Figure 2

Reelin binds to the extracellular domain of EphB proteins. (A) Supernatant of Reelin-expressing HEK-293 cells was incubated with the ectodomains of different transmembrane receptors. The Fc-fused ectodomains were precipitated with protein A/G-agarose beads. Reelin binds to the ectodomains of the lipoprotein receptors ApoER2 and VLDLR (positive controls), and to the ectodomain of EphB2. The ectodomain of platelet-derived growth factor receptor-β (Fc-PDGFRβ) and protein A/G-agarose beads without Fc-coupled ectodomain (control) served as negative controls. (B) Biochemical interaction of Reelin with members of the EphB and ephrin B gene families. Recombinant Reelin was incubated with the ectodomains of ephrin A5, ephrin B1 and ephrin B3, EphB1, EphB2 and EphB3, and of PDGFRβ as a negative control. The Fc-fused ectodomains were precipitated with protein A/G-beads, and bound Reelin was detected by immunoblotting. Fc-ApoER2 served as a positive control. Reelin was bound by the ectodomains of EphB1-3. Moderate binding was also observed for Fc-ephrin B3. (C) GST-RAP (30 μg/ml) and the calcium chelator EDTA (30 mmol/l) blocked binding of Reelin to its lipoprotein receptors, shown here for ApoER2. (D) The EphB2-Reelin interaction was not blocked by GST-RAP or EDTA.