Inhibition of the EGF-induced activation of phospholipase C-gamma 1 by a single chain antibody fragment

Abstract

Phospholipase C-gamma (PLC-gamma1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-gamma1 -specific inhibitors are commonly employed to investigate the mechanism of the PLC-gamma1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-gammal with high affinity (K-d= 1.9 x 10(-8) M) in vitro. F7-scFv also bound to PLC-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing sequestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.