Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules

The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.     

Dab1 binds to Fe65 and diminishes the effect of Fe65 or LRP1 on APP processing

Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine‐binding (PTB) domain and that affect APP processing and Aβ production. Co‐expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co‐immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C‐terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low‐density lipoprotein receptor‐related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP‐CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP–APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions. J. Cell. Biochem. 111: 508–519, 2010. © 2010 Wiley‐Liss, Inc.     

The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing

The low-density lipoprotein receptor-related protein (LRP) has recently been implicated in numerous intracellular signaling functions, as well as in Alzheimer's disease pathogenesis. Studies have shown that the beta-amyloid precursor protein (APP) interacts with LRP and that this association may impact the production of amyloid beta-protein (Abeta). In this report, we provide evidence that LRP regulates trafficking of intracellular proteins independently of its lipoprotein receptor functions. We show that in the absence of LRP, Abeta production, APP secretion, APP internalization, turnover of full-length APP and stability of APP C-terminal fragments are affected. Importantly, these changes are not APP isoform dependent. Using deletion constructs, the critical region in LRP that modulates APP processing was mapped to a seven peptide domain around the second NPXY domain (residues 4504-4510). Therefore, we propose a model by which LRP functionally modulates APP processing, including those steps critical for Abeta production, through interactions of the cytosolic domains.     

Sequences from the low density lipoprotein receptor-related protein (LRP) cytoplasmic domain enhance amyloid beta protein production via the beta-secretase pathway without altering amyloid precursor protein/LRP nuclear signaling

Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) affects the processing of amyloid precursor protein (APP) and amyloid beta (Abeta) protein production as well as mediates the clearance of Abeta from the brain. Recent studies indicate that the cytoplasmic domain of LRP is critical for this modulation of APP processing requiring perhaps a complex between APP, the adaptor protein FE65, and LRP. In this study, we expressed a small LRP domain consisting of the C-terminal 97 amino acids of the cytoplasmic domain, or LRP-soluble tail (LRP-ST), in CHO cells to test the hypothesis that the APP.LRP complex can be disrupted. We anticipated that LRP-ST would inhibit the normal interaction between LRP and APP and therefore perturb APP processing to resemble a LRP-deficient state. Surprisingly, CHO cells expressing LRP-ST demonstrated an increase in both sAPP secretion and Abeta production compared with control CHO cells in a manner reminiscent of the cellular effects of the APP "Swedish mutation." The increase in sAPP secretion consisted mainly of sAPPbeta, consistent with the increase in Abeta release. Further, this effect is LRP-independent, as the same alterations remained when LRP-ST was expressed in LRP-deficient cells but not when the construct was membrane-anchored. Finally, deletion experiments suggested that the last 50 amino acid residues of LRP-ST contain the important domain for altering APP processing and Abeta production. These observations indicate that there are cellular pathways that may suppress Abeta generation but that can be altered to facilitate Abeta production.     

Regulated proteolytic processing of LRP6 results in release of its intracellular domain

Low-density lipoprotein receptor-related protein 6 (LRP6) is a member of low-density lipoprotein receptor (LDLR) family which cooperates with Frizzled receptors to transduce the canonical Wnt signal. As a critical component of the canonical Wnt pathway, LRP6 is essential for appropriate brain development, however, the mechanism by which LRP6 facilitates Wnt canonical signaling has not been fully elucidated. Interestingly, LRP6 which lacks its extracellular domain can constitutively activate TCF/LEF and potentiate the Wnt signal. Further, the free cytosolic tail of LRP6 interacts directly with glycogen synthase kinase (GSK3) and inhibits GSK3's activity in the Wnt canonical pathway which results in increased TCF/LEF activation. However, whether these truncated forms of LRP6 are physiologically relevant is unclear. Recent studies have shown that other members of the LDLR family undergo gamma-secretase dependent regulated intramembrane proteolysis (RIP). Using independent experimental approaches, we show that LRP6 also undergoes RIP. The extracellular domain of LRP6 is shed and released into the surrounding milieu and the cytoplasmic tail is cleaved by gamma-secretase-like activity to release the intracellular domain. Furthermore, protein kinase C, Wnt 3a and Dickkopf-1 modulate this process. These findings suggest a novel mechanism for LRP6 in Wnt signaling: induction of ectodomain shedding of LRP6, followed by the gamma-secretase involved proteolytic releasing its intracellular domain (ICD) which then binds to GSK3 inhibiting its activity and thus activates the canonical Wnt signaling pathway.     

Low density lipoprotein receptor-related protein (LRP) interacts with presenilin 1 and is a competitive substrate of the amyloid precursor protein (APP) for gamma-secretase

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a gamma-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for gamma-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that gamma-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a gamma-secretase docking site located in close proximity to PS1. This is analogous to APP-gamma-secretase interactions. Finally, we show that LRP competes with APP for gamma-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the Abeta40, Abeta42, and p3 peptides without changing the total level of APP expression. In addition, transfection with the beta-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.     

Evidence for a role of the nerve growth factor receptor TrkA in tyrosine phosphorylation and processing of beta-APP

The cytoplasmic tail of the beta-amyloid precursor protein (APP) contains a Y(682)ENPTY(687) sequence through which APP associates with phosphotyrosine binding (PTB) domain containing proteins in a tyrosine phosphorylation-independent manner. We have recently found that tyrosine phosphorylation of APP-Y(682) promotes docking of Shc proteins that modulate growth factor signaling to the ERK and PI3K/Akt pathways. We have also shown that APP is phosphorylated on Y(682) in cells that overexpress a constitutively active form of the tyrosine kinase abl. Here we present evidence that the nerve growth factor receptor TrkA may also promote phosphorylation of APP. Overexpression of TrkA, but not of mutated, kinase inactive TrkA resulted in tyrosine phosphorylation of APP. Site-directed mutagenesis studies showed that TrkA overexpression was associated with phosphorylation of APP-Y(682). Moreover, overexpression of TrkA also affected APP processing reducing the generation of the APP intracellular domain (AID). Thus, tyrosine phosphorylation of APP may functionally link APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival.     

Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes

Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta 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 Abeta 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 Abeta. 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 Abeta.     

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.     

Notch, epidermal growth factor receptor, and beta1-integrin pathways are coordinated in neural stem cells

Notch1 and beta1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and beta1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that beta1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the beta1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of beta1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate beta1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches.     

Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.     

Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent gamma-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for gamma-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that gamma-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid beta-peptide 40 (Abeta40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that gamma-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that gamma-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.     

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

Background

The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.

Methodology/Principal Findings

To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.

Conclusions/Significance

Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.