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Vol. 13, Issue 5, 1522-1535, May 2002
Department of Pharmacology, University of Colorado Health Sciences
Center, Denver, Colorado 80111
Activation of the epidermal growth factor receptor (EGFR) triggers
multiple signaling pathways and rapid endocytosis of the epidermal
growth factor (EGF)-receptor complexes. To directly visualize the
compartmentalization of molecules involved in the major signaling
cascade, activation of Ras GTPase, we constructed fusions of Grb2, Shc,
H-Ras, and K-Ras with enhanced cyan fluorescent protein (CFP) or yellow
fluorescent protein (YFP), and used live-cell fluorescence imaging
microscopy combined with the fluorescence resonance energy transfer
(FRET) technique. Stimulation of cells by EGF resulted in the
accumulation of large pools of Grb2-CFP and YFP-Shc in endosomes, where
these two adaptor proteins formed a complex with EGFR. H-Ras and K-Ras
fusion proteins were found at the plasma membrane, particularly in
ruffles and lamellipodia, and also in endosomes independently of
GTP/GDP loading and EGF stimulation. The relative amount of endosomal
H-Ras was higher than that of K-Ras, whereas K-Ras predominated at the
plasma membrane. On application of EGF, Grb2, and Ras converge in the
same endosomes through the fusion of endosomes containing either Grb2
or Ras or through the joint internalization of two proteins from the plasma membrane. To examine the localization of the GTP-bound form of
Ras, we used a FRET assay that exploits the specific interaction of
GTP-bound CFP-Ras with the YFP-fused Ras binding domain of c-Raf. FRET
microscopy revealed that GTP-bound Ras is located at the plasma
membrane, mainly in ruffles and at the cell edges, as well as in
endosomes containing EGFR. These data point to the potential for
endosomes to serve as sites of generation for persistent signaling
through Ras.
Online
version of this article contains video material for some figures.
Online version available at www.molbiolcell.org.
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