Inhibition of the EGF-induced activation of phospholipase C-gamma1 by a single chain antibody fragment.
Phospholipase C-gamma1(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-gamma1 with high affinity (K(d)=1.9x10(-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.
Yi
K S
KS
Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, South Korea.
Chung
J H
JH
Lee
Y H
YH
Chung
H G
HG
Kim
I J
IJ
Suh
B C
BC
Kim
E
E
Cocco
L
L
Ryu
S H
SH
Suh
P G
PG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D000915:Antibody Affinity; D001483:Base Sequence; D019556:COS Cells; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D021381:Protein Transport / Q000187:drug effects; D013816:Thermodynamics; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007295:Inositol Phosphates; D007527:Isoenzymes; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D016650:Fluorescein-5-isothiocyanate