EXPRESSION OF HUMAN P140(TRK) RECEPTORS IN P140(TRK)-DEFICIENT, PC12 ENDOTHELIAL CELLS RESULTS IN NERVE GROWTH FACTOR-INDUCED SIGNAL-TRANSDUCTION AND DNA-SYNTHESIS/

Nerve growth factor (NGF) regulates proliferation, differentiation, an d survival of sympathetic and sensory neurons through the tyrosine kin ase activity of its receptor, p140(trk). These biological effects of N GF depend upon the signal-mediating function of pi 40(trk) substrates which are likely to differ from cell to cell. To define pi 40(trk) rec eptor substrates and the details of signalling by NGF in the hybrid ce ll PC12EN, we stably transfected cultures with a vector encoding a ful l-length human pi 40(trk) cDNA sequence. Two stably transfected clones , one expressing p140(trk) with higher affinity (PC12EN-trk3; K-d 57.4 pM, B-max 9.7 pmole/mg) and one expressing p140(trk) with a lower aff inity (PC12 EN-trk1; K-d 392.4 pM, B-max 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140(trk) receptors sh ow 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) re ceptors. NGF stimulates p140(trk) tyrosine phosphorylation in a dose-( 0.01-10 ng/ml) and time-(5-120 min) dependent manner, and tyrosine pho sphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk sti mulation for 60 min was assessed using myelin basic protein as a subst rate. NGF treatment also led to an increased phosphorylation of p70(S6 k), SNT, and phospholipase C gamma, demonstrating that the major NGF-s timulated signalling pathways found in other cells are activated in PC 12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP(1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-tr k cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- a nd 2.3-fold increase in DNA synthesis measured by [H-3]thymidine incor poration in PC12EN-trk1 and PC12EN-trk3, respectively. These data high light the functionality of the transfected p140(trk) receptors and ind icate 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. (C) 199 7 Wiley-Liss, Inc.