Functional interactions of APP with the apoE receptor family

The beta-amyloid precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, but its normal functions in the brain are poorly understood. A number of APP-interacting proteins have been identified: intracellularly, APP interacts with adaptor proteins through its conserved NPXY domain; extracellularly, APP interacts with a component of the extracellular matrix, F-spondin. Interestingly, many of these APP-interacting proteins also interact with the family of receptors for apolipoprotein E (apoE), the Alzheimer's disease risk factor. apoE receptors also share with APP the fact that they are cleaved by the same secretase activities. apoE receptors are shed from the cell surface, a cleavage that is regulated by receptor-ligand interactions, and C-terminal fragments of apoE receptors are cleaved by gamma-secretase. Functionally, both APP and apoE receptors affect neuronal migration and synapse formation in the brain. This review summarizes these numerous interactions between APP and apoE receptors, which provide clues about the normal functions of APP.     

F-spondin interaction with the apolipoprotein E receptor ApoEr2 affects processing of amyloid precursor protein

A recent study showed that F-spondin, a protein associated with the extracellular matrix, interacted with amyloid precursor protein (APP) and inhibited beta-secretase cleavage. F-spondin contains a thrombospondin domain that we hypothesized could interact with the family of receptors for apolipoprotein E (apoE). Through coimmunoprecipitation experiments, we demonstrated that F-spondin interacts with an apoE receptor (apoE receptor 2 [ApoEr2]) through the thrombospondin domain of F-spondin and the ligand binding domain of ApoEr2. Full-length F-spondin increased coimmunoprecipitation of ApoEr2 and APP in transfected cells and primary neurons and increased surface expression of APP and ApoEr2. Full-length F-spondin, but none of the individual F-spondin domains, increased cleavage of APP and ApoEr2, resulting in more secreted forms of APP and ApoEr2 and more C-terminal fragments (CTF) of these proteins. In addition, full-length F-spondin, but not the individual domains, decreased production of the beta-CTF of APP and Abeta in transfected cells and primary neurons. The reduction in APP beta-CTF was blocked by receptor-associated protein (RAP), an inhibitor of lipoprotein receptors, implicating ApoEr2 in the altered proteolysis of APP. ApoEr2 coprecipitated with APP alpha- and beta-CTF, and F-spondin reduced the levels of APP intracellular domain signaling, suggesting that there are also intracellular interactions between APP and ApoEr2, perhaps involving adaptor proteins. These studies suggest that the extracellular matrix molecule F-spondin can cluster APP and ApoEr2 together on the cell surface and affect the processing of each, resulting in decreased production of Abeta.     

Differential glycosylation regulates processing of lipoprotein receptors by gamma-secretase

The low density lipoprotein (LDL) receptor-related protein 1 (LRP1) belongs to a growing number of cell surface proteins that undergo regulated proteolytic processing that culminates in the release of their intracellular domain (ICD) by the intramembranous protease gamma-secretase. Here we show that LRP1 is differentially glycosylated in a tissue-specific manner and that carbohydrate addition reduces proteolytic cleavage of the extracellular domain and, concomitantly, ICD release. The apolipoprotein E (apoE) receptor-2 (apoER2), another member of the LDL receptor family with functions in cellular signal transmission, also undergoes sequential proteolytic processing, resulting in intracellular domain release into the cytoplasm. The penultimate processing step also involves cleavage of the apoER2 extracellular domain. The rate at which this cleavage step occurs is determined by the glycosylation state of the receptor, which in turn is regulated by the alternative splicing of an exon encoding several O-linked sugar attachment sites. These findings suggest a role for differential and tissue-specific glycosylation as a physiological switch that modulates the diverse biological functions of these receptors in a cell-type specific manner.     

Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.     

FE65 interaction with the ApoE receptor ApoEr2

The adaptor protein FE65 interacts with the beta-amyloid precursor protein (APP) via its C-terminal phosphotyrosine binding (PTB) domain and affects APP processing and Abeta production. Our previous data demonstrate that the apoE receptor ApoEr2 co-precipitated with APP and suggest that there are extracellular and intracellular interactions between these two transmembrane proteins. We hypothesized that FE65 acts as an intracellular link between ApoEr2 and APP. Co-immunoprecipitation experiments in COS7 cells demonstrated an interaction between ApoEr2 and FE65 that depended on the N-terminal PTB domain of FE65. Full-length FE65 increased co-immunoprecipitation of ApoEr2 and APP. Full-length FE65 also increased surface expression of ApoEr2, as determined by surface protein biotinylation and live cell surface staining. Constructs containing both the C- and N-terminal PTB domains of FE65 increased secreted APP, secreted ApoEr2, APP C-terminal fragment, and ApoEr2 C-terminal fragment, but constructs containing only single PTB domains did not affect APP or ApoEr2 processing. In addition, full-length FE65 decreased Abeta to a significantly greater extent than individual FE65 domains. These data suggest that FE65 can bind APP and ApoEr2 at the same time and affect the processing of each.     

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated.To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.     

Laminin 5 processing and its integration into the ECM.

Laminins are a family of multi-functional basement membrane proteins. Their C-terminal domain binds to cell surface receptors and is thereby responsible for cell anchorage and the initiation of specific outside-in and inside-out signals. With their N-terminal parts, laminins interact with proteins of the extracellular matrix scaffold to secure the basement membrane to the underlying mesenchymal tissue. Laminins 5A (alpha3Abeta3gamma2), 5B (alpha3Bbeta3gamma2) and 6 (alpha3Abeta1gamma1) are isoforms specific of the basement membrane underneath the epidermis and they undergo a sequential series of extracellular proteolytic changes, which might successively turn on and off one or several of their biological and mechanical functions. Under physiological conditions, such as in adult human skin, epithelial laminins have lost part of the C- and N-terminal domains of the alpha3 and gamma2 chains, respectively. In contrast, in cylindromatosis, a rare inherited disease characterised by major ultrastructural alterations of the basement membrane and altered expression/distribution of integrin receptors, laminin processing has not been completed. Together, these results suggest that laminin processing may regulate signalling pathways and the architecture of the basement membrane by restricting the repertoire of interactions with cell surface receptors and extracellular matrix components.     

DAB1 and Reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11alpha, affect amyloid precursor protein (APP) processing and Abeta production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP beta-C-terminal fragment and secreted Abeta. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the beta-C-terminal fragment of APP and Abeta. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.     

Transmembrane signalling by integrins

Recent work has shown that integrin receptors serve not only as structural receptors that connect the extracellular matrix to the cytoskeleton, but also as signalling receptors that regulate intracellular pH, intracellular free calcium, phosphorylation of proteins on tyrosine and inositol lipid turnover. The ability of extracellular matrix to influence growth, differentiation and other cell functions is very likely related to their effects on signaling pathways inside the cell.     

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.     

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Genetic Analyses of Integrin Signaling

The development of multicellular organisms, as well as maintenance of organ architecture and function, requires robust regulation of cell fates. This is in part achieved by conserved signaling pathways through which cells process extracellular information and translate this information into changes in proliferation, differentiation, migration, and cell shape. Gene deletion studies in higher eukaryotes have assigned critical roles for components of the extracellular matrix (ECM) and their cellular receptors in a vast number of developmental processes, indicating that a large proportion of this signaling is regulated by cell-ECM interactions. In addition, genetic alterations in components of this signaling axis play causative roles in several human diseases. This review will discuss what genetic analyses in mice and lower organisms have taught us about adhesion signaling in development and disease.Almost all cells in multicellular organisms are surrounded by a three-dimensional organized meshwork of macromolecules that constitute the extracellular matrix (ECM). The ECM is a dynamic structure that is generated and constantly remodeled by cells that secrete and manipulate its components into a precise configuration. It functions as a structural framework that provides cells with positional and environmental information, but also forms specialized structures such as cartilage, tendons, basement membranes (BM), bone, and teeth. In addition to its structural properties, the ECM acts as a signaling platform that regulates a large number of cellular functions. It is capable of binding growth factors, chemokines, and cytokines thereby modulating their bioavailability and activity. On the other hand, the ECM is recognized by multiple cell surface receptors that transmit information from the extracellular environment by propagating intracellular signals (for a review, see Hynes 2009).The major cell surface receptors that recognize and assemble the ECM are integrins. Integrins are heterodimeric transmembrane proteins composed of α and β subunits. Eighteen α subunits and eight β subunits can assemble in 24 different combinations with overlapping substrate specificity and cell-type-specific expression patterns (Hynes 2002; Humphries et al. 2006). This, together with the ability of different heterodimers to assemble specific intracellular signaling complexes, provides multiple layers of signaling specificity to these receptors. Conversely, the integrin expression profile of a given cell type determines which ECM components it can bind. Signals arising from integrins regulate virtually all aspects of cell behavior, including cell migration, survival, cell cycle progression, and differentiation.Genetics has proven to be a powerful tool to dissect the functions of ECM–cell interactions in complex organisms. To date, all of the integrin subunits and their major ligands have been deleted in mice. Given the large variety of cellular processes regulated by adhesion signaling, it is not surprising that a significant subset of these proteins has proven to be essential for embryonic development and/or tissue maintenance. However, in addition to underlining the importance of cell-ECM interactions in development, genetic studies also revealed critical roles for tissue- and cell-type-specific modes of adhesion signaling and provided important insights into human disease.     

Apolipoprotein E and apolipoprotein E receptors modulate A beta-induced glial neuroinflammatory responses

Large numbers of activated glia are a common pathological feature of many neurodegenerative disorders, including Alzheimer's disease (AD). Several different stimuli, including lipopolysaccharide (LPS), dibutyryl (db)cAMP, and aged amyloid-β 1–42 (Aβ), can induce glial activation in vitro, as measured by morphological changes and the production of pro-inflammatory cytokines and oxidative stress molecules. Only Aβ-induced activation is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. In addition, only Aβ also induces an increase in the amount of endogenous apoE, the primary apolipoprotein expressed by astrocytes in the brain. The functional significance of the increase in apoE appears to be to limit the inflammatory response. Indeed, compared to wild type mice, glial cells cultured from apoE knockout mice exhibit an enhanced production of several pro-inflammatory markers in response to treatment with Aβ and other activating stimuli. The mechanism for both the Aβ-induced glial activation and the increase in apoE appears to involve apoE receptors, a variety of which are expressed by both neurons and glia. Experiments using receptor associated protein (RAP), an inhibitor of apoE receptors with a differential affinity for the low-density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), revealed that LRP mediates Aβ-induced glial activation, while LDLR mediates the Aβ-induced changes in apoE levels. In summary, both an apoE receptor agonist (apoE) and an antagonist (RAP) inhibit Aβ-induced glial cell activation. Thus, apoE receptors appear to translate the presence of extracellular Aβ into cellular responses, both initiating glial cell activation and limiting its scope by inducing apoE, an anti-inflammatory agent.     

Apolipoprotein E modulates gamma-secretase cleavage of the amyloid precursor protein

Polymorphisms in the apolipoprotein E (APOE) gene affect the risk of Alzheimer disease and the amount of amyloid beta-protein (Abeta) deposited in the brain. The apoE protein reduces Abeta levels in conditioned media from cells in culture, possibly through Abeta clearance mechanisms. To explore this effect, we treated multiple neural and non-neural cell lines for 24 h with apoE at concentrations similar to those found in the cerebrospinal fluid (1-5 microg/mL). The apoE treatment reduced Abeta40 by 60-80% and Abeta42 to a lesser extent (20-30%) in the conditioned media. Surprisingly, apoE treatment resulted in an accumulation of amyloid precursor protein (APP)-C-terminal fragments in cell extracts and a marked reduction of APP intracellular domain-mediated signaling, consistent with diminished gamma-secretase processing of APP. All three isoforms of apoE, E2, E3 and E4, had similar effects on Abeta and APP-C-terminal fragments, and the effects were independent of the low-density lipoprotein receptor family. Apolipoprotein E had minimal effects on Notch cleavage and signaling in cell-based assays. These data suggest that apoE reduces gamma-secretase cleavage of APP, lowering secreted Abeta levels, with stronger effects on Abeta40. The apoE modulation of Abeta production and APP signaling is a potential mechanism affecting Alzheimer disease risk.     

细胞外基质与基质金属蛋白酶

细胞外基质（ECM）是存在于细胞之间的动态网状结构,由胶原、蛋白聚糖及糖蛋白等大分子物质组成.这些大分子物质可与细胞表面上的特异性受体结合,通过受体与细胞骨架结构直接发生联系或触发细胞内的一系列信号传导而引起不同的基因表达,从而导致细胞的生长和分化.作为降解ECM成分最重要的酶-基质金属蛋白酶（MMPs）及其组织抑制因子(TIMPs)在这一过程中起着非常重要的作用.MMPs是一类依赖金属离子锌并以ECM成分为水解底物的蛋白水解酶.其在转录水平的表达受到生长因子、细胞因子及激素等因素的严格调控,在蛋白质水平其活性也受到其激活剂和抑制剂的调节. MMPs通过对ECM成分的水解来影响其降解与重组的动态平衡而参与多种细胞的生理和病理过程.     

Recent advances in research on fibronectin and other cell attachment proteins

Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [1–10]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. we shall also present various popular or provocative hypotheses and speculations about future work in the field.     

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments.     

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.