Characterization of surface expression levels and PTPσ-binding properties of TrkC point mutants used in the current study. A, Schematic depiction of the major intracellular signaling pathways downstream of TrkC. Tyrosine 516 (Y516) is located in the activation loop of the TrkC tyrosine kinase domain, which forms a PTB-binding motif (NPXpY) known to bind to Shc and FRS2 adaptor proteins. Tyrosine 820 (Y820) binds to PLC-γ1. Mutation of aspartate (D) to asparagine (N) at residue 679 abolishes the kinase activity of TrkC. Mutation of D240 and D242 to alanines eliminates binding to PTPσ, whereas mutation of N366 and T369 to alanines abolishes binding to NT-3. B, HEK293T cells expressing HA-tagged TrkC WT or its various point mutants were immunostained with mouse anti-HA antibodies (green) and detected with FITC-conjugated anti-mouse secondary antibodies under nonpermeabilized conditions, followed by permeabilization of cells. Cells were stained with rabbit anti-HA antibodies (red), followed by Cy3-conjugated anti-rabbit secondary antibodies. Scale bar, 10 μm (applies to all images). C, Representative images of cell-surface-binding assays. HEK293T cells expressing HA-tagged TrkC WT or its point mutants were incubated with 10 μg/ml control IgC or Ig-PTPσ and then analyzed by immunofluorescence imaging of Ig-fusion proteins (red) and HA antibodies (green). Scale bar, 10 μm (applies to all images).