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.     

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.     

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.     

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.     

The Pafah1b complex interacts with the reelin receptor VLDLR

Reelin is an extracellular protein that directs the organization of cortical structures of the brain through the activation of two receptors, the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2), and the phosphorylation of Disabled-1 (Dab1). Lis1, the product of the Pafah1b1 gene, is a component of the brain platelet-activating factor acetylhydrolase 1b (Pafah1b) complex, and binds to phosphorylated Dab1 in response to Reelin. Here we investigated the involvement of the whole Pafah1b complex in Reelin signaling and cortical layer formation and found that catalytic subunits of the Pafah1b complex, Pafah1b2 and Pafah1b3, specifically bind to the NPxYL sequence of VLDLR, but not to ApoER2. Compound Pafah1b1(+/-);Apoer2(-/-) mutant mice exhibit a reeler-like phenotype in the forebrain consisting of the inversion of cortical layers and hippocampal disorganization, whereas double Pafah1b1(+/-);Vldlr(-/-) mutants do not. These results suggest that a cross-talk between the Pafah1b complex and Reelin occurs downstream of the VLDLR receptor.     

Signaling through Disabled 1 requires phosphoinositide binding

The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled 1 (Dab1) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1.     

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.     

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.     

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.     

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.     

Origins of peptide selectivity and phosphoinositide binding revealed by structures of disabled-1 PTB domain complexes

Formation of the mammalian six-layered neocortex depends on a signaling pathway that involves Reelin, the very low-density lipoprotein receptor, the apolipoprotein E receptor-2 (ApoER2), and the adaptor protein Disabled-1 (Dab1). The 1.5 A crystal structure of a complex between the Dab1 phosphotyrosine binding (PTB) domain and a 14-residue peptide from the ApoER2 tail explains the unusual preference of Dab1 for unphosphorylated tyrosine within the NPxY motif of the peptide. Crystals of the complex soaked with the phosphoinositide PI-4,5P(2) (PI) show that PI binds to conserved basic residues on the PTB domain opposite the peptide binding groove. This finding explains how the Dab1 PTB domain can simultaneously bind PI and the ApoER2 tail. Recruitment of the Dab1 PTB domain to PI-rich regions of the plasma membrane may facilitate association with the Reelin receptor cytoplasmic tails to transduce a critical positional cue to migrating neurons.     

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.     

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.     

Reelin activates SRC family tyrosine kinases in neurons

BACKGROUND: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown. RESULTS: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs. CONCLUSIONS: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.     

The Reelin signaling pathway in mouse cortical development

Most of the cerebral cortex derives from the cortical plate which, in all mammals, is radially organized and develops from inside to outside. Several genes involved in the organization and inside-outside development of the embryonic cortical plate in the mouse form the so-called Reelin signaling pathway. Biochemical and genetic arguments show that the extracellular matrix protein Reelin binds to two lipoprotein receptors (VLDLR and ApoER2), which relay the Reelin signal inside target neurons by docking the tyrosine kinase adapter disabled-1 (Dab1). In addition, biochemical evidence suggests that the integrins alpha 3/beta 1 and protocadherins of the CNR family may also modulate the Reelin signal. The mechanisms by which the presence of Reelin stops migration and instructs the radial organization of cortical plate cells remains unknown.     

Reelin Together with ApoER2 Regulates Interneuron Migration in the Olfactory Bulb

One pathway regulating the migration of neurons during development of the mammalian cortex involves the extracellular matrix protein Reelin. Reelin and components of its signaling cascade, the lipoprotein receptors ApoER2 and Vldlr and the intracellular adapter protein Dab1 are pivotal for a correct layer formation during corticogenesis. The olfactory bulb (OB) as a phylogenetically old cortical region is known to be a prominent site of Reelin expression. Although some aspects of Reelin function in the OB have been described, the influence of Reelin on OB layer formation has so far been poorly analyzed. Here we studied animals deficient for either Reelin, Vldlr, ApoER2 or Dab1 as well as double-null mutants. We performed organotypic migration assays, immunohistochemical marker analysis and BrdU incorporation studies to elucidate roles for the different components of the Reelin signaling cascade in OB neuroblast migration and layer formation. We identified ApoER2 as being the main receptor responsible for Reelin mediated detachment of neuroblasts and correct migration of early generated interneurons within the OB, a prerequisite for correct OB lamination.     

Up-regulation of ATP binding cassette transporter A1 expression by very low density lipoprotein receptor and apolipoprotein E receptor 2

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.     

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.     

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.     

Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway

Reelin is a secreted glycoprotein that regulates neuronal positioning in cortical brain structures through the VLDLR and ApoER2 receptors and the adaptor protein Dab1. In addition to cellular disorganization, dendrite abnormalities are present in the brain of reeler mice lacking Reelin. It is unclear whether these defects are due primarily to cellular ectopia or the absence of Reelin. Here we examined dendrite development in the hippocampus of normal and mutant mice and in dissociated cultures. We found that dendrite complexity is severely reduced in homozygous mice deficient in Reelin signaling both in vivo and in vitro, and it is also reduced in heterozygous mice in the absence of cellular ectopia. Addition of Reelin interfering antibodies, receptor antagonists, and Dab1 phosphorylation inhibitors prevented dendrite outgrowth from normal neurons, whereas addition of recombinant Reelin rescued the deficit in reeler cultures. Thus, the same signaling pathway controls both neuronal migration and dendrite maturation.