Figure 4.

Deletion of the KFG domain from TrkA alters NGF-induced trafficking of TrkA. A, HEK293T cells stably expressing WT or KFG deleted TrkA as in Figure 3 were treated with 100 ng/ml NGF for the indicated time (1–6 h) followed by biotinylation. Cell lysates were immunoprecipitated (IP) with Streptavidin beads (Strept) and blotted with an anti-TrkA and actin (used as a negative control) antibodies. Note the increased levels of TrkAΔKFG suggesting reduced degradation and/or internalization of the mutant receptors. B, Recycling of TrkA receptors was analyzed by cleavable biotinylation assay as described in Materials and Methods and immunoblotted as in A. Note the higher level of mutant TrkAΔKFG receptors following internalization suggesting increased recycling. Input lysates in A and B were blotted with anti-TrkA, phospho-Erk (as a control of NGF activity), and GAPDH (as a loading control) antibodies. C, Quantification of surface TrkA levels in A was performed by determining the band intensity of the different biotinylated TrkA samples relative to the non-NGF-treated WT sample. D, Quantification of biotinylated TrkA levels in IP samples in B was determined by comparing the GAPDH-normalized band intensities of the IP TrkA samples at various time points relative to the value of the TrkA WT sample treated with NGF for 5 min. All values are the mean ± SEM from three independent experiments. Statistical significance was calculated with the Student's t test using GraphPad Prism; *p < 0.05.