Effects of CapG overexpression on agonist-induced motility and second messenger generation

Actin modulating proteins that bind poly-phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP₂), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca²⁺- and polyphosphoinostide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control- transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca²⁺ signaling. These responses are consistent with enhanced PLCγ activity. Direct measurements of PIP₂ mass showed that the CapG effect on PLCγ was not due primarily to an increase in the PIP₂ substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP₂-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.