Sun H Q, Kwiatkowska K, Wooten D C, Yin H L
University of Texas Southwestern Medical Center, Department of Physiology, Dallas 75235-9040.
J Cell Biol. 1995 Apr;129(1):147-56. doi: 10.1083/jcb.129.1.147.
Actin modulating proteins that bind polyphosphoinositides, 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, Ca(2+)- and polyphosphoinositide-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 PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma 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 PIP(2)-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.
结合多磷酸肌醇（如磷脂酰肌醇4,5-二磷酸，PIP2）的肌动蛋白调节蛋白可能通过重构膜细胞骨架和通过磷酸肌醇循环调节第二信使的产生来参与受体信号传导。我们通过过表达CapG来研究这些可能性，CapG是凝溶胶蛋白家族的一种肌动蛋白丝末端封端、Ca(2+)和多磷酸肌醇结合蛋白。高水平的瞬时过表达减少了细胞中心而非细胞周边的肌动蛋白丝染色。在克隆选择的细胞系中适度过表达对肌动蛋白丝含量或组织没有可检测到的影响。然而，它促进了伤口愈合和趋化作用速率的剂量依赖性增加。运动表型与凝溶胶蛋白过表达时观察到的相似，凝溶胶蛋白除了封端外，还能切断和引发肌动蛋白丝。过表达CapG的克隆对血小板衍生生长因子的反应比对照转染的克隆更敏感。它们形成更多圆形的背膜褶皱，具有更高的磷酸肌醇周转率、肌醇1,4,5-三磷酸生成和Ca2+信号传导。这些反应与增强的PLCγ活性一致。对PIP2质量的直接测量表明，CapG对PLCγ的作用主要不是由于PIP2底物浓度的增加。观察到的细胞运动性和膜信号传导的变化与PIP(2)结合的肌动蛋白调节蛋白在体内调节磷酸肌醇周转和第二信使产生的假设一致。我们推断CapG和相关蛋白在细胞刺激后准备好协调膜信号传导与肌动蛋白丝动力学。
