LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages.

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.

The LDL receptor-related protein 1 (LRP1) regulates the PDGF signaling pathway by binding the protein phosphatase SHP-2 and modulating SHP-2- mediated PDGF signaling events.

BACKGROUND: The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor ß (PDGFRß) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRß pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRß signaling pathway by binding SHP-2 and competing with the PDGFRß for this molecule. METHODOLOGY/PRINCIPAL FINDINGS: To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRß kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRß. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that phosphorylated forms of LRP1 and PDGFRß compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.

LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1(-/-)) on a low-density lipoprotein receptor-deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. APPROACH AND RESULTS: We found smLRP1(-/-) mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1(-/-) vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. CONCLUSIONS: Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

Adoptive transfer of IL-4Rα+ macrophages is sufficient to enhance eosinophilic inflammation in a mouse model of allergic lung inflammation.

BACKGROUND: The IL-4 receptor α (IL-4Rα) chain has a broad expression pattern and participates in IL-4 and IL-13 signaling, allowing it to influence several pathological components of allergic lung inflammation. We previously reported that IL-4Rα expression on both bone marrow-derived and non-bone marrow-derived cells contributed to the severity of allergic lung inflammation. There was a correlation between the number of macrophages expressing the IL-4Rα, CD11b, and IA(d), and the degree of eosinophilia in ovalbumin challenged mice. The engagement of the IL-4Rα by IL-4 or IL-13 is able to stimulate the alternative activation of macrophages (AAM). The presence of AAM has been correlated with inflammatory responses to parasites and allergens. Therefore, we hypothesized that IL-4Rα⁺ AAM play an active role in allergic lung inflammation. To directly determine the role of AAM in allergic lung inflammation, M-CSF-dependent macrophages (BMM) were prepared from the bone-marrow of IL-4Rα positive and negative mice and transferred to IL-4RαxRAG2(-/-) mice. Wild type TH2 cells were provided exogenously. RESULTS: Mice receiving IL-4Rα(+/+) BMM showed a marked increase in the recruitment of eosinophils to the lung after challenge with ovalbumin as compared to mice receiving IL-4Rα(-/-) BMM. As expected, the eosinophilic inflammation was dependent on the presence of TH2 cells. Furthermore, we observed an increase in cells expressing F4/80 and Mac3, and the AAM marker YM1/2 in the lungs of mice receiving IL-4Rα(+/+) BMM. The BAL fluid from these mice contained elevated levels of eotaxin-1, RANTES, and CCL2. CONCLUSIONS: These results demonstrate that transfer of IL-4Rα + macrophages is sufficient to enhance TH2-driven, allergic inflammation. They further show that stimulation of macrophages through IL-4Rα leads to their alternative activation and positive contribution to the TH2-driven allergic inflammatory response in the lung. Since an increase in AAM and their products has been observed in patients with asthma exacerbations, these results suggest that AAM may be targeted to alleviate exacerbations.

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes.

While testing the effect of the (ß15-66)(2) fragment, which mimics a pair of fibrin ßN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (ß15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its ßN-domains, and this interaction is inhibited by RAP and (ß15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing ßN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.

Tissue transglutaminase promotes PDGF/PDGFR-mediated signaling and responses in vascular smooth muscle cells.

Although the pivotal role of platelet derived growth factor (PDGF)-mediated signaling in vascular diseases was demonstrated, the pathophysiological mechanisms driving its over-activation remain incompletely understood. Tissue transglutaminase (tTG) is a multifunctional protein expressed in the vasculature, including smooth muscle cells (SMCs), and implicated in several vascular pathologies. The goal of this study is to define the regulation of PDGF-BB/PDGFRß-induced signaling pathways and cell responses by tTG in vascular SMCs. We find that in human aortic SMCs, shRNA-mediated depletion and over-expression of tTG reveals its ability to down-regulate PDGFRß levels and induce receptor clustering. In these cells, tTG specifically amplifies the activation of PDGFRß and its multiple downstream signaling targets in response to PDGF-BB. Furthermore, tTG promotes dedifferentiation and increases survival, proliferation, and migration of human aortic SMCs mediated by this growth factor. Finally, PDGF-BB stimulates tTG expression in human aortic SMCs in culture and in the blood vessels in response to injury. Together, our results show that tTG in vascular SMCs acts as a principal enhancer within the PDGF-BB/PDGFRß signaling axis involved in phenotypic modulation of these cells, thereby suggesting a novel role for this protein in the progression of vascular diseases.

LRAD3, a novel low-density lipoprotein receptor family member that modulates amyloid precursor protein trafficking.

We have identified a novel low-density lipoprotein (LDL) receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an ∼50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain, and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW-domain-containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal-derived cell line HT22, LRAD3 partially colocalizes with amyloid precursor protein (APP) and interacts with APP as revealed by coimmunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we used solid-phase binding assays that demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released after α-secretase cleavage. In contrast, C99, the ß-secretase product that remains cell associated, coprecipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aß peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-life of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons, including its downstream signaling.

Murine missing in metastasis (MIM) mediates cell polarity and regulates the motility response to growth factors.

BACKGROUND: Missing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined. PRINCIPAL FINDINGS: We have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM(-/-) MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM(-/-) cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoA(Q63L), a constitutively activated RhoA mutant. MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM(-/-) cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM(-/-) cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM(-/-) cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment. CONCLUSIONS: Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.

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 ß1 integrins inside the recycling endosomes and is secreted as a complex with recycled ß1 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.

Low density lipoprotein receptor-related protein 1 (LRP1) forms a signaling complex with platelet-derived growth factor receptor-beta 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 degrees C but not at 4 degrees 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.

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-gammadelta 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.

Microglial low-density lipoprotein receptor-related protein 1 modulates c-Jun N-terminal kinase activation.

Apolipoprotein E (apoE)-induced activation of low-density lipoprotein receptor (LDL) family members reduces inflammatory responses by suppressing c-Jun N-terminal kinase (JNK) activation. We aimed to identify which specific receptor family member mediates the effect of apoE on inflammation in primary cultures of microglia. Low-density lipoprotein receptor-related protein 1 (LRP1)-deficient (LRP1-/-) microglia were derived from mice using tissue-specific loxP/Cre recombination. Using a peptide formed from the receptor-binding region of apoE (EP), we found that LRP1 mediates the effects of apoE on microglial inflammation. Microglial LRP1 was also essential for EP to suppress JNK activation induced by lipopolysaccharide.

Regulation of platelet-derived growth factor receptor function by integrin-associated cell surface transglutaminase.

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.

Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.

Catabolism of coagulation factor VIII (FVIII) is mediated by low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complement-type repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25-90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3-4, 4-5, 5-6, 6-7 and 7-8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay, LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3-8 in cluster II and CR 23-29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

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.

Cell-surface transglutaminase undergoes internalization and lysosomal degradation: an essential role for LRP1.

Tissue transglutaminase functions as a protein crosslinking enzyme and an integrin-binding adhesion co-receptor for fibronectin on the cell surface. These activities of transglutaminase and the involvement of this protein in cell-matrix adhesion, integrin-mediated signaling, cell migration and matrix organization suggest a precise and efficient control of its cell-surface expression. We report a novel mechanism of regulation of surface transglutaminase through internalization and subsequent lysosomal degradation. Constitutive endocytosis of cell-surface transglutaminase depends on plasma membrane cholesterol and the activity of dynamin-2, and involves both clathrin-coated pits and lipid rafts or caveolae. Furthermore, the key matrix ligands of transglutaminase, fibronectin and platelet-derived growth factor, promote its endocytosis from the cell surface. Our results also indicate that transglutaminase interacts in vitro and on the cell surface with the major endocytic receptor, low-density lipoprotein receptor-related protein 1, and demonstrate the requirement for this receptor in the endocytosis of transglutaminase. Finally, a deficiency of this endocytic receptor or blockade of endo-lysosomal function upregulate transglutaminase expression on the cell surface, leading to increased cell adhesion and matrix crosslinking. These findings characterize a previously unknown pathway of transglutaminase internalization and degradation that might be crucial for regulation of its adhesive and signaling functions on the cell surface and reveal a novel functional link between cell-matrix adhesion and endocytosis.

Fluorescence lifetime imaging microscopy (FLIM) detects stimulus-dependent phosphorylation of the low density lipoprotein receptor-related protein (LRP) in primary neurons.

The low-density lipoprotein receptor-related protein (LRP) is a large, endocytic receptor involved in intracellular signalling. LRP acts as a co-receptor with the PDGF-receptor (PDGF-r) for platelet-derived growth factor (PDGF). PDGF-r and Src-kinases induce tyrosine-phosphorylation of LRP. We used fluorescence lifetime imaging microscopy (FLIM) to specifically detect LRP phosphorylation, measure its extent and localization in intact cells, and assess its effects upon LRP-APP interaction. Robust phosphorylation of LRP throughout the cell was observed after overexpression of Src-kinase. This depended on LRP's distal NPXY domain. By contrast, activation of the PDGF-r resulted in phosphorylation of the subpopulation of LRP at or near the cell surface. PDGF activation triggered phosphorylation of endogenous LRP in primary neurons. LRP is also a trafficking receptor for the Alzheimer-related molecule amyloid-precursor-protein (APP). PDGF stimulation did not affect LRP-APP interactions. This approach allows exquisite subcellular resolution of specific LRP post-translational changes and protein-protein interactions of endogenous proteins in intact cells.

RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi.

The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.