Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells

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.