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Journal of Cell Science 114, 2437-2447 (2001)
© 2001 The Company of Biologists Limited

RESEARCH ARTICLE

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

Roland Brock* and Thomas M. Jovin{ddagger}

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
* Present address: Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany (e-mail: roland.brock{at}uni-tuebingen.de)

{ddagger}Author for correspondence (e-mail: tjovin{at}mpc186.mpibpc.gwdg.de)

Accepted April 2, 2001

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 µm 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 the two events and variation in the kinetics of signal transduction for different subcellular locations.

Key words: Tyrosine kinase inhibitor PD153035, Confocal laser scanning microscopy, Immunofluorescence, Digital image processing, QMRA


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