on which these receptors function during development. Key words: APP, endocytosis, gene transcription, lipopro- tein receptor, LRP, neurodevelopment, protein ...
Traffic 2003; 4: 291–301 Blackwell Munksgaard
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Blackwell Munksgaard 2003 ISSN 1398-9219
Review
LDL Receptor-Related Proteins in Neurodevelopment Petra May and Joachim Herz* Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390–9046, USA *Corresponding author: Joachim.Herz @UTSouthwestern.edu Low-density lipoprotein receptor-related proteins (LRPs) are evolutionarily ancient cell-surface receptors with diverse biological functions. All are expressed in the central nervous system and, for most receptors, animal models have shown that they are indispensable for successful neurodevelopment. The mechanisms by which they regulate the formation of the nervous system are varied and include the transduction of extracellular signals and the modulation of intracellular signal propagation, as well as cargo transport, the function most commonly attributed to this gene family. Here, we will summarize recent advances in our understanding of the molecular basis on which these receptors function during development. Key words: APP, endocytosis, gene transcription, lipoprotein receptor, LRP, neurodevelopment, protein kinase, Reelin, signal transduction, Wnt Received 13 January 2003, revised and accepted for publication 2 February 2003
Introduction to the LDL Receptor Family of Lipoprotein Receptors The low-density lipoprotein receptor (LDLR) gene family in mammals consists of seven core members of structurally closely related cell-surface proteins (Figure 1). The first to be identified and the namesake of the family is the lowdensity lipoprotein receptor, which plays a central role in lipid metabolism and cholesterol homeostasis by mediating the cellular uptake of cholesterol-rich low-density lipoprotein particles. It was initially assumed that the other family members would also act mainly in lipid transport and some of the receptors’ names still reflect this concept. It has now become clear, however, that LDLR-related proteins exert diverse biological functions beyond the endocytosis of lipoproteins (1) (Table 1).
structurally virtually indistinguishable from their mammalian counterparts. The core of the gene family in mammals consists of the LDL receptor, the LDL receptor-related protein1 (LRP1), the structurally and in size most similar LRP1b and LRP2 (Megalin/gp330), the very-low-density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (ApoER2, LRP8) and MEGF7 (a Multiple Epidermal Growth Factor repeat containing protein 7) (Figure 1A). They all share the same structural motifs in the same repeating arrangement, where ligand binding (complement) type cysteine-rich repeats are always followed by a YWTD-motif containing b-propeller domain that is flanked by EGF-like cysteine-rich repeats (EGF precursor homology domain). The receptors are anchored in the plasma membrane by a single membrane-spanning segment. A short cytoplasmic tail contains one or more NPxY (AsnPro-any amino acid-Tyr) motifs, which serve as docking sites for phosphotyrosine binding (PTB) domain containing adaptor proteins and as an endocytosis signal. Some of the receptors also contain an additional O-linked sugar domain that precedes the transmembrane segment [reviewed in (2)]. Other more distantly related receptors share some, but not all, of the structural hallmarks that define the core of the family. In addition, they may also contain structural and functional domains, which are not present in the core members. LRP5, LRP6 and LR11 (also called SORLA) belong to this group of proteins (Figure 1B). All the core members of the LDLR family are expressed in the central nervous system. The LDL receptor does not appear to be essential for neural development, since the brain develops normally in humans, rabbits and mice that lack functional LDL receptors. ApoER2 and VLDLR, by contrast, are important signaling receptors that regulate neuronal migration in the developing brain, while megalin (lRP2)-deficient mice exhibit a classical defect of forebrain development known as holoprosencephaly. LRP1, a multifunctional receptor with more than 30 known ligands, is essential for embryonic development. Its functions in the central nervous system are now beginning to emerge. Finally, we will address the roles of LRP5 and LRP6 as coreceptors in the canonical Wnt signaling pathway.
ApoER2 (LRP8) and VLDLR The LDLR gene family arose early during the evolution of metazoans. For most family members orthologues have been identified in the nematode Caenorhabditis elegans and in the fruitfly Drosophila melanogaster, where they are
Roles in embryonic neural development have so far been studied in greatest detail for ApoER2 and VLDLR. Both receptors were cloned as close homologues of the LDLR 291
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Figure 1: Panel A shows the seven core members of the LDLR family, Panel B the more distantly related receptors LRP5, LRP6 and SorLA. Common structural motifs are highlighted. The receptors have been labeled with their most commonly used names. Alternative denominations are: LRP ¼ LRP1, Megalin ¼ LRP2, ApoER2 ¼ LRP8. LRP3, LRP4 and LRP9 are proteins that merely contain LDLR-type ligand binding type repeats, but otherwise share no structural similarities to the shown family members. LRP5 has received a duplicate denomination (LRP7). The VLDLR has so far not been included in this nomenclature.
(3–5), leading to the initial hypothesis that they might also function in systemic lipoprotein metabolism. Neither receptor, however, is expressed in the liver [see (1) and references therein], and genetic studies with mice harboring targeted deletions of the vLDLR or the apoER2 did not show any major disturbance in lipid homeostasis (6,7). A breakthrough in the understanding of their function arose from the finding that mice that were deficient for both apoer2 and vldlr suffered from a severe developmental neurological disorder that is characterized by improper layering of neurons in the cerebral cortex and ectopic localization of neurons in the cerebellum and in the hippocampus (7). The clinical and histological findings in the receptor double knock-out mice were virtually indistinguishable from the defects that are present in the naturally occurring mutant mouse strains reeler, scrambler, and yotari. Reeler animals lack the large extracellular protein Reelin, which is secreted during brain development, for instance by Cajal-Retzius cells, and confers a positional cue to migrating neurons in the neocortex. By contrast, scrambler and yotari mice both lack the intracellular adap292
tor protein Disabled-1 (Dab1) [reviewed in (8)]. Furthermore, dab1 knock-out mice display the same characteristic phenotypic and structural abnormalities (9), and combination of the reelin and dab1 defects does not compound the phenotype of either single homozygous mutation (10). Reelin continues to be secreted by CajalRetzius neurons of dab1-deficient mice (11). Furthermore, VLDLR and ApoER2 were found to bind Dab1 on their cytoplasmic tails (7). Taken together, these results suggested that Reelin and dab1 function in a linear pathway, with Dab1 acting downstream of Reelin, and that VLDLR and ApoER2 serve as receptors that transmit the Reelin signal across the neuronal plasma membrane (Figure 2A). Indeed, both receptors bind Reelin on their extracellular domains with high affinity, and this interaction results in tyrosine phosphorylation of Dab1 in cultured neurons (12,13). Tyrosine phosphorylation of Dab1 is critical for its function. Mice in which the endogenous dab1 gene has been modified by a knock-in approach, to the effect that all possible tyrosine phosphorylation sites had been changed to phenylalanine, are phenotypically very similar to reeler mice (14). Traffic 2003; 4: 291–301
Ligands
Apolipoprotein B, apolipoprotein E, low-density lipoproteins
Apolipoprotein E, chylomicron remnants, a2-macroglobulin, amyloid precursor protein, protease/ protease inhibitor complexes, lipoprotein lipase, hepatic lipase, sphingolipid activator protein, Factor VIIa/tissue factor pathway inhibitor, plasminogen activators/plasminogen activator inhibitor-1, Factor XIa, Factor VIIIa, MMP9, MMP13, pregnancy zone protein, complement C3, C1-inhibitor, antithrombin III, heparin cofactor II, a1-antitrypsin, thrombospondin 1 and 2, Pseudomonas exotoxin A, rhinovirus, lactoferrin, heat shock protein 96, HIV tat protein
Apolipoprotein B, apolipoprotein E, apolipoprotein J, apolipoprotein H, albumin, cubilin, plasminogen activators/plasminogen activator inhibitor-1, parathyroid hormone, retinol binding protein, vitamin D binding protein
Apolipoprotein E, Reelin, lipoprotein lipase, tissue factor pathway inhibitor
Apolipoprotein E, Reelin
unknown
unknown
Wnt proteins, dickkopf proteins (?)
Wnt proteins, dickkopf proteins
Apolipoprotein E, head activator peptide
Receptor
LDL Receptor
LRP1
LRP2 (Megalin/gp330)
VLDLR
LRP8 (ApoER2)
LRP1b
MEGF7
LRP5 (¼LRP7)
LRP6
LR11/SorLa
Table 1: LDL receptor family members, their ligands and known functions
Traffic 2003; 4: 291–301 Head regeneration in hydra, presumably function in neurodevelopment
Wnt signal transduction, generation of caudal paraxial mesoderm, mid- and hindbrain development, anteroposterior and dorsoventral patterning of the developing limbs
Regulation of bone formation and ocular embryonic development, presumably as Wnt coreceptor
Unknown
Unknown
Regulation of neuronal migration during embryonic development (predominantly hippocampus and neocortex)
Regulation of neuronal migration during embryonic development (predominantly cerebellum)
Vitamin homeostasis, renotubular reabsorption of proteins, regulation of thyroid and parathyroid functions, embryonic cholesterol homeostasis?
Lipoprotein and protease uptake, modulation of APP processing, modulation of intracellular signaling, synaptic transmission?
Lipoprotein/cholesterol uptake
Functions
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Figure 2: (a) Reelin binds to ApoER2 or VLDLR and clusters several receptor molecules at the membrane. Other Reelin coreceptors may be part of this complex. Reelin-binding induces tyrosine phosphorylation of Dab1. The activity of Src family kinases (SFK) is necessary for efficient Dab1 phosphorylation. Phosphorylated Dab1 further activates SFKs, presumably by releasing their intramolecular autoinhibition. Activation of PI3 Kinase and PKB/Akt results in the inhibition of the tau kinase GSK3b. Reelin signaling has been shown to enhance LTP formation. The underlying molecular mechanism is not known. Interaction of the receptor tails with the scaffold protein JIP may regulate cytoskeletal organization, axonal transport, and JNK activation. (b) LRP1 affects proteolytical processing of APP and Ab production. Both proteins interact through their extra- and intracellular domains. The latter interaction has been proposed to occur via the adaptor protein FE65. Like APP, LRP1 can also be cleaved by the presenilin-containing g-secretase complex, resulting in the release of its intracellular domain. LRP1 is present in caveolae, where LRP1 can be phosphorylated in response to PDGF signaling. This requires the PDGFRb, Src, and PI3 kinase. PDGF-BB binds directly to LRP. One possible function of LRP in PDGF signaling might be the downregulation of PDGF receptors. In addition to FE65, LRP1 interacts with the adaptor molecules JIP1 and 2. Tyrosine-phosphorylated LRP1 binds Shc, resulting in possible modulation of MAPK signaling by LRP1. (c) LRP5 and LRP6 are necessary components of the canonical Wnt signaling pathway, presumably by acting as Wnt coreceptors. The interaction of Wnt with its receptors results in the activation of the intracellular molecule Dishevelled. This in turn leads to the inactivation of GSK3b, which phosphorylates b-catenin in an Axin-containing multiprotein complex, thereby targeting it for proteasomal degradation. Stabilized b-catenin activates target gene transcription by interrupting the interaction of TCF/LEF-1 transcription factors with certain corepressors. LRP5 interacts with Axin through its cytoplasmic tail, thereby possibly directly influencing b-catenin stabilization. Dickkopf proteins are regulators of Wnt signaling, which bind to LRP6 (and likely also to LRP5) and another transmembrane molecule, Kremen. The resulting protein complex is endocytosed, leading to the downregulation of LRP coreceptors.
An unresolved question, however, concerned the nature of the tyrosine kinase that is activated by Reelin signaling. Neither VLDLR nor ApoER2 possesses a tyrosine kinase domain, nor do they interact directly with nonreceptor kinases. Dab1, however, had been identified originally as a Src-interacting protein in a yeast-two-hybrid screen (15), 294
and genetic experiments had shown that Src family kinases (SFKs), specifically Fyn, are involved in the proper positioning of neurons during brain development (16). Two recent studies have now revealed that Reelin-induced tyrosine-phosphorylation of Dab1 does indeed depend on SFK activation, and that both, Reelin receptors and Dab1, Traffic 2003; 4: 291–301
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are required for Reelin-induced activation of SFKs. Fyn appears to be the main SFK that mediates the Reelin signal, since Dab1 protein expression is greatly increased in fyn knock-out mice. A similar increase in Dab1 expression occurs in reeler and in receptor double knock-out mice in an apparent attempt by the cell to compensate for reduced Reelin signal input (17,18). However, other SFKs can compensate to a large degree even in the complete absence of fyn. Coexpression of SFKs and Dab1 in transfected cells results in activation of the SFKs and strong phosphorylation of Dab1. This suggests that Dab1 and SFKs mutually activate each other when their local concentration exceeds a critical threshold. Physiologically, this may be caused by Reelin-induced receptor clustering at the plasma membrane. Post-translational modification of Dab1 is not restricted to tyrosine phosphorylation, but also involves serine phosphorylation by cyclin-dependent kinase 5 (Cdk5), which occurs independent of Reelin signaling (19). Cdk5 is neuronally expressed and also plays an important role during the migration events that occur during the embryonic development of the brain. Mice that lack the catalytic domain of Cdk5 or both of its regulatory subunits p35 and p39 exhibit cortical lamination defects that resemble, but are also clearly distinct from the reeler phenotype (20,21). This suggests cross-talk between these two different signaling pathways, both of which regulate steps that are critical for proper neuronal migration and positioning. The precise mechanisms through which these pathways interact, however, remain to be elucidated. More insights have recently been gained into the cytosolic signaling cascades that are activated directly by Reelin and that involve VLDLR, ApoER2 and Dab1. Reelin was found to increase phosphatidylinositol3-kinase (PI3-kinase) and Protein Kinase B (PKB/Akt) activity in a manner that is dependent upon the receptors and upon Dab1 tyrosine phosphorylation. Activation of this cascade in turn leads to the phosphorylation and thus inhibition of glycogen synthase kinase 3b (GSK3b), one of the major kinases that phosphorylate the microtubule stabilizing protein tau (22). This negative regulation of a tau kinase is consistent with the observation that tau-phosphorylation is increased in animals with defects in the Reelin signaling pathway, i.e. in reeler mice and in dab1 as well as receptor double knockout animals (13). The physiological significance of the effect of Reelin on tau phosphorylation in humans remains to be elucidated, but this is clearly of great clinical importance, since tau hyperphosphorylation occurs in various neurodegenerative disorders, resulting in the formation of a characteristic kind of intraneuronal aggregate called neurofibrillary tangles (23). The regulation of tau phosphorylation is one possible function for Reelin signaling in the adult brain, where it could be of importance for regulating the neuronal cytoskeleton. This would be consistent with the recent finding that the components of the Reelin signaling pathway are enriched in axonal growth cones (22). Traffic 2003; 4: 291–301
Another function of Reelin in the adult nervous system is its role in modulating long-term potentiation (LTP). Both ApoER2 and VLDLR are necessary for this effect (24). These findings indicate that the Reelin pathway plays a role in the regulation of synaptic transmission and learning and, indeed, receptor knock-out mice exhibited a learning deficit when subjected to contextual fear conditioning. A better understanding of the biological effects of Reelin signaling is certain to arise from the identification of additional players in this pathway. Other molecules that have been connected to Reelin signaling include putative coreceptors. Both integrins, specifically a3 and a(v), and cadherin-related neuronal receptors (CNRs) have been proposed to interact with Reelin (25–27). These molecules are attractive candidates, since they are also known to directly or indirectly associate with Src family kinases via their cytosolic domains. The defects in neuronal migration in the brains of integrin a3 knock-out mice, however, are distinct from that of reeler mice, leaving the exact nature of their involvement in Reelin signaling subject to further exploration. No genetic data exist to confirm the role of CNRs in Reelin signaling. The role of coreceptors in this pathway could explain the apparent specificity of ApoER2 and VLDLR. Both receptors seem to be functionally redundant to a certain extent, since only double-deficient mice show the same phenotype as the reeler mice. However, in specific regions of the brain they obviously have separate functions, as both vldlr and apoer2 single knock-out animals display subtle, but distinctive, migration defects in different areas of the central nervous system (7). An alternative explanation for their nonredundancy may come from the different sets of intracellular interacting partners. Both receptors recruit various adaptor and scaffold proteins via the NPxY motifs of their cytoplasmic tails (28). In addition, ApoER2 exists in splice variants with or without a 59-amino-acid insert within its cytosolic domain. This insert can recruit members of the family of JNK interacting proteins (JIP) (29) to the cytoplasmic tail. JIPs assemble mitogen-activated protein kinase (MAPK) containing complexes, which function in stress and growth factor signaling (30). Furthermore, JIPs and their Drosophila orthologues have been shown to bind to kinesin light chains, giving them a function in axonal transport (31,32).
Megalin (LRP2) Megalin is widely expressed on the apical surfaces of epithelial cells. Among other ligands, it binds and can mediate the endocytic uptake of apolipoprotein E and B and vitamin/vitamin binding protein complexes (33). In the adult, Megalin functions in the renotubular reabsorption of urinary proteins and in vitamin D and Ca2þ metabolism. During embryonic development Megalin plays an 295
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important role in the formation of the central nervous system. Mice lacking this receptor due to targeted gene inactivation suffer from holoprosencephaly, a specific malformation of the forebrain and cephalic midline structures (34). The molecular mechanism that underlies this developmental defect is not fully understood, but both impaired endocytosis of nutrients (specifically cholesterol) and defects in signal transduction processes like those seen in apoER2 and vLDLR mutants might play a role. Failure of neurodevelopment also occurs in animals lacking apolipoprotein B, the protein component of cholesterolrich low-density lipoproteins, and in humans and mice with genetic defects in cholesterol biosynthesis (reviewed in 35). Insufficient cholesterol supply to the developing brain seems to be the common pathogenetic factor in these disorders. By a similar mechanism, Megalin deficiency might result in inadequate cholesterol uptake, primarily by the most rapidly expanding anterior neuroepithelium, where it is normally expressed abundantly. Holoprosencephalic developmental abnormalities of varying severity are also caused by defects in the sonic hedgehog signaling molecule (reviewed in 35). Sonic hedgehog signals by binding to a cell-surface receptor, patched, which then releases the inhibition of the seven transmembrane protein smoothened. Smoothened in turn activates an intracellular signaling cascade that results in the regulation of target gene transcription (36). Intriguingly, sonic hedgehog is modified by the addition of a cholesterol moiety to its carboxyl terminus for full activity, and it has also been shown to be a ligand for Megalin (37). These findings suggest a role for Megalin in the modulation of sonic hedgehog signal transduction. More evidence for a role of Megalin in cellular signaling comes from studies on the interaction of cellular adaptor molecules with the cytoplasmic tail of Megalin (28,38). One of these, the recently discovered Megalin binding protein, not only associates with the intracellular domain of Megalin, but also binds the vitamin D receptor coactivator SKIP, possibly enabling Megalin directly to influence vitamin D-dependent gene expression (38). The adaptor protein Dab2, a paralogue of Dab1 that interacts with the tails of Megalin and other lipoprotein receptors as well as with the clathrin adaptor protein AP-2 (39,41), plays a crucial role in embryonic development. Dab2-deficient mouse embryos fail during gastrulation and show a phenotype that is morphologically reminiscent of a defect of the transforming growth factor b (TGFb) family member nodal. Dab2 is required in the visceral endoderm of the pregastrulation embryo but not in the embryo proper, suggesting a function in extra-embryonic signaling (40). Megalin-deficient mice do not show this phenotype, possibly because other lipoprotein receptors can mediate or compensate for the dab2-dependent modulation of this signaling pathway. By contrast, dab2 deletion only in the embryo 296
proper by conditional gene inactivation resulted in an endocytosis defect of the renal proximal tubular epithelium, suggesting a role of Dab2 in intracellular trafficking (41).
LRP (LRP1) Disruption of the lrp1 gene in mice by homologous recombination showed that LRP1 plays a crucial role in embryogenesis. Lrp1-deficient animals fail to develop normally and die during early to mid-gestation (42). The early embryonic lethality of the lrp1 knock-out forestalled more detailed insights into the role of the receptor in central nervous system development. Next to the liver, however, LRP1 is most abundantly expressed in the brain, and mRNA expression peaks around birth (43). Possible functions of LRP1 in the brain may involve interactions with the amyloid precursor protein (APP) and the g-secretase complex, its demonstrated role in the modulation of protein kinase signaling, and finally its possible function in the regulation of Ca2þ currents (Figure 2B). LRP and APP metabolism APP is a single-pass transmembrane protein, from which the (amyloid b) Ab peptide, the main component of the cerebral amyloid plaques that occur in Alzheimer’s disease, is generated by proteolytic processing. Its central role in the pathogenesis of the disease is underlined by the fact that certain mutations in APP itself or in critical components of the proteolytically active g-secretase complex, i.e. presenilin 1 and 2, enhance Ab production and almost invariably lead to early onset Alzheimer’s disease (44). Both LRP1 (45) and its ligands apolipoprotein E (46), and possibly also a2-macroglobulin (47), have been linked genetically to late-onset Alzheimer’s disease. Moreover, LRP has been found to directly interact with APP, via both their extracellular and their intracellular domains (48–50). The intracellular interaction has been suggested to occur via a scaffold protein, FE65, which possesses two PTB domains, one of which has been shown to interact with the NPTY motif of the LRP cytoplasmic tail, the other one with the YENPTY motif in the APP intracellular domain (49). Intriguingly, the interaction between LRP and APP appears to directly influence the trafficking and proteolytical processing of APP. Ulery et al. (51) showed that Ab production was impaired in cells lacking functional LRP1, while APP cell-surface levels were increased. This finding was attributed to the interaction of the extracellular domains of both proteins, involving the Kunitz proteinase inhibitor domain in APP, and subsequent endocytosis by LRP1. Recently, however, Pietrzik et al. (52) demonstrated that the defect in Ab production of LRP-deficient cells is independent of the Kunitz domain, and that this defect can be Traffic 2003; 4: 291–301
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corrected by reintroduction of an LRP1 mutant, which lacks most of the extracellular domain of the receptor. Further mapping studies showed that the region around the second NPxY motif of the LRP tail was critical for the restoration of this LRP1 function, suggesting that it might be mediated through an interaction of the cytosolic domains. In addition to the effect on Ab production, Pietrzik et al. also found increased cell-surface levels of APP, increased production of non-amyloidogenic soluble APP (APPs), and increased turnover of the C-terminal fragments (CTF), which are generated by the processing of APP. Interestingly, mutations in critical aspartate residues of PS1 result in increased stability of both full-length APP and APP CTFs, indicating that LRP1 differentially affects the PS1dependent metabolism of these APP proteins (53). How exactly the cytoplasmic domain of LRP1 exerts its function on APP metabolism remains unclear at present. On one hand, the critical region mapped by Pietrzik et al. contains a putative endocytosis motif; on the other hand, it is also the site of interaction with several adaptor proteins (28,49). The trivalent scaffolding protein FE65 binds to LRP1 via the first NPxY motif in the LRP tail, but other adaptors, e.g. Dab1 and the JNK interacting proteins 1 and 2, interact with the second one. Both Dab1 and JIP1b also bind the APP cytoplasmic tail. Moreover, it has been described that the APP cytoplasmic tail can be phosphorylated by JNK3 at Thr668 (54), and that the phosphorylation of this residue impairs the interaction of APP with FE65 (55). Several groups recently reported that the APP-CTF can be released from the plasma membrane and enter the nucleus, where it can regulate target gene transcription (56–59). Nuclear translocation of APP-CTF is facilitated by FE65. The interplay between APP and LRP1 might be more complex still, as evidence is emerging that the g-secretasedependent release of the cytoplasmic tail of membrane proteins is a widespread principle, and that LRP1 itself can also undergo proteolytic processing by g-secretase (60). Whether FE65 in conjunction with the LRP1 cytoplasmic domain also directly regulates gene transcription under physiological conditions, and what the transcriptional targets would be remains to be elucidated. The possibility that LRP1 might be a transcriptional regulator, however, sheds new light on its essential role in embryonic development and offers new perspectives regarding its interactions with developmentally or neuronally important signaling pathways. LRP and protein kinase signaling Src can mediate tyrosine phosphorylation of the second NPxY motif in the cytoplasmic tail of LRP1, converting it into a binding site for the adaptor protein Shc, a mediator of MAP kinase activation (61). Studies in nontransformed cell lines showed that LRP1 is tyrosine-phosphorylated in response to platelet-derived growth factor (PDGF) treatTraffic 2003; 4: 291–301
ment (62,63), and this requires cellular Src family kinases, PDGF receptor b-chain, and phosphatidylinositol 3-kinase. Moreover, this tyrosine phosphorylation of LRP1 takes place in caveolae, specialized cholesterol-rich membrane compartments that contain preassembled signaling complexes that can be activated by extracellular stimuli (64). This intriguing finding raises the possibility that LRP1 may have coreceptor functions, similar to those of the VLDLR and the ApoER2. Indeed, PDGF-BB binds directly to LRP (63), and the PDGF-induced tyrosine phosphorylation of LRP is inhibited by the LRP ligands a2-macroglobulin and ApoE-enriched b-VLDL (62). This is particularly interesting, since Swertfeger et al. described an LRP-dependent inhibition of PDGF-induced smooth muscle cell migration by ApoE (65), suggesting a physiological role for LRP in PDGF receptor signaling. This also shows how ApoE may regulate LRP function, a mechanism that could be employed more generally in different tissues and possibly different signaling pathways. Recently, Gianni et al. (66) reported that PDGF BB positively regulates APP processing via a Src- and Rac1dependent pathway. This raises the possibility that LRP1 could exert its regulatory function in APP metabolism by modulating the PDGF signal. However, Src activity depends on the input of many different signaling pathways. For instance, as described above, Reelin activates Src family kinases through its interaction with ApoER2 and VLDLR. Thus, it is possible that Reelin could have an additional function in the regulation of APP processing, which also might be modulated by the interaction of its receptors with ApoE isoforms.
LRP and calcium signaling Recently, several investigators reported a function for LRP in the regulation of calcium currents in neuronal cells. Bacskai et al. (67) found that the LRP ligand activated a2-macroglobulin induced Ca2þ influx in neurons, and that this effect could be blocked by the chaperone receptor associated protein (RAP). Another study revealed that, in cultured hippocampal neurons, activated a2-macroglobulin modulates the calcium response to the neurotransmitter N-methyl-D-aspartate (NMDA); however, this time a negative regulation was observed. Treatment with RAP again blocked this effect. Down-regulation of an NMDA receptor subunit in response to activated a2-macroglobulin was noted and proposed as a possible mechanism for its modulatory function (68). A role for LRP in neurotransmission is supported by the finding that the LRP tail interacts with the adaptor protein PSD95, which also binds the NMDA receptor, and could thus mediate the interaction between the two molecules (28). Similar mechanisms might play a role in the reported effect of the LRP ligand tissue-type plasminogen activator on long-term potentiation and synaptic plasticity. This effect was shown to involve LDLR family members, as it could be inhibited by receptor-associated protein (69). 297
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Figure 3: LRPs play important roles during various stages of neurodevelopment. Indicated dates are approximate and reflect the time-point at which genetic deficiency results in the first morphologically detectable abnormalities.
LRP5/6 lrp5, lrp6 and their orthologue in Drosophila, arrow, play an important role as components of the canonical Wnt signaling pathway in embryonic development (70–72) (Figure 2C). Wnt/wingless proteins are extracellular glycoproteins that regulate a number of developmental processes through different signaling mechanisms [reviewed in (73,74)]. In the so-called canonical pathway, Wnts bind to seven transmembrane domain receptors, the Frizzled receptors, and activate by an as yet unknown mechanism the intracellular mediator Dishevelled. This in turn leads to the stabilization of cytosolic b-catenin via the inhibition of the enzyme glycogen synthase kinase 3b (GSK3b), which targets b-catenin for proteasomal degradation by phosphorylating it. This interaction takes place in a large protein complex that also comprises the proteins Axin, Adenomatous Polyposis Coli (APC), Protein Phosphatase 2 A and the F-box protein b-transducin repeat containing protein (b-TrCP). If not targeted for degradation, b-catenin interacts with transcription factors of the lymphoid enhancer factor 1(LEF-1)/ T-cell factor (TCF) family, which finally leads to the transcription of target genes (75). Recently, arrow was shown to be an essential component of Drosophila wingless (wg) signaling, where it acts in wingless responsive cells upstream of Dishevelled (70). At the same time, mice lacking lrp6 were found to exhibit complex developmental defects that resembled a composite phenotype of deficiency in several wnt genes, which also include neural tube defects (72). Overexpression of LRP6 in Xenopus eggs induced Wnt signaling responses (71), which required the carboxyl terminus of LRP6. Expression of a truncated LRP6 protein lacking the cyto298
plasmic domain actually inhibited Wnt signaling. Direct interaction of LRP6 with Wnt proteins and the formation of a ternary complex that includes frizzled receptors suggested a role of LRP6 as a Wnt coreceptor (71), although this is still subject to debate (76). The closely related receptor LRP5 was recently shown to participate in canonical Wnt signaling by recruiting Axin upon stimulation with Wnt, thereby destabilizing the protein complex in which b-catenin is targeted for degradation (77). Furthermore, the sequence of the LRP5 cytosolic tail that was necessary for the recruitment of Axin was also critical for LEF1/TCF activation. Interestingly, a truncated form of LRP5 lacking the extracellular domain was constitutively active. This is in concordance with the discovery that the activity of the LRPs is regulated by dickkopf proteins (Dkks), secreted molecules that modulate Wnt signaling by binding to the extracellular domains of the receptors (78–80). In addition to LRP, Dkks also bind the transmembrane protein Kremen, which mediates endocytosis of the ternary complex and thereby the downregulation of the LRP coreceptors (81). Unlike the core members of the LDL receptor gene family, LRP5 and 6 do not possess intracellular NPxY motifs. Their internalization is thus dependent upon the interaction with Kremen. Endocytic uptake of extracellular ligands may thus be a function of LDLR family members that arose in parallel or in addition to their roles in cellular signaling.
Conclusion Our understanding of lipoprotein receptor physiology is rapidly expanding and the mechanisms by which LDL Traffic 2003; 4: 291–301
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receptor family members control neurodevelopment and synaptic function (Figure 3) have begun to be elucidated. Ever more refined insights into the diverse molecular mechanisms by which this remarkable gene family regulates development, as well as chemical and electrophysiological signal transduction in vivo, will come from the analysis of mice with conditional and functionally selective receptor mutations.
Acknowledgments We thank H.H. Bock for critical reading of the manuscript. P.M. is recipient of an Emmy-Noether fellowship of the Deutsche Forschungsgemeinschaft. J.H. is supported by grants from the NIH, the Alzheimer Association, the Perot Family Foundation and the Humboldt Foundation.
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