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Keywords:

  • TrkA;
  • Nerve growth factor receptor;
  • Neurotrophin;
  • Signaling;
  • Mutagenesis;
  • Survival

Abstract

  1. Top of page
  2. Abstract

Abstract: To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14-amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC(Ki14) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild-type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Ki14). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)-mediated SHC tyrosine phosphorylation and a loss of high-affinity TrkA-SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF-dependent tyrosine phosphorylation of the signaling molecules phospholipase Cγ-1, MAP kinase/ERK-1, and SNT. TrkAKi receptors internalize NGF comparable to wild-type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3-kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF-dependent cell survival under serum-free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate-early genes (NGF1A, NGF1B, and c-fos), suggesting that these gene products are not required for NGF-dependent cell survival responses.

Abbreviations used: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; HA, hemagglutinin; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; NP-40, Nonidet P-40; PBS, phosphate-buffered saline; PI-3 kinase, phosphatidylinositol 3-kinase; PLCγ-1, phospholipase Cγ-1; SDS, sodium dodecyl sulfate.