Abstract
We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.
Highlights
Demonstration of Epidermal Growth Faetor-induced Receptor Dimerization in Living Cells Using Ca hemical Covalent Cross-linking Agent”
An interesting and unreagent l-ethyl-3-[3-(dimethylamino)propyl]carbodi- solved question isthe mechanism of signal transduction beimide (EDAC) to examine the capacity of epidermal tween the hormone Iigand-binding domaitno the cytoplasmic growth factor (EGF) to stimulate the dimerizationof domainleading to the stimu~tionof the protein tyrosine purified Epidermal growth factor (EGF) receptor, of EGF receptor in membrane preparations andin intact A431 cells
EGF induced rapid dimerization of EGF receptor in mobilization of EGF-R on various solid matrices prevents living cells, suggesting that this process may play a EGF from activating the kinase function; and fourth, crossrole in transmembrane signalling mediated by EGF. linking of EGF-R increases their affinity towards EGF
Summary
We have shown previously that EGF can induce the formation of receptor oligomers as detected by nondenaturing gel electrophoresis @).To stabilize the interaction of receptors, in the present study we have used the covalent crosslinker EDAC. This reagent links free amino groups to carboxyl groups which approach eachother within the distance of a peptide bond. A431 cells were labeled with [35S]methioninefor 16 h and incubated with 500 ng/ml EGF for 90 minat 4 "C, treated with the cross-linkingagent (EDAC), lysed, immunoprecipitated with anti-EGF-R antibodies, and analyzed by SDS-PAGE. A small but reproducible amount of EGF-R dimers was detected in the absence of EGF (Fig. 4, lane B )
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