title: Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation.
abstract: 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 microm 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.
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany. roland.brock@uni-tuebingen.de
authors.forename: R
authors.initials: R
authors.lastname: Brock
authors.forename: T M
authors.initials: TM
authors.lastname: Jovin
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D046508:Culture Techniques; D066246:ErbB Receptors / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D019751:Intramolecular Transferases / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008863:Microspheres; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase