title: Characterization of the epidermal growth factor receptor from Ehrlich ascites tumor cells.
abstract: We have identified the 170 kDa epidermal growth factor (EGF) receptor in crude membrane fractions isolated from Ehrlich ascites tumor cells by its EGF-dependent phosphorylation with [gamma-32P]ATP. An apparent affinity constant for the ligand of 40-50 nM, based on the extent of its EGF-dependent phosphorylation, was calculated. [125I]EGF binds to the 170 kDa receptor and Scatchard plot analysis shows high affinity and low affinity Kds of 1.7 nM and 24 nM, respectively, in whole cells, and 0.2-0.8 nM and 39-116 nM, respectively, in isolated non-phosphorylated membrane fractions. We have estimated the presence of 48 x 10(3) high affinity and 275 x 10(3) low affinity EGF binding sites per tumor cell. Phosphoamino acid analysis shows EGF-dependent phosphorylation of tyrosine and serine residues. A polyclonal antibody to a human EGF receptor/c-erbB-2 product common cytoplasmic domain epitope immunoprecipitates a 45 kDa phosphopolypeptide from the tumor membrane fractions and from whole cell lysates. Phosphoamino acid analysis of the immunoprecipitated 45 kDa phosphopolypeptide shows the presence of phosphoserine. The immunoprecipitated 45 kDa polypeptide is able to undergo EGF-independent phosphorylation, although no significant protein kinase activity towards exogenous substrates is detected.
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas Arturo Duperier 4, Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Elexpuru
authors.forename: M
authors.initials: M
authors.lastname: Soriano
authors.forename: A
authors.initials: A
authors.lastname: Villalobo
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000493:pharmacokinetics; D066246:ErbB Receptors / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D004815:Epidermal Growth Factor; D066246:ErbB Receptors