Browsing by Author "Bu, GJ"

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    Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains
    (2003) Marzolo, MP; Yuseff, MI; Retamal, C; Donoso, M; Ezquer, F; Farfán, P; Li, YH; Bu, GJ
    Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts .
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    Expression of α2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cells
    (2000) Marzolo, MP; von Bernhardi, R; Bu, GJ; Inestrosa, NC
    Low density lipoprotein receptor-related protein (LRP) participates in the uptake and degradation of several ligands implicated in neuronal pathophysiology including apolipoprotein E (apoE), activated alpha(2)-macroglobulin (alpha(2)M*) and beta-amyloid precursor protein (APP). The receptor is expressed in a variety of tissues. in the brain LRP is present in pyramidal-type neurons in cortical and hippocampal regions and in astrocytes that are activated as a result of injury or neoplasmic transformation. As LRP is expressed in the monocyte/macrophage cell system, we were interested in examining whether LRP is expressed in microglia. We isolated glial cells from the brain of neonatal rats and LRP was immunodetected both in microglial cells and in astrocytes expressing glial fibrillar acidic protein (GFAP). Microglial cells were able to bind and internalize LRP-specific ligand, alpha(2)M*. The internalization was inhibitable by RAP, with a Kd of 1.7 nM. The expression of LRP was upregulated by dexamethasone, and down-regulated by lipopolysaccharide (LPS), gamma interferon (IFN-gamma) or a combination of both. LRP was less sensitive to dexamethasone in activated astrocytes than in microglia. We provided the first analysis of LRP expression and regulation in microglia. Our results open the possibility that microglial cells could be related to the participation of LRP and its ligands in different pathophysiological states in brain. (C) 2000 Wiley-Liss, Inc.