Endocytosis of megalin by visceral endoderm cells requires the Dab2 adaptor protein

Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109, USA.
Journal of Cell Science (Impact Factor: 5.43). 12/2005; 118(Pt 22):5345-55. DOI: 10.1242/jcs.02650
Source: PubMed


Rapid endocytosis of lipoprotein receptors involves NPxY signals contained in their cytoplasmic tails. Several proteins, including ARH and Dab2, can bind these sequences, but their importance for endocytosis may vary in different cell types. The lipoprotein receptor megalin is expressed in the visceral endoderm (VE), a polarized epithelium that supplies maternal nutrients to the early mammalian embryo. Dab2 is also expressed in the VE, and is required for embryo growth and gastrulation. Here, we show that ARH is absent from the VE, and Dab2 is required for uptake of megalin, its co-receptor cubilin, and a cubilin ligand, transferrin, from the brush border of the VE into intracellular vesicles. By making isoform-specific knock-in mice, we show that the p96 splice form of Dab2, which binds endocytic proteins, can fully rescue endocytosis. The more abundant p67 isoform, which lacks some endocytic protein binding sites, only partly rescues endocytosis. Endocytosis of cubilin is also impaired in VE and in mid-gestation visceral yolk sac when p96 is absent. These studies suggest that Dab2 p96 mediates endocytosis of megalin in the VE. In addition, rescue of embryonic viability correlates with endocytosis, suggesting that endocytosis mediated by Dab2 is important for normal development.

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    • "DAB2 splice forms are expressed in a tissue-specific pattern suggesting that they possess non-overlapping cellular activities. In particular, in renal tissue, p96 is equally or more abundant than p67 [34]. Our analysis confirm this data as we measured a percentage of about 50% of inclusion of the CE in normal kidney tissues (Figure 4A). "
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    ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response.
    PLoS ONE 10/2013; 8(10):e78452. DOI:10.1371/journal.pone.0078452 · 3.23 Impact Factor
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    • "There are two predominant Dab2 splice variants encoding 96 kD (p96) and 67kD (p67) proteins [9]. The p96 Dab2 isoform is the predominant isoform found in the adult, whereas, the p67 isoform is predominantly expressed during embryogenesis [10]. Disabled-2 expression within the visceral endoderm during embryonic development is necessary for survival of the early embryo [8,10,11]. "
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    ABSTRACT: Background Neuroinflammation regulates both disease pathogenesis and repair in multiple sclerosis. In early multiple sclerosis lesion development, neuroinflammation causes demyelination and axonal injury, the likely final common determinant of disability. Here we report the identification of a novel neuroinflammatory mediator, Disabled-2 (Dab2). Dab2 is an intracellular adaptor protein with previously unknown function in the central nervous system. Results We report that Dab2 is up-regulated in lesional macrophages/microglia in the spinal cord in murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. We demonstrate that dab2 expression is positively correlated with experimental autoimmune encephalomyelitis disease severity during the acute disease phase. Furthermore, dab2-deficient mice have a less severe experimental autoimmune encephalomyelitis disease course and suffer less neuroinflammation and less axonal injury than their wild-type littermates. We demonstrate that dab2 expression is strongly associated with the expression of inducible nitric oxide synthase. We further demonstrate that Dab2 is expressed at the protein level by macrophages in early acute human multiple sclerosis lesions and that this correlates with axonal injury. Conclusions Together, these results suggest that endogenous Dab2 exacerbates central nervous system inflammation, potentially acting to up-regulate reactive oxygen species expression in macrophages and microglia, and that it is of potential pathogenic relevance in Multiple Sclerosis.
    07/2013; 1(1). DOI:10.1186/2051-5960-1-32
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    • "Moreover, DAB2 is a negative regulator of integrin αIIbβ3-mediated fibrinogen adhesion and cell signaling and is secreted to display on platelet surface in both sulfatide-and integrin αIIb-bound states to control the extent of clotting response upon platelet activation [19] [27] [28]. In addition to playing a role in cell signaling [30] [31] [32] [33] [34], DAB2 is an NPXY sequence-specific adaptor protein that regulates clathrin-coated vesicles trafficking involving binding with myosin VI [35] [36] and receptor recycling of LDLR [37], apolipoprotein E receptor 2 (ApoER2) [38], LRP1 [39], megalin [40], intrinsic factor-vitamin B12 receptor cubam [41], integrin β1 [42] [43] [44], and type II transforming growth factor-beta receptor [45]. This task is mainly mediated by engaging the N-terminal phosphotyrosine binding (PTB) domain of DAB2 with the NPXY motif of the receptor [18] [19]. "
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    ABSTRACT: Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.
    Biochimica et Biophysica Acta 06/2012; 1823(10):1778-88. DOI:10.1016/j.bbamcr.2012.06.008 · 4.66 Impact Factor
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