FIGURE 9:

GIV depletion increases the membrane association of EEA1 and prolongs and enhances EGFR signaling from EEA1 endosomes. (A–F) Before EGF stimulation (0 min), little pY1068 staining for activated receptors (green) is observed at the PM or EEA1 endosomes (red) in either control (A) or GIV-depleted (D) cells. At 10 min after stimulation some activated EGFRs are associated with EEA1 endosomes in both control (B) and GIV-depleted (E) cells (yellow, arrowheads). By 30 min, activated EGFRs are barely detectable in EEA1 endosomes in controls (C), whereas GIV-depleted cells show a striking accumulation of activated EGFRs in EEA1 endosomes (F; yellow, arrowheads). GIV-depleted HeLa cells and controls were serum starved, stimulated with EGF, and stained for pY1068-EGFR (green) and EEA1 (red). Bar, 10 μm. (G, H) After GIV depletion, 24% of the total EEA1 is associated with membrane fractions, ∼11% in controls. The distribution of EEA1, GIV, Gαs, and actin in membrane (120,000 × g pellet, P100) and cytosolic (120,000 × g supernatant, S100) fractions prepared from control (lanes 1 and 2) or GIV-depleted (lanes 3 and 4) HeLa cells was assessed by immunoblotting. EEA1 bands such as those in A were quantified from three different experiments and averaged, and the percentage of EEA1 on membrane fractions calculated and plotted as in Figure 5B (*p < 0.01). (I, J) Gαs and GIV cooperatively facilitate the loss of EEA1 from membranes. The amount of EEA1 on membranes after depletion of both Gαs and GIV (lanes 5 and 6) is similar to that seen after depletion of Gαs alone (lanes 7 and 8). HeLa cells were depleted of GIV, Gαs, or both GIV and Gαs and fractions prepared and immunoblotted as in A. Results are shown as the mean ± SEM (p = 0.02; n/s, no significant difference).