Claudio Retamal

Adaptor Protein Sorting Nexin 17 Regulates Amyloid Precursor Protein Trafficking and Processing in the Early Endosomes

Accumulation of extracellular amyloid β peptide (Aβ), generated from amyloid precursor protein (APP) processing by β- and γ-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Aβ 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 Aβ production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state 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 Aβ. 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 Aβ.

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains.

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.

ApoER2 is Endocytosed by a Clathrin-Mediated Process Involving the Adaptor Protein Dab2 Independent of its Rafts' Association

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 receptors 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.

The low density lipoprotein receptor-related protein functions as an endocytic receptor for decorin

Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type β and other growth factors and thereby influences the processes of proliferation and differentiation in a wide array of physiological and pathological reactions. Hence, understanding the regulatory mechanisms of decorin activity has broad implications. Here we report that the extracellular levels of decorin are controlled by receptor-mediated catabolism, involving the low density lipoprotein receptor family member, low density lipoprotein receptor-related protein (LRP). We show that decorin is endocytosed and degraded by C2C12 myoblast cells and that both processes are blocked by suppressing LRP expression using short interfering RNA. The same occurs with CHO cells, but not with CHO cells genetically deficient in LRP. Finally, we show that LRP-null CHO cells, transfected to express mini-LRP polypeptides containing either the second or fourth LRP ligand-binding domains, carry out decorin endocytosis and lysosomal degradation. These findings point to LRP-mediated catabolism as a new control pathway for the biological activities of decorin, specifically for its ability to influence extracellular matrix signaling.

Polarized traffic of LRP1 involves AP1B and SNX17 operating on Y-dependent sorting motifs in different pathways.

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 receptors 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.

The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

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.

Anti–Ribosomal P Protein Autoantibodies From Patients With Neuropsychiatric Lupus Impair Memory in Mice

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.

The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release.

ABSTRACT 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 post-endocytic 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.

Pathogenicity of Lupus Anti–Ribosomal P Antibodies: Role of Cross‐Reacting Neuronal Surface P Antigen in Glutamatergic Transmission and Plasticity in a Mouse Model

To assess whether autoantibodies against ribosomal P (anti‐P), which are possibly pathogenic in neuropsychiatric systemic lupus erythematosus (NPSLE), alter glutamatergic synaptic transmission and to what extent the cross‐reacting neuronal surface P antigen (NSPA) is involved.

Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation

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.