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.Correct positioning of neurons of the cortical plate depends on Reelin, an extracellular matrix protein produced by CajalRetzius cells (10), on the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) (35), and on the intracellular adaptor protein disabled 1 (Dab1) (30). Mutations in the corresponding genes, i.e., the Reelin gene (as in the reeler mouse) (12) and the Dab1 gene (as in the scrambler and yotari mice) (16,32,37), and deletions of the genes for both ApoER2 and VLDLR (35) result in identical cortical layering defects, suggesting that the gene products are part of the same signaling pathway. The current working model proposes that Reelin binds to ApoER2 and VLDLR (11,14). Subsequent phosphorylation of Dab1 is a key event leading to the ultimate cell responses required for correct positioning of newly generated neurons (17, 18). Dab1 was originally identified as an interaction partner of Src (15) and contains a phosphotyrosine binding domain which interacts with the unphosphorylated NPXY motif present in the cytoplasmic domains of ApoER2 and VLDLR (19,34). Phosphorylation of Dab1 induced by Reelin is dependent on the presence of ApoER2 and VLDLR (5) and occurs on Tyr198 and Tyr220 (20). Recent studies demonstrated that members of the Src family of nonreceptor tyrosine kinases (SFKs) are involved in Dab1 phosphorylation in neurons (2, 6). Coreceptors, such as members of the family of cadherin-related neuronal receptors (CNRs), have been proposed to be involved in this pathway (31). Neuronal migration is also regulated by cyclin-dependent kinase 5 (27, 28), but whether t...
Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to speci®c membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane traf®cking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and de®ne a novel mechanism for modulation of their endocytic activity.
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.
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. IntroductionThe Reelin signalling pathway is indispensable for the development of laminated structures in the brain (Tissir and Goffinet, 2003). The best studied role of Reelin is its function in radial neuronal migration during development of the neocortex and positioning of granule cells in the hippocampus (Forster et al, 2006). Key players of the Reelin signalling pathway are apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR) that relay the Reelin signal into radially migrating neurons. The molecular basis of this signalling cascade was recently summarized (Herz and Chen, 2006) and involves binding of oligomeric Reelin to ApoER2 and/or VLDLR, subsequent activation of Src family kinases, and phosphorylation of Disabled 1 (Dab1).Reelin also has an important function in the integration of neuronal precursors into the olfactory bulb (OB) during postnatal development of the olfactory system in rodents. Neuronal precursors within the subventricular zone (SVZ) migrate tangentially through the rostral migratory stream (RMS) towards the OB forming chains ensheathed by glial cells (Lois et al, 1996). Reelin produced by mitral cells in the OB promotes the detachment of neuronal precursors from the incoming chains so as to allow proper radial migration of the precursors within the bulb (Hack et al, 2002). As recently discovered in our laboratory, however, ApoER2 and Dab1 within the RMS function independently of Reelin in postnatal tangential neuronal migration (Andrade et al, 2007). In mice lacking either ApoER2 or Dab1, chain formation is severely compromised, and neuronal precursors accumulate in the SVZ unable to migrate into the OB and thereby failing to form an RMS. As Reelin-producing cells are absent from both the SVZ and the RMS, it seems likely that ligands other than Reelin interact with ApoER2. The following observations prompted us to consider thrombospondin-1 (THBS-1) as such a ligand. THBSs are widely expressed in the developing central nervous system (O'Shea and Dixit, 1988;O'Shea et al, 1990). Cell culture experiments demonstrated...
Binding of low density lipoprotein (LDL) to platelets enhances platelet responsiveness to various aggregation-inducing agents. However, the identity of the platelet surface receptor for LDL is unknown. We have previously reported that binding of the LDL component apolipoprotein B100 to platelets induces rapid phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK ). Here, we show that LDL-dependent activation of this kinase is inhibited by receptor-associated protein (RAP), an inhibitor of members of the LDL receptor family. Confocal microscopy revealed a high degree of co-localization of LDL and a splice variant of the LDL receptor family member apolipoprotein E receptor-2 (apoER2) at the platelet surface, suggesting that apoER2 may contribute to LDL-induced platelet signaling. Indeed, LDL was unable to induce p38 MAPK activation in platelets of apoER2-deficient mice. Furthermore, LDL bound efficiently to soluble apoER2, and the transient LDL-induced activation of p38 MAPK was mimicked by an anti-apoER2 antibody. Association of LDL to platelets resulted in tyrosine phosphorylation of apoER2, a process that was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated but not native apoER2 co-precipitated with the Src family member Fgr. This suggests that exposure of platelets to LDL induces association of apoER2 to Fgr, a kinase that is able to activate p38 MAPK . In conclusion, our data indicate that apoER2 contributes to LDL-dependent sensitization of platelets. Platelets and low density lipoproteins (LDL)1 are key elements in the development of atherothrombotic complications.The interplay between both elements is apparent from the notion that LDL particles markedly enhance the responsiveness of platelets to various aggregation-inducing agents (1-4). These agonists mediate the release of growth factors, vasoactive substances, and chemotactic agents that are known to stimulate atherosclerotic plaque formation. Sensitization of platelets by LDL involves the major LDL constituent apolipoprotein B100 (apoB100) (5), a 4563 amino acid protein that is wrapped around the LDL particle (6). LDL particles are recognized by the classical hepatic LDL receptor (LDL-R) through the apoB100 moiety, and in particular through a region within the apoB100 protein that is enriched in positively charged amino acids, the so-called B-site (7). Like LDL, a synthetic peptide mimicking this B-site associates to the platelet surface (5). Moreover, this peptide interferes with binding of LDL to platelets (5), suggesting that both elements share similar binding sites. This possibility is supported by the observation that binding of either LDL or the B-site peptide to the platelet results in a near immediate activation of the intracellular enzyme p38 mitogen-activated protein kinase (p38 MAPK ) (5, 8). Activation of this Ser/Thr kinase is associated with downstream phosphorylation and activation of cytosolic phospholipase A 2 , which leads to the formation of thromboxane A 2 (9, 10). Fi...
Transport, biological action, and clearance of leptin are subject to modulation by plasma components responsible for the formation of the so-called "bound" fraction of serum leptin. Candidates for modulators have been identified previously, but mechanisms for their action, and thus their physiological roles, have remained unclear. Here we have obtained evidence for a role of serum-borne clusterin in leptin biology and have delineated a possible mechanism for its action. We demonstrate complex formation between clusterin and leptin by several approaches and show that the binary complex retains the ability to transduce the leptin signal via binding to the leptin receptor and activation of the Janus kinase/signal transducer and activator of transcription pathway. The interaction of leptin with clusterin does not require additional serum components. Furthermore, and importantly for modulation of the bioactivity of leptin, uptake of leptin present in the complex can be mediated by members of the low density lipoprotein (LDL) receptor family, i.e., apolipoprotein receptor type-2 and the very LDL receptor, which here are shown to efficiently endocytose both free and leptin-associated clusterin. Thus, bioavailability of leptin at a given tissue site may be determined by the levels of clusterin and/or by the relative distribution of certain relatives of the LDL receptor vis-à-vis active leptin receptors.
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.
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