusing on the LRP pathway using a cell line deficient in LDLR. In this study, we show that LRP and HSPG are part of a co-immunoprecipitable complex at the cell surface demonstrating a novel association for these two cell surface receptors. Cell surface binding assays show that this complex can be disrupted by an LRP-specific ligand binding antagonist, which in turn leads to increased VLDL binding and degradation. The increase in VLDL binding results from an increase in the availability of HSPG sites as treatment with heparinase or competitors of glycosaminoglycan chain addition eliminated the augmented binding. From these results we propose a model whereby LRP regulates the availability of VLDL binding sites at the cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.