FIGURE 6:

Inactive Gαs (Gαs-GA) reverses the effects of Gαs depletion on EGFR autophosphorylation and the membrane association of EEA1. (A, B) Transfection of Gαs-depleted cells with an inactive srGαs-GA mutant (lanes 10–12) but not an active srGαs-QL mutant (lanes 7–9) reverses the effects of Gαs depletion (lanes 4–6) and restores EGFR autophosphorylation to levels comparable to controls (lanes 1–3). Control (lanes 1–3) or Gαs siRNA–treated (lanes 4–12) HeLa cells were transfected with pCDNA3.1 (vector; lanes 1–6), constitutively active srGαs-QL (lanes 7–9), or inactive srGαs-GA (lanes 10–12) and then serum starved, stimulated with EGF, and immunoblotted as in Figure 3A. pY1068 and pY1045 bands such as those in A were quantified from four different experiments, averaged, normalized, and plotted as in Figure 3B (*p < 0.05). Black bar, control siRNA + vector; dark gray bar, Gαs siRNA + vector; light gray bar, Gαs siRNA + srGαs-Q227L; open bar, Gαs siRNA + srGαs-G226A. (C, D) An inactive Gαs mutant reverses the effect of Gαs depletion on the membrane distribution of EEA1. In Gαs-depleted cells transfected with srGαs-GA (lanes 5 and 6) and in controls transfected with vector alone (lanes 1 and 2), ∼16% of the total EEA1 is associated with the membrane fraction, whereas in Gαs-depleted cells (lanes 3 and 4), ∼29% of EEA1 is in the membrane fraction. Membrane and cytosol fractions were prepared from control siRNA–treated HeLa cells transfected with vector (lanes 1 and 2), Gαs siRNA–treated cells transfected with vector (lanes 3 and 4), or inactive srGαs-GA (lanes 5 and 6) and immunoblotted for EEA1, actin, and Gαs as in Figure 5A. (D) The percentage of EEA1 on membranes was calculated and plotted as in Figure 5B (n = 7, *p < 0.05). Light gray bar, control siRNA + vector; black bar, Gαs siRNA + vector; dark gray bar, Gαs siRNA + srGαs-GA.