title: A 40-kDa epidermal growth factor/transforming growth factor alpha-binding domain produced by limited proteolysis of the extracellular domain of the epidermal growth factor receptor.
abstract: Elucidation of the three-dimensional structure of the complex of the epidermal growth factor (EGF) and its receptor is essential for understanding the molecular mechanisms of the EGF-receptor interaction and EGF-induced receptor-receptor interaction. NMR is useful to investigate interactions in solution between macromolecules at atomic resolution, but has a limitation in molecular masses of target proteins: less than 300 residues. We have prepared a fragment with apparent molecular mass of 40 kDa in SDS gels from the soluble extracellular domain of the EGF receptor (sEGFR, 619 residues) by sequential limited proteolysis with proteinase K and bromelain. This fragment is a monomeric structural domain consisting of 202 amino acid residues (Cys302-Arg503) and 18-kDa sugar chains, and binds EGF and transforming growth factor-alpha (TGF alpha). This 40-kDa domain has a dissociation constant of about 1 microM for human TGF alpha, which is similar to that of the parental sEGFR. sEGFR oligomerizes in response to EGF and TGF alpha, while the 40-kDa domain does not, suggesting that the sequences other than this domain is required for receptor oligomerization. The 40-kDa ligand-binding domain described in this report is suitable for analysis by various physico-chemical approaches such as NMR.
authors.affiliation: Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Kohda
authors.forename: M
authors.initials: M
authors.lastname: Odaka
authors.forename: I
authors.initials: I
authors.lastname: Lax
authors.forename: H
authors.initials: H
authors.lastname: Kawasaki
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
authors.forename: F
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001963:Bromelains / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019286:Endopeptidase K; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016211:Transforming Growth Factor alpha / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D001963:Bromelains; D066246:ErbB Receptors; D012697:Serine Endopeptidases; D019286:Endopeptidase K