vGPCR induces activation of Rac but not Rho. (A) Activated mutants of all 3 small G proteins (RhoA, Rac1, and Cdc42) can induce NF-κB activation and IL-6 transcription, similar to vGPCR, in COS-7 cells. (B) 293T cells expressing vGPCR or vGPCR R143Q (R143Q) did not demonstrate elevated levels of active Rho in the absence (-) or presence (+) of 50 nM IL-8 (1 minute) with respect to GFP-expressing cells (control). Similar results were obtained using longer exposure to agonist. The DH/PH domain of PDZ-Rho-GEF was used as a positive control. (C) Transcriptional activation through the κB site by vGPCR in COS-7 cells is not inhibited by treatment with C3 toxin. Gα13 was used as a positive control for C3 toxin effects. Data in panels A and C represent the mean ± SEM of triplicate samples from a typical experiment, expressed as fold induction with respect to control. (D) 293T cells expressing vGPCR had elevated levels of active Rac in the absence (-) or presence (+) of 50 nM IL-8 (1 minute). Agonist-dependent vGPCR mutant (R143Q) only induced activation of Rac in presence of IL-8. The constitutively active Rac GEF, truncated TIAM (TIAM1 C1199), was used as a positive control. (E-F) PAE cells transfected with vGPCR and Rac WT had elevated levels of active Rac (E) and demonstrated Rac-like morphology (F). Cells were fixed and stained with phalloidin-specific antibodies to label the actin cytoskeleton. Arrows indicate membrane ruffling (vGPCR and RacQL) or filopodia (Cdc42QL). Pictures are representative of 3 independent experiments. PAE cells transfected with expression vectors for RhoAQL, Rac1QL, or Cdc42QL were used as controls.