The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx17 expression by short interfering RNA. We conclude that Snx17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.

Apolipoprotein E (apoE), a chaperone for the amyloid ␤ (A␤) peptide, regulates the deposition and structure of A␤ that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr ؊/؊ ) mice had a significant, ϳ50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr ؊/؊ background, we did not observe a significant change in brain A␤ levels either before or after the onset of A␤ deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr ؊/؊ background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect A␤ levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD. Alzheimer disease (AD)1 is a progressive neurodegenerative disease and is the most common cause of dementia. One of the key pathological hallmarks of AD is the deposition of the 39 -43-amino acid amyloid ␤ (A␤) peptide in the form of both diffuse (thioflavine-S/Congo red-negative) and fibrillar (thioflavine-S/Congo red-positive) plaques. An abundance of data suggests that conversion of A␤ from soluble to insoluble forms is an early event in the pathogenesis of AD. The A␤ peptide is generated from cleavage of the larger amyloid precursor protein (APP) with the predominant species being A␤ 40 and ,to a lesser extent, A␤ 42 (1). Although accounting for Ͻ1% of all cases, early-onset, autosomal-dominant forms of AD have been identified that share the common feature of an overall increase in A␤ levels or a relative elevation in the more fibrillogenic A␤ 42 throughout life, ultimately resulting in early A␤ deposition and plaque formation. Identification of these familial AD cases has led to the generation of several APP transgenic mouse models that recapitulate many aspects of A␤ deposition and associated pathology (2).In 1993, the ⑀4 allele of apoE was found to be a genetic risk factor for the most common form of AD (late-onset AD) as well as for cerebral amyloid angiopathy, whereas the ⑀2 allele was shown to be protective (3). Abundant data suggest that apoE is linked to AD and cerebral amyloid angiopathy due to its ability to act as ...

Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.

Wnt co-receptors LRP5 and LRP6 are two members of the low-density lipoprotein receptor family. Receptor-associated protein is not only a specialized chaperone but also a universal antagonist for members of the low-density lipoprotein receptor family. Here we test whether Mesd, a newly identified chaperone for members of the low-density lipoprotein receptor family, also binds to mature receptors at the cell surface and antagonizes ligand binding. We found that Mesd binds to cell surface LRP5 and LRP6, but not to other members of the low-density lipoprotein receptor family. Scatchard analysis revealed that Mesd binds cell surface LRP6 with high affinity (Kd ∼3.3 nM). Interestingly, the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6, and is required for LRP6 folding. We also found that LRP6 is not a constitutively active endocytosis receptor and binding of the receptor-associated protein to LRP6 partially competes for Mesd binding. Finally, we demonstrated that Mesd antagonizes ligand binding to LRP6 at the cell surface. Together our results show that in addition to serving as a folding chaperone, Mesd can function as a receptor antagonist by inhibiting ligand binding to mature LRP6.

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA⅐PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.The plasminogen activation system consists of a cascade of enzymes and plays a central role in many physiological processes requiring the degradation of basement membrane and components of the extracellular matrix. When the regulation of this system is disrupted, as occurs in the pathogenesis of cancer, malignant cells are able to invade surrounding tissue and metastasize to distant body regions (1-3). Urokinase plasminogen activator (uPA) 1 catalyzes the formation of plasmin from its inactive precursor, plasminogen. The activity of uPA is regulated by two proteins, the glycosylphosphatidylinositollinked uPA receptor (uPAR) and plasminogen activator inhibitor type 1 (PAI-1). The binding of uPA to uPAR at the cell surface greatly increases its catalytic rate. In contrast, uPA is inactivated by binding to PAI-1. When active uPA is bound to uPAR, it is not internalized but remains at the cell surface. However, when receptor-bound uPA is complexed to its inhibitor, PAI-1, the complex is rapidly internalized and degraded. The mechanism by which this occurs was unknown until it was determined that low density lipoprotein receptor-related protein (LRP) is responsible for this process (4). Following the internalization, uPAR and LRP recycle back to the cell surface, while uPA and PAI-1 are degraded in lysosomes (4 -7). The regeneration of unoccupied uPAR at the cell surface is thus critical for the maintenance of plasminogen activation and for regulation of cellular migration and invasion (8 -10).LRP1B is a recently discovered member of the LDLR family (11, 12). The LDLR family previously contained two large members, LRP (LRP1), a dimer of 515-and...