Figure 5.

Ca ²⁺ -triggered Ras deactivation by CAPRI in live cells. (A) GFP-CAPRI translocation correlates with HcRed-RBD dissociation. HeLa cells cotransfected with GFP-CAPRI, HcRed-RBD, and H-Ras. Time after 50 μM histamine stimulation of nonstarved cells is indicated. Nuclear HcRed fluorescence is masked. To enhance red signal, curves were adjusted simultaneously across the images so that no single image was enhanced over another. (B) ATP-induced deactivation of H-Ras in COS-7 cells cotransfected with CAPRI, GFP-RBD, and H-Ras. Time after 50 μM ATP stimulation of nonstarved cells indicated. Arrows highlight GFP-RBD ruffles. G = Golgi apparatus; inset is Golgi at lower image intensity. The experiment is best viewed in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200504167/DC1). (C) Relative dissociation of GFP-RBD by ATP-induced activation of CAPRI in COS-7 cells (black trace). Nonstarved cells were cotransfected with CAPRI, H-Ras, and GFP-RBD, and were stimulated with 50 μM ATP (n = 9 cells, n = 6 experiments ± SD). Gray trace indicates GFP-RBD dissociation in the absence of CAPRI transfection (n = 4 cells, n = 2 experiments). (D) Relative dissociation of GFP-RBD by histamine-induced activation of CAPRI in HeLa cells. Nonstarved HeLa cells were cotransfected with CAPRI, H-Ras, and GFP-RBD, and were stimulated by 100 μM histamine (n = 4 cells, n = 2 experiments ± SD). (E) Comparison of the GFP-CAPRI translocation induced by 50 μM ATP (average n = 8 cells, n = 3 experiments) to dissociation of GFP-RBD from the membrane in C. (F) Comparison of the GFP-CAPRI translocation induced by 100 μM histamine in HeLa cells (Fig. 1 B) to dissociation of GFP-RBD from the membrane in D.