Figure 5.

G-protein–coupled receptor-2–interacting protein (GIT1) is required for extracellular signal–regulated kinases 1 and 2 (ERK1/2) and Rac1/Cdc42-mediated activation and localization of cortactin. A, Human umbilical vein endothelial cells (HUVEC) were transfected with either scrambled control (Con) or GIT1-specific small interfering RNA (siRNA) for 36 hours and then serum starved for 2 hours. Cells were stimulated with vascular endothelial growth factor (VEGF; 10 ng/mL) and pS405-cortactin and pERK1/2 were measured by Western blot (n=3). B, Wounds were created in the monolayer of HUVEC cultured on fibronectin-coated cover glasses and serum starved overnight. Next, cells were treated with VEGF (10 ng/mL) and incubated for 6 hours. Cells were fixed and triple stained for pERK1/2 (green), GIT1 (red) and cortactin (blue). As shown by arrowhead, GIT1–ERK1/2–cortactin complex formation was detected by appearance of purple (colocalization) in the leading edge (n=3). C, GIT1-depleted HUVECs were serum starved for 2 hours followed by stimulating with VEGF (10 ng/mL) for 30 minutes. Cells were lysed and equal amounts of protein (400 µg) were incubated with 20 µg p21-associated kinase (PAK) 1-p21 binding domain (PBD) agarose beads for 60 minutes at 4°C. Active Rac1/Cdc42 (GTP bound Rac1/Cdc42) was precipitated with PAK1-PBD agarose beads and the amount of active Rac1/Cdc42 was measured by Western blotting. D, Quantification by densitometric analysis using ImageJ software and the active Rac1/Cdc42 were normalized with the total Rac1/Cdc42 in whole cell lysates. *Vs control siRNA (n=3; *P<0.05).