Biomedical subjects
David Benhayon
Publications and source records attributed to David Benhayon.
Components of the reelin signaling pathway are expressed in the spinal cord.
The Reelin signaling pathway in the brain involves the binding of Reelin to very-low-density lipoprotein receptors (VLDLR) and apolipoprotein E receptor 2 (ApoER2). After Reelin binds the lipoprotein receptors on migrating neurons, the intracellular adaptor protein Disabled-1 (Dab1) becomes phosphorylated, ultimately resulting in the proper positioning of cortical neurons. Previous work showed that Reelin also affects the positioning of sympathetic preganglionic neurons (SPN) in the spinal cord (Yip et al. [2000] Proc Natl Acad Sci USA 97:8612-8616). We asked in the present study whether components of the Reelin signaling pathway in the brain also function to control SPN migration in developing spinal cord. Results showed that Reelin and reelin mRNA are found adjacent to migrating SPN. In addition, dab1 mRNA and protein are expressed by migrating SPN, and dab1-null mice show abnormal SPN migration similar to that seen in reeler. Finally, vldlr and apoER2 are also expressed in migrating SPN, and mice lacking both vldlr and apoER2 show aberrant SPN location that is identical to that of reeler and dab1-null mice. Because molecules known to be involved in Reelin signaling in the brain are present in the developing spinal cord, it is likely that the Reelin signaling pathways in the brain and spinal cord function similarly. The relative simplicity of the organization of the spinal cord makes it a potentially useful model system with which to study the molecular and cellular function of the Reelin signaling pathway in control of neuronal migration.
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.
RETRACTED: Reelin promotes peripheral synapse elimination and maturation.
Reelin is an extracellular protein that is crucial for layer formation in the embryonic brain. Here, we demonstrate that Reelin functions postnatally to regulate the development of the neuromuscular junction. Reelin is required for motor end-plate maturation and proper nerve-muscle connectivity, and it directly promotes synapse elimination. Unlike layer formation, neuromuscular junction development requires a function of Reelin that is not mediated by Disabled1 or very-low-density lipoprotein receptors and apolipoprotein E receptor 2 receptors but by a distinct mechanism involving its protease activity.
Binding of purified Reelin to ApoER2 and VLDLR mediates tyrosine phosphorylation of Disabled-1.
Reelin, Disabled-1 (Dab1), apolipoprotein E receptor 2 (ApoER2), and very low density lipoprotein receptor (VLDLR) participate in a signaling pathway required for layer formation during mammalian brain development. Binding of Reelin to ApoER2 and VLDLR induces a rapid increase in tyrosine phosphorylation of Dab1, an adaptor protein that associates with the cytoplasmic domain of the receptors. However, Reelin has also been proposed to signal through integrin and protocadherin. Here we compare the roles of ApoER2 and VLDLR in Reelin signaling. We used layer-specific markers to identify the final positions of early- and late-born neurons in the cortices of mice lacking ApoER2, VLDLR, or both ApoER2 and VLDLR. Subtle alterations were observed in mice lacking VLDLR, whereas more severe abnormalities were detected in the absence of ApoER2, and major disruptions were obvious in mice lacking both receptors. Purified Reelin associated more readily with ApoER2 than with VLDLR and no synergy was observed in the presence of both receptors. Consistent with the binding data, the level of Reelin-induced Dab1 phosphorylation was more severely reduced in neurons lacking ApoER2 than in neurons lacking VLDLR. However, similarly low levels of Dab1 tyrosine phosphorylation were observed in ApoER2(-/-) and VLDLR(-/-) mice in vivo. Finally, there was a complete absence of Reelin-induced tyrosine phosphorylation of Dab1 in cortical neurons from mice lacking both ApoER2 and VLDLR. These findings demonstrate that ApoER2 and VLDLR are essential for Reelin signaling and that no other receptor molecules can compensate for their role in mediating tyrosine phosphorylation of Dab1.
Cyclin-dependent kinase 5 phosphorylates disabled 1 independently of Reelin signaling.
Two major signaling pathways that control neuronal positioning during brain development have been uncovered as a result of genetic and biochemical studies on neurological mouse mutants. Mice deficient in Reelin, Disabled 1 (Dab1), or both the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2) exhibit identical neuroanatomic defects in laminar structures throughout the brain. These proteins function as components of the Reelin signaling pathway. Reelin is a secreted glycoprotein that binds to VLDLR and ApoER2, inducing tyrosine phosphorylation of Dab1, an intracellular adapter protein. Neuronal migration is also regulated by cyclin-dependent kinase 5 (Cdk5) and its activating subunits p35 and p39. Mice deficient in Cdk5, p35, or both p35 and p39 exhibit lamination defects that are similar but not identical to those observed in mice with a defect in the Reelin signaling pathway. Cdk5 phosphorylates proteins that maintain cytoskeletal structures and promote cell motility. To explore the possibility that Cdk5 influences the Reelin pathway, we sought to determine whether Dab1 is a substrate for Cdk5. Here we show that Cdk5 phosphorylates Dab1 on serine 491 in vitro and in vivo, independently of Reelin signaling. We also show that ectopic neurons in Cdk5-deficient mice exhibit reduced levels of Reelin signaling during later stages of cortical development, although Cdk5 is not required for Reelin-induced tyrosine phosphorylation of Dab1. Although the functional significance of Dab1 serine phosphorylation is unclear, our results suggest that there is biochemical cross-talk between two signaling pathways that control cell positioning.