Figure 4.

p75NTR inhibits ligand-induced tyrosine kinase (Trk) receptor ubiquitination. (A) Human embryonic kidney 293 cells were transfected with expression plasmids encoding TrkA and p75 neurotrophin receptor (p75NTR), and after 2 days were exposed to nerve growth factor (NGF; 50 ng/ml) at 37°C for 2–15 min, as indicated. Cellsurface proteins were then biotinylated at 4°C, biotinylated proteins were precipitated from lysed cells using streptavidin beads and levels of TrkA and the TrkA phosphotyrosine (pTyr) content were determined by immunoblotting. Lysate levels of TrkA and p75NTR are shown in the lower panels. SA PD, streptavidin pullot. (B) The PC12^p75kd line and the control PC12 line were biotinylated at 4°C, exposed to 50 ng/ml NGF and then incubated at 37°C for the durations indicated. Biotinylated proteins were recovered from lysed cells using streptavidin-coated beads and levels of TrkA in the precipitates were determined by immunoblotting using RTA. Lysate levels of p75NTR and Erk are shown in the lower panels. GFP, green fluorescent protein; RNAi, RNA interference. (C) Quantification of data shown in (B) from three separate experiments determined by densitometry, pooled and analysed for significance by one-way analysis of variance. ^*P<0.05. The experiment in (A) was performed three times, all with identical results.