The E3 Ubiquitin Ligase IDOL Induces the Degradation of the Low Density Lipoprotein Receptor Family Members VLDLR and ApoER2

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.     

Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation

The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.     

A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling

Specialized neurons throughout the developing central nervous system secrete Reelin, which binds to ApoE receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), triggering a signal cascade that guides neurons to their correct position. Binding of Reelin to ApoER2 and VLDLR induces phosphorylation of Dab1, which binds to the intracellular domains of both receptors. Due to differential splicing, several isoforms of ApoER2 differing in their ligand-binding and intracellular domains exist. One isoform harbors four binding repeats plus an adjacent short 13 amino acid insertion containing a furin cleavage site. It is not known whether furin processing of this ApoER2 variant actually takes place and, if so, whether the produced fragment is secreted. Here we demonstrate that cleavage of this ApoER2 variant does indeed take place, and that the resulting receptor fragment consisting of the entire ligand-binding domain is secreted as soluble polypeptide. This receptor fragment inhibits Reelin signaling in primary neurons, indicating that it can act in a dominant-negative fashion in the regulation of Reelin signaling during embryonic brain development.     

Reelin induces EphB activation

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the ''classical'' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.     

Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.

Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.     

Receptor clustering is involved in Reelin signaling

The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.     

The reelin receptor ApoER2 recruits JNK-interacting proteins-1 and -2

Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice.     

Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration

Apolipoprotein E receptor 2 (ApoER2), very low-density lipoprotein receptor (VLDLR), and Dab1 are the main components of the Reelin signalling cascade. Reelin is the sole ligand defined so far in signalling through this pathway. Postnatal migration of neuronal precursors from the subventricular zone (SVZ) to the olfactory bulb (OB), however, depends on ApoER2 and Dab1, but functions independently of Reelin. Here, we show that thrombospondin-1 (THBS-1) is a novel physiological ligand for ApoER2 and VLDLR. THBS-1 is present in the SVZ and along the entire rostral migratory stream (RMS). It binds to ApoER2 and VLDLR and induces phosphorylation of Dab1. In contrast to Reelin, it does not induce Dab1 degradation or Akt phosphorylation, but stabilizes neuronal precursor chains derived from subventricular explants. Lack of THBS-1 results in anatomical abnormalities of the RMS and leads to a reduction of postnatal neuronal precursors entering the OB.     

Differential binding of ligands to the apolipoprotein E receptor 2

Apolipoprotein E receptor 2 (apoER2) is an important participant in the Reelin signaling pathway that directs cell positioning during embryogenesis. ApoER2 is a cell surface molecule that elicits intracellular signal transduction through binding of Reelin. The structural requirements for Reelin binding to apoER2 and the receptor domains involved in this process are unclear at present. Using a series of receptor mutants, we characterized the interaction of apoER2 with Reelin and compared this interaction to that of apoER2 with the receptor-associated protein (RAP), an apoER2 ligand that does not induce signaling. By surface plasmon resonance we demonstrate that apoER2 exhibits 6-fold higher affinity for Reelin than the very low density lipoprotein receptor (VLDLR), which also functions as a Reelin receptor (K(D) 0.2 nM versus K(D) 1.2 nM). Acidic amino acid residues in complement-type repeat domains 1 and 3 of apoER2 are required for Reelin binding. The same regions of the receptor are also bound by RAP with a 25-fold lower affinity (K(D) 5 nM). Whereas RAP binds to apoER2 with a 1:1 stoichiometry, experimental evidence suggests that Reelin associates with two or more receptor molecules simultaneously to achieve high-affinity interaction. This finding indicates that aggregation of apoER2 by multivalent ligands such as Reelin may be the structural basis for signal transduction.     

Reelin binds alpha3beta1 integrin and inhibits neuronal migration

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.     

ApoER2 and VLDLr Are Required for Mediating Reelin Signalling Pathway for Normal Migration and Positioning of Mesencephalic Dopaminergic Neurons

The migration of mesencephalic dopaminergic (mDA) neurons from the subventricular zone to their final positions in the substantia nigra compacta (SNc), ventral tegmental area (VTA), and retrorubral field (RRF) is controlled by signalling from neurotrophic factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM). Reelin and the cytoplasmic adaptor protein Disabled-1 (Dab1) have been shown to play important roles in the migration and positioning of mDA neurons. Mice lacking Reelin and Dab1 both display phenotypes characterised by the failure of nigral mDA neurons to migrate properly. ApoER2 and VLDLr are receptors for Reelin signalling and are therefore part of the same signal transduction pathway as Dab1. Here we describe the roles of ApoER2 and VLDLr in the proper migration and positioning of mDA neurons in mice. Our results demonstrate that VLDLr- and ApoER2-mutant mice have both a reduction in and abnormal positioning of mDA neurons. This phenotype was more pronounced in VLDLr-mutant mice. Moreover, we provide evidence that ApoER2/VLDLr double-knockout mice show a phenotype comparable with the phenotypes observed for Reelin- and Dab1- mutant mice. Taken together, our results demonstrate that the Reelin receptors ApoER2 and VLDLr play essential roles in Reelin-mediated migration and positioning of mDA neurons.     

A mouse homologue of Strawberry Notch is transcriptionally regulated by Reelin signal

Reelin is a glycoprotein secreted by specific neuronal populations of the adult and developing nervous system of vertebrates. The morphological abnormalities in the brain of reeler, the Reelin deficient mutant mice, indicate that Reelin is essential for the brain morphogenesis. However, biochemical function of Reelin signal is not well understood. Here, we examined possible function of Reelin signal in regulation of gene expression by performing a microarray analysis. We found that expression level of a mouse homologue of Strawberry Notch (mSno1) is markedly reduced in the reeler embryos. In situ hybridization showed that mSno1 is expressed in the developing nervous system colocalizing with expression of ApoER2, a Reelin receptor. Treatment of P19 cells with Reelin protein enhanced mSno1 expression. Overexpression of ApoER2 with Reelin treatment gave a synergistic effect on mSno1 expression level. These observations suggest that Reelin signal is involved in embryonic expression of a novel vertebrate gene, mSno1.     

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.     

Divergent roles of ApoER2 and Vldlr in the migration of cortical neurons

Reelin, its lipoprotein receptors [very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (ApoER2; also known as Lrp8)], and the cytoplasmic adaptor protein disabled 1 (Dab1) are important for the correct formation of layers in the cerebral cortex. Reeler mice lacking the reelin protein show altered radial neuronal migration resulting in an inversion of cortical layers. ApoER2 Vldlr double-knockout mutants and Dab1 mutants show a reeler-like phenotype, whereas milder phenotypes are found if only one of the two lipoprotein receptors for reelin is absent. However, the precise role of the individual reelin receptors in neuronal migration remained unclear. In the study reported here, we performed fate mapping of newly generated cortical neurons in single and double receptor mutants using bromodeoxyuridine-labeling and layer-specific markers. We present evidence for divergent roles of the two reelin receptors Vldlr and ApoER2, with Vldlr mediating a stop signal for migrating neurons and ApoER2 being essential for the migration of late generated neocortical neurons.     

Reconstitution of the Reelin signaling pathway in fibroblasts demonstrates that Dab1 phosphorylation is independent of receptor localization in lipid rafts

The Reelin signaling pathway operates in migrating neurons and is indispensable for their correct positioning during embryonic brain development. Many biochemical and cell biological studies to dissect the Reelin pathway at the molecular level are hampered by the lack of a cell line harboring a functional Reelin signaling pathway. Here we present fibroblast cell lines in which all required functional components of the pathway have been reconstituted. These cells react upon Reelin treatment in the same way as primary neurons. We have subsequently used these cell lines to study the subcellular localization of ApoER2 and the VLDL receptor and could demonstrate that receptor-mediated Dab1 phosphorylation does not depend on lipid rafts and that phosphorylated Dab1 remains bound to the receptor tail when the pathway is activated by Reelin.     

Clusterin Is a Ligand for Apolipoprotein E Receptor 2 (ApoER2) and Very Low Density Lipoprotein Receptor (VLDLR) and Signals via the Reelin-signaling Pathway

Clusterin, also known as apolipoprotein J, is a multifunctional glycoprotein with the capacity to interact with a wide range of molecules. Although clusterin has been implicated in a broad spectrum of physiological and pathological processes, such as Alzheimer disease or cancer, its precise functions remain elusive. Here we report, that clusterin binds to apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) and is internalized by cells expressing either one of these receptors. Binding of clusterin to these receptors triggers a Reelin-like signal in cells expressing disabled-1 (Dab1). It induces phosphorylation of Dab1, which leads to activation of PI3K/Akt and n-cofilin. Cell proliferation and neuroblast chain formation in subventricular zone (SVZ) explants are compromised when clusterin, which is present in the subventricular zone, is blocked in vitro. These data suggest that in the subventricular zone where Reelin is not present but ApoER2, VLDLR, and Dab1, clusterin might be involved in maintaining neurogenesis in vivo.     

Localization of apolipoprotein E receptor 2 to caveolae in the plasma membrane

The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.     

Ligand-induced Homotypic and Heterotypic Clustering of Apolipoprotein E Receptor 2

ApoE Receptor 2 (ApoER2) and the very low density lipoprotein receptor (VLDLR) are type I transmembrane proteins belonging to the LDLR family of receptors. They are neuronal proteins found in synaptic compartments that play an important role in neuronal migration during development. ApoER2 and VLDLR bind to extracellular glycoproteins, such as Reelin and F-spondin, which leads to phosphorylation of adaptor proteins and subsequent activation of downstream signaling pathways. It is thought that ApoER2 and VLDLR undergo clustering upon binding to their ligands, but no direct evidence of clustering has been shown. Here we show strong clustering of ApoER2 induced by the dimeric ligands Fc-RAP, F-spondin, and Reelin but relatively weak clustering with the ligand apoE in the absence of lipoproteins. This clustering involves numerous proteins besides ApoER2, including amyloid precursor protein and the synaptic adaptor protein PSD-95. Interestingly, we did not observe strong clustering of ApoER2 with VLDLR. Clustering was modulated by both extracellular and intracellular domains of ApoER2. Together, our data demonstrate that several multivalent ligands for ApoER2 induce clustering in transfected cells and primary neurons and that these complexes included other synaptic molecules, such as APP and PSD-95.     

Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning

Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for Reelin, a signaling protein that regulates neuronal migration during brain development. In the adult brain, Reelin is expressed by GABA-ergic interneurons, suggesting a potential function as a modulator of neurotransmission. ApoE receptors have been indirectly implicated in memory and neurodegenerative disorders because their ligand, apoE, is genetically associated with Alzheimer disease. We have used knockout mice to investigate the role of Reelin and its receptors in cognition and synaptic plasticity. Mice lacking either the VLDL receptor or the apoER2 show contextual fear conditioning deficits. VLDL receptor-deficient mice also have a moderate defect in long term potentiation (LTP), and apoER2 knockouts have a pronounced one. The perfusion of mouse hippocampal slices with Reelin has no effect on baseline synaptic transmission but significantly enhances LTP in area CA1. This Reelin-dependent augmentation of LTP is abolished in VLDL receptor and apoER2 knockout mice. Our results reveal a role for Reelin in controlling synaptic plasticity in the adult brain and suggest that both of its receptors are necessary for Reelin-dependent enhancement of synaptic transmission in the hippocampus. Thus, the impairment of apoE receptor-dependent neuromodulation may contribute to cognitive impairment and synaptic loss in Alzheimer disease.     

Reelin is a ligand for lipoprotein receptors

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.