NMR solution structure of complement-like repeat CR3 from the low density lipoprotein receptor-related protein. Evidence for specific binding to the receptor binding domain of human alpha(2)-macroglobulin

We have used NMR methods to determine the structure of the calcium complex of complement-like repeat 3 (CR3) from the low density lipoprotein receptor-related protein (LRP) and to examine its specific interaction with the receptor binding domain of human alpha(2)-macroglobulin. CR3 is one of eight related repeats that constitute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous complement-like repeat CR8 from LRP and complement-like repeats LB1, LB2, and LB5 from the low density lipoprotein receptor and contains a short two-strand antiparallel beta-sheet, a one turn alpha-helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and topography are, however, quite distinct from each of these other repeats. Two-dimensional (1)H,(15)N-heteronuclear single quantum coherence spectra provide evidence for a specific, though relatively weak (K(d) approximately 140 microM), interaction between CR3 and human alpha2-macroglobulin receptor binding domain that involves a contiguous patch of surface residues in the central region of CR3. This specific interaction is consistent with a mode of LRP binding to ligands that uses contributions from more than one domain to generate a wide array of different binding sites, each with overall high affinity.     

Receptor-associated protein binding blocks ubiquitinylation of the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000. LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands.     

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.     

Three complement-like repeats compose the complete alpha2-macroglobulin binding site in the second ligand binding cluster of the low density lipoprotein receptor-related protein

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of alpha2-macroglobulin (alpha2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity alpha2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for alpha2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nM) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.     

Regulation of Rac1 activation by the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.     

The single ligand-binding repeat of Tva, a low density lipoprotein receptor-related protein, contains two ligand-binding surfaces

The receptor for avian sarcoma/leukosis virus subtype A (ASLV-A), Tva, is the simplest member of the low density lipoprotein receptor family containing a single ligand-binding repeat (LBR). Most LBRs contain a central Trp (Trp33 in Tva) that is important for ligand binding and, for the low density lipoprotein receptor, is associated with familial hypercholesterolemia. The Tva ligand-binding module contains a second Trp (Trp48) that is part of a DEW motif present in a subset of LBRs. Trp48 is important for ASLV-A infectivity. A soluble Tva (sTva) ligand-binding module is sufficient for ASLV-A infectivity. Tva interacts with the viral glycoprotein, and a soluble receptor-binding domain (SUA) binds sTva with picomolar affinity. We investigated whether Tva, a retroviral receptor, could behave as a classic LBR by assessing sTva interactions with the universal receptor-associated protein (RAP) and comparing these interactions with those between sTva and its viral ligand (SUA). To address the role of the two Trp residues in Tva function, we prepared sTva harboring mutations of Trp33, Trp48, or both and determined the binding kinetics with RAP and SUA. We found that sTva behaved as a "normal" receptor toward RAP, requiring both calcium and Trp33 for binding. However, sTva binding to SUA required neither calcium nor Trp33. Furthermore, sTva could bind both RAP and SUA simultaneously. These results show that the single LBR of Tva has two ligand-binding sites, raising the possibility that other LBRs may also.     

A protective role of the low density lipoprotein receptor-related protein against amyloid beta-protein toxicity

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid beta-protein toxicity, studies were performed in C6 cells challenged with amyloid beta-protein in the presence or absence of activated alpha(2)-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid beta-protein toxicity in the presence of activated alpha(2)-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid beta-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid beta-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid beta-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimer's disease.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

The low density lipoprotein receptor-related protein functions as an endocytic receptor for decorin

Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type beta and other growth factors and thereby influences the processes of proliferation and differentiation in a wide array of physiological and pathological reactions. Hence, understanding the regulatory mechanisms of decorin activity has broad implications. Here we report that the extracellular levels of decorin are controlled by receptor-mediated catabolism, involving the low density lipoprotein receptor family member, low density lipoprotein receptor-related protein (LRP). We show that decorin is endocytosed and degraded by C2C12 myoblast cells and that both processes are blocked by suppressing LRP expression using short interfering RNA. The same occurs with CHO cells, but not with CHO cells genetically deficient in LRP. Finally, we show that LRP-null CHO cells, transfected to express mini-LRP polypeptides containing either the second or fourth LRP ligand-binding domains, carry out decorin endocytosis and lysosomal degradation. These findings point to LRP-mediated catabolism as a new control pathway for the biological activities of decorin, specifically for its ability to influence extracellular matrix signaling.     

Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.     

Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.     

The alpha -chains of C4b-binding protein mediate complex formation with low density lipoprotein receptor-related protein.

C4b-binding protein (C4BP) is a heparin-binding protein that participates in both the complement and hemostatic system. We investigated the interaction between C4BP and low density lipoprotein receptor-related protein (LRP), an endocytic receptor involved in the catabolism of various heparin-binding proteins. Both plasma-derived C4BP and recombinant C4BP consisting of only its alpha-chains (rC4BPalpha) bound efficiently to immobilized LRP, as determined by surface plasmon resonance analysis. Complementary, two distinct fragments of LRP, i.e. clusters II and IV, both associated to immobilized rC4BPalpha, and binding could be inhibited by the LRP antagonist receptor-associated protein. Further analysis showed that association of rC4BPalpha to LRP was inhibited by heparin or by anti-C4BP antibody RU-3B9, which recognizes the heparin-binding region of the C4BP alpha-chains. In cellular degradation experiments, LRP-expressing fibroblasts effectively degraded (125)I-labeled rC4BPalpha, whereas their LRP-deficient counterparts displayed a 4-fold diminished capacity of degrading (125)I-rC4BPalpha. Finally, initial clearance of C4BP in mice was significantly delayed upon co-injection with receptor-associated protein. In conclusion, our data demonstrate that the alpha-chains of C4BP comprise a binding site for LRP. We propose that LRP mediates at least in part the catabolism of C4BP and, as such, may regulate C4BP participation in complement and hemostatic processes.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein.

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.     

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization.

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.     

Serum deprivation increases the expression of low density lipoprotein receptor-related protein in primary cultured rat astrocytes

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and "ciliogenic" cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.     

Proteolytic processing of low density lipoprotein receptor-related protein mediates regulated release of its intracellular domain

The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts through its cytoplasmic tail with adaptor and scaffold proteins that participate in cellular signaling. Its extracellular domain, like that of the signaling receptor Notch and of amyloid precursor protein (APP), is proteolytically processed at multiple positions. This similarity led us to investigate whether LRP, like APP and Notch, might also be cleaved at a third, intramembranous or cytoplasmic site, resulting in the release of its intracellular domain. Using independent experimental approaches we demonstrate that the cytoplasmic domain is released by a gamma-secretase-like activity and that this event is modulated by protein kinase C. Furthermore, cytoplasmic adaptor proteins that bind to the LRP tail affect the subcellular localization of the free intracellular domain and may regulate putative signaling functions. Finally, we show that the degradation of the free tail fragment is mediated by the proteasome. These findings suggest a novel role for the intracellular domain of LRP that may involve the subcellular translocation of preassembled signaling complexes from the plasma membrane.     

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.