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© The Rockefeller University Press, 0021-9525/2001//599 $5.00
The Journal of Cell Biology, Volume 153, Number 3, , 2001 599-612

Real Time Fluorescence Imaging of Plc Translocation and Its Interaction with the Epidermal Growth Factor Receptor

^a Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London SW3 6JB, United Kingdom
^b Department of Anatomy and Developmental Biology, University College, London WC1 6BT, United Kingdom
CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK.44-207-352-329944-207-352-8133

matilda{at}icr.ac.uk

The translocation of fluorescently tagged PLC and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor–effector interactions. The translocation of PLC to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P₂), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P₂ substrate could also be visualized. At later times, internalization of PLC, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC to the receptor as the main site of the plasma membrane recruitment. The presence of PLC in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

Key Words: PLC • EGF receptor • translocation • real time imaging • SH2 and PH domains

© 2001 The Rockefeller University Press

Abbreviations used in this paper: EGFR, EGF receptor; SA, PLC-SA; GFP, green fluorescent protein; I, inositol; I 1,4,5-P₃, I 1,4,5-trisphosphate; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI, phosphatidylinositol; PI 4,5-P₂, PI 4,5-bisphosphate; PI 3,4,5-P₃, PI 3,4,5-trisphosphate; PI-PLC, phosphoinositide-specific PLC; RFP, red fluorescent protein; SA, specific domain array; SH, Src homology.

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