Specific membrane targeting of Reelin receptors

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 which 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. Phosphorylation of Dab1 is triggered by ligand-induced receptor clustering. Apparently both receptors are able to compensate for the lack of the other in terms of inducing Dab1 phosphorylation. Still, mice lacking either one of the receptors exhibit a subtle phenotype which is specific for the lack of each receptor. Recent studies in ou laboratory have demonstrated distinct targeting of ApoER2 and VLDL receptor to different sub-domains of the plasma membrane. These observations suggest that ApoEr2 and VLDL receptor have distinct functions in the Reelin signaling cascade. Using a novel cell model which allows thorough biochemical and cell biological experiments in respect to the Reelin pathway and using primary neurons from genetically altered mice we have planned to delineate functional differences of both receptors caused by their membrane localization. Special emphasis will be given to receptor-mediated Reelin endocytosis, receptor trafficking, and ligand induced Receptor degradation. Results of this project are expected to generate a significant step forward in understanding key aspects of the Reelin signaling pathway at the cellular level.

ApoER2 and VLDL receptor work in the central nervous system as Reelin receptors and are an integral part of the Reelin signaling pathway. During development of the cortex newly generated neurons migrate from the subventricular zone along glial fibers to their final position thus establishing a well defined laminated structure of the cortex. Correct positioning of these neurons in distinct neuronal layers is orchestrated by the Reelin signaling pathway. Reelin binding to ApoER2 and VLDL receptor leads to phosphorylation of Dab1 and subsequently to the ultimate cell responses required for the correct positioning of newly generated neurons. At the beginning of the granting period we focused on experiments which originated from a previous project where we discovered that ApoER2 and VLDLR must have a Reelin-independent function in the migration of postnatal neurons into the olfactory bulb. In rodents development of the olfactory bulb continues after birth. Most interneurons differentiate from neuroblasts generated postnatally in the subventricular zone of the cerebral cortex and migrate to the olfactory bulb along the so-called "rostral migratory stream". Neuronal migration in the RMS occurs by formation of chains that are ensheathed by glial cells and their processes. We could demonstrate that the lack of ApoER2, VLDL receptor, or Dab1 leads to a cell-autonomous migration defect in the RMS, which is independent of the Reelin signal. We discovered that in this situation thrombospondin-1 acts as ligand for ApoER2 and VLDLR and this interaction is involved in maintaining the architecture of the RMS and in replenishing the olfactory bulb with new interneurons. In the second part of the project we worked out in detail differences between ApoER2-mediated and VLDLR- mediated Reelin signaling. Despite the common ability to promote Dab-1 phosphorylation, ApoER2 and VLDLR have specific distinct functions, as corroborated by analyses of the subtle phenotypes displayed in mice lacking either ApoER2 or VLDL receptor. Using a panel of chimeric receptors, we could demonstrate that endocytosis of Reelin and the fate of the individual receptors upon stimulation are linked to their specific sorting to raft versus non-raft domains of the plasma membrane. VLDL receptor residing in the non-raft domain endocytoses and destines Reelin for degradation. Binding of Reelin to ApoER2 a resident of rafts leads to the production of specific receptor fragments with specific functions on their own. These features contribute to a receptor-specific fine-tuning of the Reelin signal, leading to a novel model which emphasizes negative feedback loops specifically mediated by ApoER2 and VLDL receptor, respectively.