Mammalian cell expression of an active site mutant of Pseudomonas exotoxin disrupts LRP1 maturation

Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEΔ553) was expressed. A permanent cell line (termed CY301) was established whereby PEΔ553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEΔ553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEΔ553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEΔ553 appears to be a valuable probe of LRP1 maturation and trafficking.     

Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein [published erratum appears in J Cell Biol 1995 Aug;130(4):1015]

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.     

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.     

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.     

Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.     

Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.     

Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-β peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-β-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.     

The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells

Lactoferrin induces osteoblast proliferation and survival in vitro and is anabolic to bone in vivo. The molecular mechanisms by which lactoferrin exerts these biological actions are not known, but lactoferrin is known to bind to two members of the low-density lipoprotein receptor family, low- density lipoprotein receptor-related proteins 1 (LRP1) and 2 (LRP2). We have examined the role(s) of these receptors in the actions of lactoferrin on osteoblasts. We show that lactoferrin binds to cultured osteoblastic cells, and that LRP1 and LRP2 are expressed in several osteoblastic cell types. In primary rat osteoblastic cells, the LRP1/2 inhibitor receptor associated protein blocks endocytosis of lactoferrin and abrogates lactoferrin-induced p42/44 MAPK signaling and mitogenesis. Lactoferrin-induced mitogenesis is also inhibited by an antibody to LRP1. Lactoferrin also induces receptor associated protein-sensitive activation of p42/44 MAPK signaling and proliferation in osteoblastic human SaOS-2 cells, which express LRP1 but not LRP2. The mitogenic response of LRP1-null fibroblastic cells to lactoferrin is substantially reduced compared with that of cells expressing wild-type LRP1. The endocytic and signaling functions of LRP1 are independent of each other, because lactoferrin can activate mitogenic signaling in conditions in which endocytosis is inhibited. Taken together, these results 1) suggest that mitogenic signaling through LRP1 to p42/44 MAPKs contributes to the anabolic skeletal actions of lactoferrin; 2) demonstrate growth-promoting actions of a third LRP family member in osteoblasts; and 3) provide further evidence that LRP1 functions as a signaling receptor in addition to its recognized role in ligand endocytosis.     

A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer

Pseudomonas exotoxin A (PE) is a highly toxic protein secreted by the opportunistic pathogen Pseudomonas aeruginosa. The modular structure and corresponding mechanism of action of PE make it amenable to extensive modifications that can redirect its potent cytotoxicity from disease to a therapeutic function. In combination with a variety of artificial targeting elements, such as receptor ligands and antibody fragments, PE becomes a selective agent for the elimination of specific cell populations. This review summarizes our current understanding of PE, its intoxication pathway, and the ongoing efforts to convert this toxin into a treatment for cancer.     

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.     

gamma-Secretase-mediated release of the low density lipoprotein receptor-related protein 1B intracellular domain suppresses anchorage-independent growth of neuroglioma cells

The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a gamma-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release.     

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.     

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

In the central nervous system (CNS), fast neuronal signals are facilitated by the oligodendrocyte-produced myelin sheath. Oligodendrocyte turnover or injury generates myelin debris that is usually promptly cleared by phagocytic cells. Failure to remove dying oligodendrocytes leads to accumulation of degraded myelin, which, if recognized by the immune system, may contribute to the development of autoimmunity in diseases such as multiple sclerosis. We recently identified low density lipoprotein receptor-related protein-1 (LRP1) as a novel phagocytic receptor for myelin debris. Here, we report characterization of the LRP1 interactome in CNS myelin. Fusion proteins were designed corresponding to the extracellular ligand-binding domains of LRP1. LRP1 partners were isolated by affinity purification and characterized by mass spectrometry. We report that LRP1 binds intracellular proteins via its extracellular domain and functions as a receptor for necrotic cells. Peptidyl arginine deiminase-2 and cyclic nucleotide phosphodiesterase are novel LRP1 ligands identified in our screen, which interact with full-length LRP1. Furthermore, the extracellular domain of LRP1 is a target of peptidyl arginine deiminase-2-mediated deimination in vitro. We propose that LRP1 functions as a receptor for endocytosis of intracellular components released during cellular damage and necrosis.     

Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL

Pseudomonas exotoxin (PE) is a 66,000 molecular weight protein secreted by Pseudomonas aeruginosa. PE is made up of three domains, and PE40 is a form of PE which lacks domain Ia (amino acids 1-252) and has very low cytotoxicity because it cannot bind to target cells. The sequence Arg-Glu-Asp-Leu-Lys (REDLK) at the carboxyl terminus of Pseudomonas exotoxin has been shown to be important for its cytotoxic activity (Chaudhary, V. K., Jinno, Y., FitzGerald, D. J., and Pastan, I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312). In this study, we tested the effect of altering the carboxyl sequence of PE from REDLK to the characteristic endoplasmic reticulum retention sequence, KDEL, or to KDEL repeated three times (KDEL)3. We also made similar changes at the carboxyl terminus of two chimeric toxins in which domain I of PE (amino acids 1-252) was either replaced with transforming growth factor alpha (TGF alpha) to make TGF alpha-PE40 or with a single chain antibody (anti-Tac) reacting with the human interleukin 2 receptor to make anti-Tac(Fv)-PE40. Statistical analyses of our results demonstrate that PE and its derivatives ending in KDEL or (KDEL)3 are significantly more active than PE or derivatives ending in REDLK. We have also found that brefeldin A, which is known to perturb the endoplasmic reticulum, inhibits the cytotoxic action of PE. Our results suggest that the altered carboxyl terminus may enable the toxin to interact more efficiently with a cellular component involved in translocation of the toxin to the cytosol.     

The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor-related protein

The low density lipoprotein receptor-related protein-deleted in tumor (LRP1B, initially referred to as LRP-DIT) was cloned and characterized as a candidate tumor suppressor. It is a new member of the low density lipoprotein receptor gene family. Its overall domain structure and large size (approximately 600 kDa) are similar to LRP and suggest that it is a multifunctional cell surface receptor. Herein, we characterize a series of ligands for the receptor using cell lines that stably express it as a domain IV minireceptor (mLRP1B4). Ligands of LRP including receptor-associated protein, urokinase plasminogen activator, tissue-type plasminogen activator, and plasminogen activator inhibitor type-1 each demonstrate binding, internalization, and degradation via mLRP1B4. Interestingly, the kinetics of ligand endocytosis is distinctly different from that of LRP, with LRP1B exhibiting a markedly diminished internalization rate. In addition, tissue expression analysis reveals that the LRP1B gene is expressed in brain, thyroid, and salivary gland. These studies thus extend the physiological roles of members of the LDL receptor family.     

Neuronal LRP1 functionally associates with postsynaptic proteins and is required for normal motor function in mice

The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-D-aspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitter-dependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.     

Insulin induces the low density lipoprotein receptor‐related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage‐derived cells

Low‐density lipoprotein receptor‐related protein 1 (LRP1) is an endocytic receptor, which binds and internalizes diverse ligands such as activated α₂‐macroglobulin (α₂M*). LRP1 promotes intracellular signaling, which downstream mediates cellular proliferation and migration of different types of cells, including macrophages. Unlike the LDL receptor, LRP1 expression is not sensitive to cellular cholesterol levels but appears to be responsive to insulin. It has been previously demonstrated that insulin increases the cell surface presentation of LRP1 in adipocytes and hepatocytes, which is mediated by the intracellular PI₃K/Akt signaling activation. The LRP1 protein distribution is similar to other insulin‐regulated cell surface proteins, including transferring receptor (Tfr). However, in macrophages, the insulin effect on the LRP1 distribution and expression is not well characterized. Considering that macrophages play a central role in the pathogenesis of atherosclerosis, herein we evaluate the effect of insulin on the cellular expression of LRP1 in J774 macrophages‐derived cells using Western blot and immunofluorescence microscopy. Our data demonstrate that insulin induces a significant decrease in the LRP1 protein content, without changing the specific mRNA level of this receptor. Moreover, insulin specifically affected the protein expression of LRP1 but not Tfr. The insulin‐induced protein degradation of LRP1 in J774 cells was mediated by the activation of the PI₃K/Akt pathway and proteasomal system by an enhanced ubiquitin–receptor conjugation. The decreased content of LRP1 induced by insulin affected the cellular internalization of α₂M*. Thus, we propose that the protein degradation of LRP‐1 induced by insulin in macrophages could have important effects on the pathogenesis of atherosclerosis. J. Cell. Biochem. 106: 372–380, 2009. © 2008 Wiley‐Liss, Inc.     

Molecular basis for the interaction of low density lipoprotein receptor-related protein 1 (LRP1) with integrin alphaMbeta2: identification of binding sites within alphaMbeta2 for LRP1

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β(2) integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes α(M)β(2) but not its homologous receptor α(L)β(2). The interaction between these two cellular receptors in macrophages is significantly enhanced upon α(M)β(2) activation by LPS and is mediated by multiple regions in both LRP1 and α(M)β(2). Specifically, we find that both the heavy and light chains of LRP1 are involved in α(M)β(2) binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For α(M)β(2), we find that the α(M)-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the α(L)β(2) receptor with that of α(M) confers the α(L)β(2) receptor with the ability to interact with LRP1. Furthermore, we show that residues (160)EQLKKSKTL(170) within the α(M)-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of α(M)β(2), our finding suggests that binding of LRP1 to α(M)β(2) could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits α(M)β(2)-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway

Members of the low density lipoprotein receptor family (LDLR), LRP5/6, were shown to interact with the Frizzled (Fz) receptors and to function as Wnt coreceptors. Here we show that mLRP4T100, a minireceptor of LRP1, another member of the LDLR family, interacts with the human Fz-1 (HFz1), previously shown to serve as a receptor transmitting the canonical Wnt-3a-induced signaling cascade. However, in contrast to LRP5/6, mLRP4T100, as well as the full-length LRP1, did not cooperate with HFz1 in transmitting the Wnt-3a signaling but rather repressed it. mLRP4T100 inhibitory effect was displayed also by endocytosis-defective mLRP4T100 mutants, suggesting that LRP1 repressive effect is not attributable to LRP1-mediated enhanced HFz1 internalization and subsequent degradation. Enforced expression of mLRP4T100 decreased the capacity of HFz1 cysteine-rich domain (CRD) to interact with LRP6, in contrast to HFz1-CRD/Wnt-3a interaction that was not disrupted by overexpressing mLRP4T100. These data suggest that LRP1, by sequestering HFz1, disrupts the receptor/coreceptor complex formation, leading to the repression of the canonical Wnt signaling. Thus, this study implies that the ability to interact with Fz receptors is shared by several members of the LDLR family. However, whereas some members of the LDLR family, such as LRP5/6, interact with Fz and serve as Wnt coreceptors, others negatively regulate Wnt signaling, presumably by sequestering Fz.