Regulation of epidermal growth factor receptor in NIH3T3/HER14 cells by antireceptor monoclonal antibodies.
The mechanism(s) by which monoclonal antibodies (mAbs) against the epidermal growth factor (EGF) receptor regulate receptor function have been investigated with NIH3T3/HER14 fibroblasts expressing human EGF receptors. Bivalent 225 mAb or monovalent 225 Fab' inhibited transforming growth factor (TGF)-alpha-induced EGF receptor tyrosine phosphorylation and cell proliferation. Culture of HER14 cells with 225 mAb or 225 Fab' did not activate EGF receptor tyrosine kinase when assayed after lysis of cells in SDS sample buffer. However, when cells were cultured with bivalent 225 mAb, but not with monovalent 225 Fab', and were subsequently lysed and further incubated in Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors, receptor phosphorylation was observed. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein after lysis, indicating that it was due to the activation of protein tyrosine kinase. The activity of bivalent 225 mAb was unphysiologic, in contrast with TGF-alpha, in that receptor kinase activation occurred only after cell lysis and with delayed kinetics; serine and threonine phosphorylation did not occur; and down-regulation of EGF receptors was slower. Selective mAb-mediated phosphorylation of tyrosine residues on EGF receptors was sufficient to activate phosphorylation of a SH2 group-bearing substrate, phospholipase C-gamma, indicating that serine/threonine phosphorylation is not required for EGF receptor kinase activity. These studies provide novel insights into the capacity of bivalent mAb to modulate EGF receptor function.
Fan
Z
Z
Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Mendelsohn
J
J
Masui
H
H
Kumar
R
R
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D066246:ErbB Receptors; D010738:Type C Phospholipases