Dimerization of internalized epidermal growth factor receptors.
Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.
Department of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
A
A
Sorkin
G
G
Carpenter
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents; D004247:DNA; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D004815:Epidermal Growth Factor; D004247:DNA; D010770:Phosphotransferases; D066246:ErbB Receptors