Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140(trk). These biological effects of NGF depend upon the signal-mediating function of p140(trk) substrates which are likely to differ from cell to cell. To define p140(trk) receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140(trk) DNA sequence. Two stably transfected clones, one expressing p140(trk) with higher affinity (PC12EN-trk3; Kd 57.4 pM, B(max) 9.7 pmole/mg) and one expressing p140(trk) with a lower affinity (PC12EN-trk1; K 392.4 pM, B(max) 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140(trk) receptors show slow NGF dissociation kinetics, are resistant to trypsin or Triton X- 100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140(trk) receptors. NGF stimulates p140(trk) tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70(s6k), SNT, and pbospholipase Cγ, demonstrating that the major NGF stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [¹H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140(trk) receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140(trk) receptor substrates.