LDL receptor-related protein as a component of the midkine receptor

Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.     

The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing

The low-density lipoprotein receptor-related protein (LRP) has recently been implicated in numerous intracellular signaling functions, as well as in Alzheimer's disease pathogenesis. Studies have shown that the beta-amyloid precursor protein (APP) interacts with LRP and that this association may impact the production of amyloid beta-protein (Abeta). In this report, we provide evidence that LRP regulates trafficking of intracellular proteins independently of its lipoprotein receptor functions. We show that in the absence of LRP, Abeta production, APP secretion, APP internalization, turnover of full-length APP and stability of APP C-terminal fragments are affected. Importantly, these changes are not APP isoform dependent. Using deletion constructs, the critical region in LRP that modulates APP processing was mapped to a seven peptide domain around the second NPXY domain (residues 4504-4510). Therefore, we propose a model by which LRP functionally modulates APP processing, including those steps critical for Abeta production, through interactions of the cytosolic domains.     

Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin-proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.     

The cytoplasmic domain of the low density lipoprotein (LDL) receptor-related protein,but not that of the LDL receptor,triggers phagocytosis

The macrophage LDL receptor and LDL receptor-related protein (LRP, CD91) mediate the phagocytic-like uptake of atherogenic lipoproteins and apoptotic cells, yet the structural basis of their phagocytic functions is not known. To address this issue, we transfected macrophages with chimeric proteins containing the cytoplasmic tails and transmembrane regions of the LDL receptor or LRP and the ectodomain of CD2, which can bind non-opsonized sheep red blood cells (SRBCs). Macrophages expressing receptors containing the LDL receptor domains were able to bind but not internalize SRBCs. In contrast, macrophages expressing receptors containing the cytoplasmic tail of LRP were able to bind and internalize SRBCs. Chimeras in which the LRP cytoplasmic tail was mutated in two di-leucine motifs and a tyrosine in an NPXYXXL motif were able to endocytose anti-CD2 antibody and bind SRBCs, but SRBC phagocytosis was decreased by 70%. Thus, the phagocytic-like functions of LRP, but not those of the LDL receptor, can be explained by the ability of the LRP cytoplasmic tail to trigger phagocytosis. These findings have important implications for atherogenesis and apoptotic cell clearance and for a fundamental cell biological understanding of how the LDL receptor and LRP function in internalization processes.     

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.     

Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands

Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.     

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.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein

Apolipoprotein E (apoE), an apoprotein involved in lipid transport in both the plasma and within the brain, mediates the binding of lipoproteins to members of the low density lipoprotein (LDL) receptor family including the LDL receptor and the LDL receptor-related protein (LRP). ApoE/LRP interactions may be particularly important in brain where both are expressed at high levels, and polymorphisms in the apoE and LRP genes have been linked to AD. To date, only apoE-enriched lipoproteins have been shown to be LRP ligands. To investigate further whether other, more lipid-poor forms of apoE interact with LRP, we tested whether lipid-free apoE in the absence of lipoprotein particles interacts with its cell-surface receptors. No detectable lipid was found associated with bacterially expressed and purified apoE either prior to or following incubation with cells when analyzed by electrospray ionization mass spectrometry. We found that the degradation of lipid-poor (125)I-apoE was significantly higher in wild type as compared to LRP-deficient cells, and was inhibited by receptor-associated protein (RAP). In contrast, (125)I-apoE-enriched beta-VLDL was degraded by both LRP and the LDL receptor. When analyzed via a single cycle of endocytosis, (125)I-apoE was internalized prior to its subsequent intracellular degradation with kinetics typical of receptor-mediated endocytosis. Thus, we conclude that a very lipid-poor form of apoE can be catabolized via cell surface LRP, suggesting that the conformation of apoE necessary for recognition by LRP can be imposed by situations other than an apoE-enriched lipoprotein.     

The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin

Toxins produced by Bacillus anthracis and other microbial pathogens require functions of host cell genes to yield toxic effects. Here we show that low density lipoprotein receptor-related protein 6 (LRP6), previously known to be a coreceptor for the Wnt signaling pathway, is required for anthrax toxin lethality in mammalian cells. Downregulation of LRP6 or coexpression of a truncated LRP6 dominant-negative peptide inhibited cellular uptake of complexes containing the protective antigen (PA) carrier of anthrax toxin moieties and protected targeted cells from death, as did antibodies against epitopes in the LRP6 extracellular domain. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with PA receptors TEM8/ATR and/or CMG2 to form a multicomponent complex that enters cells upon PA binding. Our results, which reveal a previously unsuspected biological role for LRP6, identify LRP6 as a potential target for countermeasures against anthrax toxin lethality.     

39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor with the ability to bind and endocytose several structurally and functionally distinct ligands. A 39 kDa receptor-associated protein (RAP) inhibits all ligand interactions with LRP in vitro. In the present study, we demonstrate that RAP is an endoplasmic reticulum (ER) resident protein. The tetrapepetide sequence HNEL at the C-terminus of RAP is both necessary and sufficient for RAP retention within the ER. Metabolic labeling combined with cross-linking studies show that RAP interacts with LRP in vivo. Pulse-chase analysis reveals that this association is transient early in the secretory pathway and coincides with LRP aggregation and reduced ligand binding activity. Both internal triplicated LRP binding domains on RAP and multiple RAP binding domains on LRP appear to contribute to the aggregation of LRP and RAP. Dissociation of RAP from LRP results from the lower pH encountered later in the secretory pathway and correlates with an increase in LRP ligand binding activity. Taken together, our results thus suggest that RAP functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity along the secretory pathway.     

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.     

Upregulation of hepatic LDL receptor-related protein in nephrotic syndrome: response to statin therapy

Nephrotic syndrome (N-S) is associated with elevated plasma concentration and impaired clearance of VLDL, chylomicrons (CM), and their atherogenic remnants. These abnormalities are largely due to lipoprotein lipase, hepatic triglyceride lipase, and VLDL receptor deficiencies and impaired HDL-mediated shuttling of apoE and apoC between the nascent and remnant VLDL and CM. LRP is a multifaceted endocytic receptor that is heavily expressed in the liver. LRP recognizes at least 30 different ligands including VLDL and CM remnants. These observations prompted the present study to discern the effect of N-S on hepatic LRP gene and protein expressions. The study further sought to explore the effect of lipid-lowering therapy on LRP expression in N-S. Sprague-Dawley rats were randomized to the N-S (given ip injections of puromycin aminonucleoside; 130 mg/kg on day 1, 60 mg/kg on day 14) and placebo-injected control groups. On day 14, animals were subdivided into statin-treated (rosuvastatin; 20 mg x kg(-1) x day(-1) mixed with powdered chow) and untreated groups and studied on day 28. The untreated N-S group exhibited severe proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of hepatic tissue LRP mRNA and protein abundance. Statin administration for 2 wk resulted in significant improvements of plasma lipid profile, proteinuria, and hypoalbuminemia as well as hepatic LRP mRNA and protein abundance. In contrast, statin administration had no significant effect on either plasma lipids or hepatic LRP levels in the normal control rats. In conclusion, N-S results in marked upregulation of hepatic LRP expression that is partly reversed with statin administration. These findings exclude depressed hepatic LRP expression as the primary cause of elevated plasma lipoprotein remnants in N-S.     

Low density lipoprotein (LDL) receptor-related protein 1B impairs urokinase receptor regeneration on the cell surface and inhibits cell migration

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.     

Optimizing multiple seeds for protein homology search

We present a framework for improving local protein alignment algorithms. Specifically, we discuss how to extend local protein aligners to use a collection of vector seeds or ungapped alignment seeds to reduce noise hits. We model picking a set of seed models as an integer programming problem and give algorithms to choose such a set of seeds. While the problem is NP-hard, and Quasi-NP-hard to approximate to within a logarithmic factor, it can be solved easily in practice. A good set of seeds we have chosen allows four to five times fewer false positive hits, while preserving essentially identical sensitivity as BLASTP.     

Pregnancy zone protein-tissue-type plasminogen activator complexes bind to low-density lipoprotein receptor-related protein (LRP)

Tissue-type plasminogen activator (t-PA), is a serine proteinase that catalyzes the initial and rate-limiting step in the fibrinolytic cascade. Its plasma activity is determined by the rate of release into the bloodstream, the rate of inhibition by plasminogen-activator inhibitor type 1 (PAI-1) and the rate of hepatic clearance. Two receptor systems contribute to the clearance of t-PA: the mannose receptor and the low-density lipoprotein receptor-related protein (LRP) that removes free t-PA as well as t-PA-PAI-1 complexes from the blood. During pregnancy a significant rise in the plasma levels of pregnancy zone protein (PZP) is observed, while alpha(2)-macroglobulin (alpha(2)-M) remains constant. Interestingly, the fibrinolytic activity is decreased during this period. In this context, we have recently demonstrated the in vitro formation of PZP-t-PA complexes. Here, we purified LRP from human placenta by affinity chromatography and then analyzed the binding specificity and affinity of PZP-proteinase complexes to the receptor by enzyme immunoassay (EIA). Our results clearly established that the binding of PZP-t-PA complexes to LRP was specific, saturable, and with K(d) = 337 +/- 31 nM. Moreover, by using the same EIA, we further observed that this binding was inhibited by receptor-associated protein. These data suggest that PZP, by binding to t-PA and promoting its clearance via LRP, might contribute in vivo to the downregulation of the fibrinolytic activity during pregnancy.     

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.     

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.     

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.