Irina Mikhailenko

Modulation of beta-amyloid precursor protein processing by the low density lipoprotein receptor-related protein (LRP). Evidence that LRP contributes to the pathogenesis of Alzheimer's disease.

β-Amyloid peptide (Aβ), which plays a central role in the pathogenesis of Alzheimers disease, is derived from the transmembrane β-amyloid precursor protein (APP) by proteolytic processing. Although mechanisms associated with Aβ generation are not fully understood, it is known that Aβ can be generated within endosomal compartments upon internalization of APP from the cell surface. The low density lipoprotein receptor-related protein (LRP) was previously shown to mediate the endocytosis of APP isoforms containing the Kunitz proteinase inhibitor domain (Kounnas, M. Z., Moir, R. D., Rebeck, G. W., Bush, A. I., Argraves, W. S., Tanzi, R. E., Hyman, B. T., and Strickland, D. K. (1995)Cell 82, 331–340; Knauer, M. F., Orlando, R. A., and Glabe, C. G. (1996) Brain Res. 740, 6–14). The objective of the current study was to test the hypothesis that LRP-mediated internalization of cell surface APP can modulate APP processing and thereby affect Aβ generation. Here, we show that long term culturing of cells in the presence of the LRP-antagonist RAP leads to increased cell surface levels of APP and a significant reduction in Aβ synthesis. Further, restoring LRP function in LRP-deficient cells results in a substantial increase in Aβ production. These findings demonstrate that LRP contributes to Aβ generation and suggest novel pharmacological approaches to reduce Aβ levels based on selective LRP blockade.

Role of the Low Density Lipoprotein-related Protein Receptor in Mediation of Factor VIII Catabolism

In the present study, we found that catabolism of coagulation factor VIII (fVIII) is mediated by the low density lipoprotein receptor-related protein (LPR), a liver multiligand endocytic receptor. In a solid phase assay, fVIII was shown to bind to LRP (K d 116 nm). The specificity was confirmed by a complete inhibition of fVIII/LRP binding by 39-kDa receptor-associated protein (RAP), an antagonist of all LRP ligands. The region of fVIII involved in its binding to LRP was localized within the A2 domain residues 484–509, based on the ability of the isolated A2 domain and the synthetic A2 domain peptide 484–509 to prevent fVIII interaction with LRP. Since vWf did not inhibit fVIII binding to LRP, we proposed that LRP receptor may internalize fVIII from its complex with vWf. Consistent with this hypothesis, mouse embryonic fibroblasts that express LRP, but not fibroblasts genetically deficient in LRP, were able to catabolize 125I-fVIII complexed with vWf, which was not internalized by the cells. These processes could be inhibited by RAP and A2 subunit of fVIII, indicating that cellular internalization and degradation were mediated by interaction of the A2 domain of fVIII with LRP. In vivo studies of125I-fVIII·vWf complex clearance in mice demonstrated that RAP completely inhibited the fast phase of the biphasic125I-fVIII clearance that is responsible for removal of 60% of fVIII from circulation. Inhibition of the RAP-sensitive phase prolonged the half-life of 125I-fVIII in circulation by 3.3-fold, indicating that LRP receptor plays an important role in fVIII clearance.

Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease.

Background: The immune-mediated enteropathy, celiac disease (CD), and gluten sensitivity (GS) are two distinct clinical conditions that are both triggered by the ingestion of wheat gliadin. CD, but not GS, is associated with and possibly mediated by an autoimmune process. Recent studies show that gliadin may induce the activation of IL-17-producing T cells and that IL-17 expression in the CD mucosa correlates with gluten intake. Methods: The small-intestinal mucosa of patients with CD and GS and dyspeptic controls was analyzed for expression of IL-17A mRNA by quantitative RT-PCR. The number of CD3+ and TCR-γδ lymphocytes and the proportion of CD3+ cells coexpressing the Th17 marker CCR6 were examined by in situ small-intestinal immunohistochemistry. Results: Mucosal expression of IL-17A was significantly increased in CD but not in GS patients, compared to controls. This difference was due to enhanced IL-17A levels in >50% of CD patients, with the remainder expressing levels similar to GS patients or controls, and was paralleled by a trend toward increased proportions of CD3+CCR6+ cells in intestinal mucosal specimens from these subjects. Conclusion: We conclude that GS, albeit gluten-induced, is different from CD not only with respect to the genetic makeup and clinical and functional parameters, but also with respect to the nature of the immune response. Our findings also suggest that two subgroups of CD, IL-17-dependent and IL-17-independent, may be identified based on differential mucosal expression of this cytokine.

Beyond endocytosis: LRP function in cell migration, proliferation and vascular permeability

Summary.  The low‐density lipoprotein (LDL) receptor related protein (LRP1 or LRP) is a large endocytic receptor widely expressed in several tissues and known to play roles in areas as diverse as lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. This member of the LDL receptor superfamily is constitutively endocytosed from the membrane and recycled back to the cell surface. Its many functions were long thought to involve its ability to bind over 30 different ligands and deliver them to lysosomes for degradation. However, LRP has since been shown to interact with scaffolding and signaling proteins via its intracellular domain in a phosphorylation‐dependent manner and to function as a co‐receptor partnering with other cell surface or integral membrane proteins. This multi‐talented receptor has been implicated in regulation of platelet derived growth factor receptor activity, integrin maturation and recycling, and focal adhesion disassembly. These functions may account for recent studies identifying LRPs role in protection of the vasculature, regulation of cell migration, and modulation of the integrity of the blood–brain barrier.

Cellular Internalization and Degradation of Thrombospondin-1 Is Mediated by the Amino-terminal Heparin Binding Domain (HBD) HIGH AFFINITY INTERACTION OF DIMERIC HBD WITH THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN

Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.

IL-4 promotes the formation of multinucleated giant cells from macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-cadherin.

Multinucleated giant cells (MNG) are central players in the inflammatory response to foreign materials and in adverse responses to implants. IL‐4 promotes the formation of MNG from bone marrow‐derived precursors in vitro and participates in the development of the foreign body reaction in vivo. Therefore, we investigated the mechanism by which IL‐4 promotes formation of MNG and engulfment of foreign bodies. We found that generation of MNG cells by IL‐4 was dependent on cell density and expression of STAT6; macrophages derived from STAT6−/− mice were unable to form MNG in response to IL‐4. No soluble factors including CCL2 or supernatants from IL‐4‐treated macrophages compensated for the lack of MNG cells in STAT6−/− cultures. We found that IL‐4 must remain present during the full differentiation process and that STAT6+/+ macrophage precursors retained their ability to differentiate into MNG over time. These MNG were able to internalize large particles efficiently, and the mononuclear STAT6−/− macrophages were unable to do so. Furthermore, we found that IL‐4 induced expression of E‐cadherin and dendritic cell‐specific transmembrane protein in a STAT6‐dependent manner. E‐cadherin expression was critical for the formation of MNG cells by IL‐4; an anti‐E‐cadherin antibody prevented the formation of large MNG. In addition, we found that STAT6−/− progenitors failed to fuse with STAT6+/+, revealing the need for a homotypic interaction. Thus, IL‐4 promotes the formation of MNG in a STAT6‐dependent manner by regulating cell surface expression of E‐cadherin, leading to homotypic cell fusion and the incorporation of large foreign bodies.

Platelet-derived Growth Factor Receptor-β (PDGFR-β) Activation Promotes Its Association with the Low Density Lipoprotein Receptor-related Protein (LRP) EVIDENCE FOR CO-RECEPTOR FUNCTION

Activation of the platelet-derived growth factor receptor-β (PDGFR-β) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-β was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-β, we confirmed that the kinase domain of PDGFR-β is absolutely required for LRP tyrosine phosphorylation but that PDGFR-β-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-γ, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-β may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-β co-immunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-β and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.

Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

Recognition of α2-Macroglobulin by the Low Density Lipoprotein Receptor-related Protein Requires the Cooperation of Two Ligand Binding Cluster Regions

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor α2-macroglobulin (α2M*) and amyloid precursor protein, two ligands genetically linked to Alzheimers disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as α2M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of α2M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of125I-labeled α2M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for α2M*, and they suggest a strategy for developing specific inhibitors to block α2M* binding to LRP by identifying molecules capable of binding repeats in cluster I.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Forms a Signaling Complex with Platelet-derived Growth Factor Receptor-β in Endosomes and Regulates Activation of the MAPK Pathway

In addition to its endocytic function, the low density lipoprotein receptor-related protein 1 (LRP1) also contributes to cell signaling events. In the current study, the potential of LRP1 to modulate the platelet-derived growth factor (PDGF) signaling pathway was investigated. PDGF is a key regulator of cell migration and proliferation and mediates the tyrosine phosphorylation of LRP1 within its cytoplasmic domain. In WI-38 fibroblasts, PDGF-mediated LRP1 tyrosine phosphorylation occurred at 37 °C but not at 4 °C, where endocytosis is minimized. Furthermore, blockade of endocytosis with the dynamin inhibitor, dynasore, also prevented PDGF-mediated LRP1 tyrosine phosphorylation. Immunofluorescence studies revealed co-localization of LRP1 with the PDGF receptor after PDGF treatment within endosomal compartments, whereas surface biotinylation experiments confirmed that phosphorylated LRP1 primarily originates from intracellular compartments. Together, the data reveal the association of these two receptors in endosomal compartments where they form a signaling complex. To study the contribution of LRP1 to PDGF signaling, we used mouse embryonic fibroblasts genetically deficient in LRP1 and identified phenotypic changes in these cell lines in response to PDGF stimulation by performing phospho-site profiling. Of 38 phosphorylated proteins analyzed, 8 were significantly different in LRP1 deficient fibroblasts and were restored when LRP1 was expressed back in these cells. Importantly, the results revealed that LRP1 expression is necessary for PDGF-mediated activation of ERK. Overall, the studies reveal that LRP1 associates with the PDGF receptor in endosomal compartments and modulates its signaling properties affecting the MAPK and Akt/phosphatidylinositol 3-kinase pathways.