Identification of proteins differentially expressed upon neurotrophin receptor activation using DIGE and MALDI-MS

Activation of the neurotrophin receptor TrkA by its ligand NGF mediates growth inhibition and differentiation of neuroblastoma cells, whereas TrkB/BDNF signaling promotes proliferation and therapy resistance. Nonetheless, TrkA and TrkB exhibit a high level of sequence homology and use overlapping signaling pathways. To identify novel effector molecules contributing to the characteristic phenotypes of TrkA- and TrkB-expressing neuroblastoma cells, we analyzed global protein expression in neuroblastoma SY5Y cells stably transfected with TrkA or TrkB, and activated by their specific ligands in a time course from 0–24h. The recently introduced DIGE (fluorescence 2-D difference gel electrophoresis) system allowed reproducible identification of differentially expressed proteins between two samples in the same gel. In SY5Y-TrkA cells, we detected 8 proteins regulated upon NGF-induced receptor activation. Proteomic analysis of SY5Y-TrkB cells identified 23 proteins regulated upon BDNF-stimulation. Differentially expressed proteins were identified by MALDI-PMF/PFF mass spectrometry. Functional assignment revealed that the majority of identified proteins is involved in cytoskeleton organisation. Proteomics proved to be a promising tool to identify novel target proteins of Trk signaling in neuroblastoma cells.

Supported by NGFN/BMBF and Deutsche Krebshilfe.