Figure 6: ARHGAP33 promotes the interaction between TrkB and SORT1.
(a) ARHGAP33 formed complexes with SORT1 and TrkB. ARHGAP33-immunoprecipitates (left) and hippocampal total lysates (right) were immunoblotted with the indicated antibodies. (b–d) ARHGAP33 formed complexes with SORT1 and TrkB. HEK293T cells were transfected with ARHGAP33, SORT1 and TrkB, as indicated. Immunoprecipitates were immunoblotted with the indicated antibodies. (e) Weakened interaction between TrkB and SORT1 in ARHGAP33 KO mice. SORT1 immunoprecipitates were immunoblotted with the indicated antibodies. Representative blots (left) and quantification of co-immunoprecipitated TrkB (right; each n=9; P=3.4 × 10−4, Mann–Whitney U-test). *P<0.05. The level of co-precipitated TrkB in ARHGAP33 KO mice was normalized to that in WT mice (the averaged WT value was set to 100%). Bars show median values. Western blots show representative results from nine independent experiments performed using different mice. (f) Weakened interaction between ARHGAP33 and TrkB in the SORT1 knockdown neuron. ARHGAP33-immunoprecipitates and total lysates were immunoblotted with the indicated antibodies. Representative blots (left), quantification of co-immunoprecipitated TrkB (centre) and quantification of SORT1 expression (confirmation of SORT1 knockdown; right; each n=8; TrkB, P=7.5 × 10−4, SORT1, P=7.7 × 10−4, Mann–Whitney U-test). The averaged values of the control neurons were set to 100%. *P<0.05. Bars show median values. Western blots show representative results from eight independent experiments performed using neurons from different mice; cont., control; KD, knockdown. Note that the MISSION shRNA construct (TRCN0000034496) was used. (g) In the ARHGAP33/SORT1/TrkB complex, ARHGAP33 promoted the interaction between TrkB and SORT1. In addition, ARHGAP33 is suggested to recruit Cdc42 into the complex7. (h) Colocalization of ARHGAP33, SORT1 and TrkB at the perinuclear region. Triple immunostaining for ARHGAP33, SORT1 and TrkB in dissociated hippocampal neurons. Scale bar, 5 μm. The asterisks indicate the nucleus of the neuron. The arrowhead indicates the colocalization of these three proteins at the perinuclear region. The data are representative of five independent experiments. (i) Interactions between ARHGAP33 and Rab11 or Rab27 were not detected in the hippocampal lysates. ARHGAP33-immunoprecipitates (right) and hippocampal total lysates (left) were immunoblotted with the indicated antibodies.
