High-yield trapping of EGF-induced receptor dimers by chemical cross-linking.
The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue-specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding give rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X-100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10(-7) M. When samples of the extracts were incubated with or without EGF and then treated with the high-yield cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross-linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF-induced receptor dimers were also efficiently cross-linked in highly purified receptor preparations, suggesting that EGF-induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins.
Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
B O
BO
Fanger
J E
JE
Stephens
J V
JV
Staros
D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology / Q000502:physiology*; D066246:ErbB Receptors / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D004815:Epidermal Growth Factor; D066246:ErbB Receptors