All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.

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...

Amyloid-β (Aβ) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Aβ is produced through proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by β-and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Members of the lowdensity lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy.Keywords lipoprotein receptors; cholesterol; amyloid precursor protein; amyloid-β peptide; Alzheimer's disease Amyloid-β peptide and Alzheimer's diseaseAlzheimer's disease (AD) is the most common cause of dementia in the elderly. The characteristic pathological lesions found in AD are the deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles [1]. The major component of the amyloid plaques is the ~4 kDa amyloid-β (Aβ) peptide, which is a cleavage product of the β-amyloid precursor protein (APP) [2,3]. Aβ ranges in size from 37 to 43 amino acids; however, Aβ42 (43) may act as a pathogenic seed for Aβ aggregation and amyloid plaque formation because they are more hydrophobic compared to the shorter Aβ peptides. One current hypothesis known as the "amyloid hypothesis" postulates that increased Aβ production or reduced Aβ clearance results in the formation of aggregated Aβ deposits leading to AD dementia [1,4,5].*Correspondence author: Department of Pediatrics, Washington University School of Medicine, CB 8208, 660 South Euclid Ave, St. Louis, MO 63110, USA, Phone: 314-286-2860, Fax: 314-286-2894, Email: E-mail: bu@wustl.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptSemin Cell Dev Biol. Author manuscript; available in PMC 2010 April 1. Published in final edited form as:Semin Cell Dev Biol. 2009 April ; 20(2): 191-200. doi:10.1016/j.semcdb.2008.005. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptNon-amyloid assemblies of Aβ are now considered as the primary cause of neuronal injury, synaptic loss, and the eventual dementia associated with AD. Soluble Aβ42, isolated from brain, plasma, and cerebral-spinal fluid (CSF), correlates with the severity of neurodeg...