Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis
✍ Scribed by Hao Jiang; Vilen Movsesyan; Donald W. Fink; Jr.; Monika Fasler; Michael Whalin; Yasuhiro Katagiri; Mariam Monshipouri; Geneva Dickens; Peter I. Lelkes; Gordon Guroff; Philip Lazarovici
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 386 KB
- Volume
- 66
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
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 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 affinity (PC12EN-trk1; K d 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 phospholipase Cg, 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 [ 3 H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data Abbreviations used: BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulfoxide; EDTA, ethylenediamine N, N, N8, N8-tetraacetic acid; EGF, epidermal growth factor; EGTA, ethylene glycol-bis (baminoethyl ether) N,N,N8,N8-tetraacetic acid; Erk, extracellular signal-regulated kinase; G418, geneticin; HEPES, N-2-hydroxyethyl piperazine-N8-2-ethanesulfonic acid; K-252a, 8R*, 9S*, 11S*-(2)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2, 7b, 11a-triazadibenzo (a, g) cyclo-octa (cde) trindene-1-one; MBP, myelin basic protein; NGF, nerve growth factor; NT-3, neurotrophin 3; NT-4, neurotrophin 4; p75 NGFR , low affinity NGF receptor; p140 trk , high affinity NGF receptor; PBS, phosphate-buffered saline; PCEN-trk1 and -trk3, two different p140 trk -overexpressing PC12EN clones; PC12, pheochromocytoma cells; PC12EN, hybrid cells obtained by somatic hybridization