Deoxygedunin binds TrkB ECD and elicits its dimerization.

(A) [³H]deoxygedunin binds the ECD but not ICD of TrkB receptor. In vitro binding assay with purified TrkB ECD or ICD (10 µg) and [³H]deoxygedunin (upper panel). In vitro binding assay with purified fragments of TrkB ECD (10 µg) and [³H]deoxygedunin. (B) Determination of binding constant. Binding curve (upper panel). Scatchard plot analysis revealed that deoxygedunin binds TrkB with binding constant of 1.4 µM (lower panel). (C) Deoxygedunin provokes TrkB dimerization. mGST-TrkB and HA-TrkB were cotransfected into HEK293 cells, treated with 0.5 µM various gedunins for 30 min. GST-TrkB was pulled down by glutathione beads, the coprecipitated proteins were analyzed with anti-HA HRP antibody. (D) Deoxygedunin induces TrkB autophosphorylation. HEK 293 cells were transfected with various Trk receptors, followed by deoxygedunin treatment for 30 min. Cell lysates were analyzed by immunoblotting assay.