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Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

MPG-Autoren
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Brock,  R.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Jovin,  T. M.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Zitation

Brock, R., & Jovin, T. M. (2001). Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation. Journal of Cell Science, 114, 2437-2447.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0012-F5AE-7
Zusammenfassung
Epidermal growth factor receptor (EGFR, erbB1) activation and translocation of the Shc adaptor protein to activated receptors were analyzed at the subcellular level by dual-label immunofluorescence and confocal laser scanning microscopy in conjunction with a new microsphere-based protocol. In the Quantitative Microsphere Recruitment Assay (QMRA) introduced here, epidermal growth factor-coated 1 mm diameter microspheres were distributed over the surface of adherent tissue culture cells expressing the receptor. High-resolution confocal microscopy of a fusion construct of the receptor and the green fluorescent protein expressed in Chinese hamster ovary cells demonstrated that engulfment and internalization of the microspheres occurred rapidly within minutes, and in a receptor activation-dependent manner. In human epidermoid carcinoma A431 cells, receptor activation and Shc translocation persisted over the 20-minute time course of the experiments. However, at the subcellular level the positive correlation of receptor activation and Shc translocation observed at 5-8 minutes dissipated, indicating a time-dependent decoupling of thetwo events and variation in the kinetics of signal transduction for different subcellular locations.