Figure 5
Reelin induces proteolytic processing of EphB2. (A) Treatment with Reelin induces decrease of full-length EphB2 in transfected HEK-293 cells. HEK-293 cells were transiently transfected with a plasmid encoding full-length EphB2 and treated with preclustered soluble ephrin B1 or Reelin for 4 h. Cell lysates were analyzed by western blotting. Treatment with both preclustered ephrin B1 or Reelin led to a significant decrease of full-length EphB2 protein levels. Actin served as a loading control. (B, C) Decrease of EphB2 levels in cortical neurons after stimulation with Reelin (B) or Fc-ephrin B1 (C). Cortical neurons (E15.5, DIV5) were treated for the indicated times, lysed in RIPA buffer and analyzed by western blotting for EphB2 protein levels. Treatment with Reelin induced a moderate decrease in the levels of full-length EphB2 after 2-8 h. Actin served as a loading control. (D) Quantification of the EphB2 signal intensity after treatment with Reelin (white bars) or Fc-ephrin B1 (grey bars) is shown (S.D., *P < 0.05, **P < 0.01 for Reelin and #P < 0.005 for Fc-ephrin B1 as compared to controls, n = 3). (E) Quantification of relative Ephb2 mRNA levels in primary neurons treated for 6 h with recombinant Reelin (n = 3) as determined by qRT-PCR. n.s., non-significant. (F) Cortical neurons were preincubated with bafilomycin A1 or lactacystin for 30 min and then treated with Reelin or preclustered Fc-ephrin B1 for 7.5 h. The endosomal inhibitor bafilomycin prevented degradation of the full-length form of EphB2, whereas the proteasomal inhibitor lactacystin had no effect. (G) EphB2-transfected HEK-293 cells were preincubated with lactacytin or lactacystin + DAPT, a γ-secretase inhibitor, and then treated with Reelin or preclustered Fc-ephrin B1 for 8 h. Inhibition of the proteasome led to an increase of a carboxy-terminal fragment of EphB2 (CTF2), whereas pretreatment with lactacystin and DAPT leads to the accumulation of CTF1, a slightly larger carboxy-terminal fragment of EphB2 that serves as a substrate for the γ-secretase complex. (H) Cortical neurons were pretreated with GST or GST-RAP (30 μg/ml), then stimulated with Reelin for the indicated times, lysed in RIPA buffer and analyzed by western blotting for EphB2 protein levels. GST-RAP did not prevent the Reelin-induced EphB2 degradation. Actin served as a loading control. One experiment representative of 3 is shown. (I) Cortical neurons were prepared from embryos lacking both VLDLR and ApoER2 (VR−/−;ER2−/−), stimulated with Reelin for the indicated times and analyzed as described above. Shown is one out of two independent experiments.
