Phospholipase C-γ (PLCγ) is required for EGF-induced motility (Chen, P., H. Xie, M.C. Sekar, K.B. Gupta, and A. Wells, J. Cell Biol. 1994. 127:847-857); however, the molecular basis of how PLCγ modulates the actin filament network underlying cell motility remains undetermined. We propose that one connection to the actin cytoskeleton is direct hydrolysis of PIP2 with subsequent mobilization of membrane-associated actin modifying proteins. We used signaling-restricted EGFR mutants expressed in receptor-devoid NR6 fibroblast cells to investigate whether EGFR activation of PLC causes gelsolin mobilization from the cell membrane in vivo and whether this translocation facilitates cell movement. Gelsolin anti-sense oligonucleotide (20 μM) treatment of NR6 cells expressing the motogenic full-length (WT) and truncated c'1000 EGFR decreased endogenous gelsolin by 30-60%; this resulted in preferential reduction of EGF (25 nM)-induced cell movement by >50% with little effect on the basal motility. As 14 h of EGF stimulation of cells did not increase total cell gelsolin content, we determined whether EGF induced redistribution of gelsolin from the membrane fraction. EGF treatment decreased the gelsolin mass associated with the membrane fraction in motogenic WT and c'1000 EGFR NR6 cells but not in cells expressing the fully mitogenic, but nonmotogenic c'973 EGFR. Blocking PLC activity with the pharmacologic agent U73122 (1 μM) diminished both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Concomitantly observed was reorganization of submembranous actin filaments correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increased basal cell motility in NR6 cells expressing either motogenic (WT and c'1000) or nonmotogenic (c'973) EGFR; EGF did not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrated actin reorganization similar to that observed in EGF-treated control cells; the peptide-induced changes were unaffected by U73122. These data suggest that much of the EGF-induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2- bound state. This provides a signaling mechanism for translating cell surface receptor-mediated biochemical reactions to the cell movement machinery.