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

• Published in: Journal of cellular biochemistry 1997-08, Vol.66 (2), p.229-244
• Main Authors: Jiang, Hao, Movsesyan, Vilen, Fink, Jr, Donald W., Fasler, Monika, Whalin, Michael, Katagiri, Yasuhiro, Monshipouri, Mariam, Dickens, Geneva, Lelkes, Peter I., Guroff, Gordon, Lazarovici, Philip
• Format: Article
• Language: English
• Subjects: Animals; differentiation; DNA - biosynthesis; DNA synthesis; Endothelium - chemistry; Endothelium - cytology; Endothelium - metabolism; fibroblast growth factor; Humans; K-252a; Mitogens - pharmacology; nerve growth factor; Nerve Growth Factors - pharmacology; overexpression; p140trk; PC12 Cells; PC12/endothelial hybrid cells; Phosphorylation; Phosphotyrosine - metabolism; proliferation; Proto-Oncogene Proteins - biosynthesis; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - deficiency; Rats; receptor; Receptor Protein-Tyrosine Kinases - biosynthesis; Receptor Protein-Tyrosine Kinases - chemistry; Receptor Protein-Tyrosine Kinases - deficiency; Receptor, trkA; Receptors, Nerve Growth Factor - biosynthesis; Receptors, Nerve Growth Factor - chemistry; Receptors, Nerve Growth Factor - deficiency; signal transduction; Signal Transduction - drug effects; Signal Transduction - genetics; staurosporine; Transfection; tyrosine kinase
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(19970801)66:2<229::AID-JCB10>3.0.CO;2-C

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal‐mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk 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 p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN‐trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN‐trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk 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 p140trk receptors. NGF stimulates p140trk 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 p70S6k, SNT, and phospholipase 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 [3H]thymidine incorporation in PC12EN‐trk1 and PC12EN‐trk3, respectively. These data highlight the functionality of the transfected p140trk 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 p140trk receptor substrates. J. Cell. Biochem. 66:229‐244. © 1997 Wiley‐Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.