title: Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.
abstract: The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic kinase domain by a single transmembrane (TM) region. To explore the role of the TM region in the process of receptor activation, we have generated EGF-receptor mutants with altered TM regions by utilizing in vitro site-directed mutagenesis. The TM regions of two mutant receptors were either extended (designated i626-3) or shortened (designated d625.3) by three hydrophobic amino acid residues. In the other two mutant receptors, hydrophobic amino acids were substituted by charged residues--i.e., Val-627 was replaced by glutamic acid (designated V627E) or Leu-642 was replaced by an arginine residue (designated L642R). NIH 3T3 cells lacking endogenous EGF receptors were transfected with constructs encoding either wild-type or mutant receptors and shown to express the receptor molecules using 125I-labeled EGF binding and immunoprecipitation experiments. The mutant receptors were expressed on the cell surface as polypeptides of Mr 170,000 exhibiting typical high- and low-affinity binding sites for 125I-labeled EGF. Similar to its effect on wild-type receptors, phorbol 12-myristate 13-acetate abolished the mutant-receptor high-affinity binding sites for EGF. Moreover, EGF was able to stimulate the kinase activities of wild-type and mutant receptors both in vitro and in living cells. The mutant receptors were also able to undergo EGF-induced receptor dimerization as revealed by cross-linking experiments with a bifunctional covalent cross-linking agent. These results are compatible with an intermolecular allosteric oligomerization model for receptor activation rather than with a model based on an intramolecular mechanism for receptor activation.
authors.affiliation: Rorer Biotechnology, King of Prussia, PA 19406.
authors.forename: O
authors.initials: O
authors.lastname: Kashles
authors.forename: D
authors.initials: D
authors.lastname: Szapary
authors.forename: F
authors.initials: F
authors.lastname: Bellot
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
authors.forename: A
authors.initials: A
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D008024:Ligands / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
chemical_list: D008024:Ligands; D004815:Epidermal Growth Factor; D008715:Methionine; D011494:Protein Kinases; D066246:ErbB Receptors