Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor

The translocation of fluorescently tagged PLC gamma and requirements for th is process in cells stimulated with EGF were analyzed using real time fluor escence microscopy applied for the first time to monitor growth factor rece ptor-effector interactions. The translocation of PLC gamma to the plasma me mbrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidy linositol (PI) 3-kinase. An array of domains specific for PLC gamma isoform s was sufficient for this translocation. The dynamics of translocation to t he plasma membrane and redistribution of PLC gamma, relative to localizatio n of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P-2), were shown. Colo calization with the receptor was observed in the plasma membrane and in mem brane ruffles where PI 4,5-P-2 substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from t he substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The pr esence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intra cellular vesicles, leading to downregulation of the PLC activity.