EXPRESSION OF HUMAN P140(TRK) RECEPTORS IN P140(TRK)-DEFICIENT, PC12 ENDOTHELIAL CELLS RESULTS IN NERVE GROWTH FACTOR-INDUCED SIGNAL-TRANSDUCTION AND DNA-SYNTHESIS/
H. Jiang et al., EXPRESSION OF HUMAN P140(TRK) RECEPTORS IN P140(TRK)-DEFICIENT, PC12 ENDOTHELIAL CELLS RESULTS IN NERVE GROWTH FACTOR-INDUCED SIGNAL-TRANSDUCTION AND DNA-SYNTHESIS/, Journal of cellular biochemistry, 66(2), 1997, pp. 229-244
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.