FIGURE 3.

Juxtamembrane region in TrkB is responsible for its interaction with LIMK1. A, schematic presentation of FLAG-TrkB and its deletion constructs. ΔCT indicates deletion of the C-terminal region of TrkB; ΔTK indicates deletion of both tyrosine kinase and the C-terminal regions of TrkB; JM0 indicates deletion of the whole intracellular region of TrkB. B and C, lack of TrkB JM region disrupts TrkB/LIMK1 interaction. Lysates of HEK293 cells transfected with FLAG-TrkB deletion mutants and HA-LIMK1 were immunoprecipitated (IP) with rabbit anti-HA antibody, followed by immunoblotting (IB) with mouse anti-FLAG or anti-HA antibodies, respectively. D, schematic representation of the deletion mutants of TrkB constructs containing juxtamembrane domain. EC, extracellular; TM, transmembrane. E and F, 9 amino acids between JM3 and JM4 (JMBox4) are required for TrkB/LIMK1 interaction. HEK293 cells were transiently transfected with the indicated constructs. Protein extracts were immunoprecipitated with rabbit anti-FLAG antibodies and immunoblotted with mouse anti-FLAG or mouse anti-HA antibodies. G, JMBox4 is sufficient for the binding between TrkB and LIMK1. Chimera FLAG-T1-JMBox4 was generated by PCR. HEK293 cells were transfected with the indicated constructs. Protein extracts were immunoprecipitated by rabbit anti-FLAG antibodies and immunoblotted with mouse anti-FLAG or mouse anti-HA antibodies. H, all three Trk receptors can associate with LIMK1. Lysates from HEK293 cells co-transfected with the indicated constructs were immunoprecipitated with rabbit anti-FLAG antibody, followed by immunoblotting with mouse anti-HA and mouse anti-FLAG antibodies. I, multiple sequence alignment on the JMBox4 region of three Trk receptors is performed by Clustal Omega.