Accumulation of extracellular amyloid ␤ peptide (A␤), generated from amyloid precursor protein (APP) processing by ␤-and ␥-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of A␤ from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in A␤ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steadystate levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to A␤. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to A␤.Mounting evidence has demonstrated that proteolytic processing of the amyloid precursor protein (APP) 4 is central to the pathogenesis of Alzheimer disease (AD) (1, 2). Many reports have shown that APP processing to A␤ is greatly affected by the subcellular localization of APP, presumably because of the specific subcellular localizations of ␤-and ␥-secretases (3). Both transmembrane receptors and cytoplasmic adaptor proteins have been shown to interact with APP and affect its trafficking. The low-density lipoprotein receptor-related protein 1 (LRP1) increases APP endocytosis and A␤ production (4), whereas SorLA decreases APP processing to A␤ by shuttling APP away from endosomes (5). Several cell surface adaptor proteins, including Fe65, X11, and Dab1, bind to the NPXY motif within the APP cytoplasmic domain and regulate its trafficking and processing to A␤ (6 -8). By overexpression or knockdown, Fe65 has been shown to affect APP processing to A␤ (9, 10). Although Dab1 has been shown to affect APP processing and A␤ production (11), the function of its homologue Dab2 in APP trafficking and processing to A␤ has not been studied (8). These studies firmly establish that APP-interacting proteins can both positively and negatively affect A␤ production by altering APP trafficking through ␤-and ␥-secretase-containing compartments. Revealing the mechanisms by which intracellular trafficking of APP is regulated may permit the development of novel therapeutic approaches for AD.Sorting nexin 17 (SNX17) is a member of the sorting nexin family characteri...

Megalin and the low-density lipoprotein (LDL) receptorrelated 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. The low-density lipoprotein (LDL) receptor gene family contains three very large members, LRP (LRP1), a dimer of 515 kDa and 85 kDa, its closely related homolog, megalin (LRP2), a single species of 600 kDa (1) and the recently discovered LRP1B, more closely related to LRP than to megalin (2). Comparison of LRP and megalin reveals that the overall protein domain structural organization of the two proteins is very similar. LRP has four ligandbinding domains (I, II, III, and IV from the N-terminus) separated from one another by clusters of EGF-precursor repeats and F/YWXD spacer repeats. LRP contains a furin endopeptidase processing site in its ectodomain that is cleaved to form the mature receptor, a noncovalently associated heterodimer, consisting of an extracellular 515-kDa subunit and a transmembrane 85-kDa subunit (3, 4). The cytoplasmic tail of LRP has 100 amino acids and harbors two NPxY motifs, one Yxxf motif, recently shown as the dominant endocytosis motif (5), and two LL motifs, with the distal one playing a small role in LRP internalization. In addition to these motifs, phosphorylation of the LRP tail by PKA also plays a role in the internalization of the receptor (6). Megalin contains four clusters of ligand-binding domains. The first ligand-binding domain is the most different from the corresponding domain in LRP, with seven ligand binding repeats instead of two (7). The t...

SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface.

Objective. To define whether anti-ribosomal P (anti-P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood.Methods. We used anti-P antibodies from patients with NPSLE and rabbit-generated anti-P and anti-NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal-dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti-P or anti-NSPA, using lipopolysaccharide (LPS) to permeate the blood-brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays.Results. Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti-P and anti-NSPA staining colocalization, and responded to both anti-P and anti-NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti-P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti-P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti-NSPA antibodies also impaired memory.Conclusion. Anti-P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti-P can access the hippocampus and impair memory without requiring neuronal death when the blood-brain barrier is disrupted. NSPA can mediate antibody-driven diffuse brain dysfunction, and anti-P might contribute to the cognitive impairment that is frequently observed in SLE.Controversy surrounds the clinical and pathogenic role of anti-ribosomal P (anti-P) protein autoantibodies in neuropsychiatric systemic lupus erythematosus (NPSLE) (1-4). Diffuse brain dysfunctions frequently occur in SLE without overt brain inflammation, and these dysfunctions thus might involve pathogenic autoantibodies, especially against neuronal surface components (5). An early study by Bonfa et al (6) revealed an association between blood circulating anti-P and lupus psychosis. Such an intriguing association has been confirmed by several studies and extended to other NPSLE manifestations but has not yet been extended to include cognitive impairment (1,2,7-10). Anti-P antibodies are also detected in the cerebrospinal fluid of patients with NPSLE, indicating blood-brain barrier permeation (9,11). However, conflicting clinical results and the as-yet little-understood mechanisms of neuropathogenesis mean that the role of anti-P is contentious (1-4).Experimental models and the identification of a neuronal surface target have pr...

The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis. Apolipoprotein E receptor 2 or apoER2 (also called LRP8) is a member of the low-density lipoprotein receptor (LDL-R) family and binds ligands including apolipoproteins E (apoE), apoB, apoJ and reelin (1-4). It is expressed predominantly in the brain, platelets, ovary, epididymis and placenta (1,5,6). Apolipoprotein E receptor 2 shares several structural characteristics with LDL-R, including an extracellular domain composed of modules of ligand-binding repeats, a single transmembrane domain and a cytoplasmic domain containing one NPXY motif, which is determinant for the clathrin-mediated endocytosis of LDL-R (1,7). As a receptor for reelin, apoER2 participates in brain development and synaptic plasticity (2,8,9). Upon reelin ligand binding, apoER2 induces the activation of Src family kinases and the phosphorylation of disabled 1 (Dab1), which binds to the NPXY motif and initiates a signaling cascade including the activation of phosphatidylinositol-3-kinase and the inhibition of glycogen synthase kinase-3b (9). Apolipoprotein E receptor 2 also has a role in the LDL-dependent sensitization of platelets by inducing the activation of p38MAPK (4). One of the interesting characteristics of apoER2 is its expression in several spliced variants (10-12). These structural variations differ in the receptor's ligand-bindi...

The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and postendocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.

Endocytic traffic can control cell surface versus intracellular distribution of empty/inactive EGFR, an thus its accessibility to external stimuli, through a pathway involving down regulation of PKA activity mediated by PA signaling towards PDE4. This novel control mechanism can trans-modulate EGFR function by heterologous stimuli of PLD.

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17).In the biosynthetic pathway, Y 29 but not N 26 from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N 26 A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y 63 ATL 66 motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL 86,87 ), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes. INTRODUCTIONEpithelial cells posses functional, morphological, and biochemically distinct apical and basolateral cell surface domains and maintain this polarized phenotype addressing specific plasma membrane proteins into each domain (Yeaman et al., 1999;Mostov, 2003;Rodriguez-Boulan et al., 2005). Apical and basolateral proteins are sorted in the biosynthetic route at the level of the trans-Golgi network (TGN; Rindler et al., 1984;Fuller et al., 1985;Griffiths and Simons, 1986), and those proteins that undergo endocytosis can be additionally sorted in recycling endosomes (RE;Mostov and Cardone, 1995;Odorizzi and Trowbridge, 1997). Evidence accumulated over a decade and consolidated in the most recent studies (Ang et al., 2004;Lock and Stow, 2005;Cancino et al., 2007;Cresawn et al., 2007;Gravotta et al., 2007) have shown that the biosynthetic route of at least some proteins includes a post-TGN transit through RE. Under this scenery, it is now important to define the relative contribution of the TGN and RE in the polarized sorting mechanisms of different cargo and in different kind of polarized cells. Neurons, for instance, have to direct distinct proteins to somato-dendritic or axonal plasma membrane domains (Rodriguez-Boulan and Powell, 1992;Winckler and Mellman, 1999), yet their protein-sorting mechanisms remain less known than in epithelial cells. A comparative analysis in epithelial cells and neurons could indeed help to understand the underlying mechanisms of the polarized phenotype.Studies in MDCK cells, the most currently used model of cell polarity, settled the basics of apical and basolateral protein sorting (Rodriguez-Boulan et al., 2005). Apical membrane proteins possess sorting information located in their extracel...