EGF domain of coagulation factor IX is conducive to exposure of phosphatidylserine.
Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulation factor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins.
Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan.
Chiaki
C
Hidai
Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan.
Yusuke
Y
Fujiwara
Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan.
Shinichiro
S
Kokubun
Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan.|Division of Oral Surgery, Department of Otorhinolaryngology Head and Neck Surgery, Nihon University School of Medicine, Tokyo, Japan.
Hisataka
H
Kitano
D017209:Apoptosis; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000502:physiology; D005164:Factor IX / Q000502:physiology*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000502:physiology; D000072417:Protein Domains
D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D004815:Epidermal Growth Factor; D005164:Factor IX
EGF motif; cell membrane; coagulation factor; lipid raft; phosphatidylserine; scramblase