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

Phospholipase C-gamma (PLC-gamma1) is known to play an essential role in va rious cellular responses, such as proliferation and tumorigenesis, and PLC- gamma1 -specific inhibitors are commonly employed to investigate the mechan ism of the PLC-gamma1-mediated signaling pathway. In this study, we develop ed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 medi ated 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 PL C-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from th e cytoplasm to the intracellular aggregates, where F7-scFv was localized. M oreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 fr om the cytosol to the membrane ruffle and attenuated EGF-induced inositol p hosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing s equestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.