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

Epidermal growth factor receptor (EGFR, erbB1) activation and translocation of the She adaptor protein to activated receptors were analyzed at the sub cellular level by dual-label immunofluorescence and confocal laser scanning microscopy in conjunction with a new microsphere-based protocol. In the Qu antitative Microsphere Recruitment Assay (QMRA) introduced here, epidermal growth factor-coated 1 mum diameter microspheres were distributed over the surface of adherent tissue culture cells expressing the receptor. High-reso lution confocal microscopy of a fusion construct of the receptor and the gr een fluorescent protein expressed in Chinese hamster ovary cells demonstrat ed that engulfment and internalization of the microspheres occurred rapidly within minutes, and in a receptor activation-dependent manner. In human ep idermoid carcinoma A431 cells, receptor activation and She translocation pe rsisted over the 20-minute time course of the experiments. However, at the subcellular level the positive correlation of receptor activation and She t ranslocation observed at 5-8 minutes dissipated, indicating a time-dependen t decoupling of the two events and variation in the kinetics of signal tran sduction for different subcellular locations.