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 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 gamma 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 gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane 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 the 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 presence of PLC gamma 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.
Matsuda
M
M
Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
Paterson
H F
HF
Rodriguez
R
R
Fensome
A C
AC
Ellis
M V
MV
Swann
K
K
Katan
M
M
D000818:Animals; D001692:Biological Transport; D001798:Blood Proteins; D019556:COS Cells; D002451:Cell Compartmentation; D002645:Chickens; D002867:Chromones / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000302:isolation & purification / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D010784:Photomicrography; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D018909:src Homology Domains
D001798:Blood Proteins; D002867:Chromones; D007527:Isoenzymes; D009025:Morpholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; C039077:platelet protein P47; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma