Controlled proteolysis of EGF receptors: evidence for transmembrane distribution of the EGF binding and phosphate acceptor sites.
A small quantity of the 125I-EGF (epidermal growth factor) bound specifically to EGF receptors on the human epidermoid carcinoma cell line A431 associates covalently. The direct linkage complex formed migrates during gel electrophoresis as a single diffuse band of MW = 160,000-170,000. In contrast, direct linkage complexes of 160,000, 145,000, and 115,000 daltons are formed when EGF is incubated with membranes isolated from these cells; these arise from EGF receptor modification during membrane isolation. None of these modifications affected the affinity of the EGF binding site for 125I-EGF. The electrophoretic mobilities of the MW = 160,000 and 145,000 direct linkage complexes were similar to those of the major 32Pi-labeled products of the EGF-stimulated phosphorylation reaction described by Carpenter et al [Nature 276:409-410, 1978], indicating that proteolytic fragments of EGF receptors are the major phosphate acceptors in this reaction. EGF receptors on intact A431 cells accepted phosphate effectively from gamma-32Pi-ATP only when the cells were permeabilized with lysolecithin. This shows that the EGF binding and phosphate acceptor sites lie on opposing faces of the membrane. When the 145,000 dalton form of receptor is labeled with EGF or 32Pi and the labeled peptides subjects to tryptic hydrolysis under identical conditions, all phosphate is lost from high molecular weight products under conditions where the EGF-receptor covalent complex is converted largely to a 115,000 dalton form. This suggests that the phosphate acceptor site lies on the cytoplasmic side of the membrane on a region of receptor extending 30,000 daltons from the 115,000 dalton fragment containing the EGF binding site.
D001665:Binding Sites; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors; D006801:Humans; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014357:Trypsin
D010710:Phosphates; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D014357:Trypsin