Solution structure of betacellulin, a new member of EGF-family ligands

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Abstract

The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five β-strands and one short 310 helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands.

Section snippets

Materials and methods

Sample preparation of BTCe. The expression and purification of recombinant human BTC protein was performed in the same manner as that described in our previous paper [3]. BTCe was obtained as a lysylendo-peptidase product of recombinant human BTC.

For the NMR experiments performed in H2O solution, 3.8 mg of the lyophilized BTCe sample was dissolved in 0.30 ml of H2O (1H2O:2H2O=9:1) containing 1% acetate buffer to give a final concentration of 2.0 mM (pH 3.5). For the experiments performed in 2H2O

Chemical shift assignments of BTCe

The chemical shift assignments of BTCe were carried out using the sequential assignment procedure [35] in two-dimensional-NMR experiments performed at 25 °C. The 1H-resonance assignments of all of the main chain atoms, except for the amino proton of the N-terminal Arg31, and most of the side chain atoms of BTCe were unambiguously assigned. The final assignment of 1H-resonances of BTCe at 25 °C has been deposited in the BioMagResBank (Accession Number 4990).

Structural calculation of BTCe

The solution structure of BTCe at 25 °C

Acknowledgements

This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) to K.K.

References (40)

  • D.A. Engler et al.

    Critical functional requirement for the guanidinium group of the arginine 41 side chain of human epidermal growth factor as revealed by mutagenic inactivation and chemical reactivation

    J. Biol. Chem.

    (1992)
  • S.M. Puddicombe et al.

    The significance of valine 33 as a ligand-specific epitope of transforming growth factor α

    J. Biol. Chem.

    (1996)
  • Y. Shing et al.

    Betacellulin: a mitogen from pancreatic β cell tumors

    Science

    (1993)
  • H. Mashima et al.

    Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells

    J. Clin. Invest.

    (1996)
  • M. Seno et al.

    Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form

    Growth Factors

    (1996)
  • J. Massague et al.

    Membrane-anchored growth factors

    Annu. Rev. Biochem.

    (1993)
  • P. Van der Geer et al.

    Receptor protein-tyrosine kinases and their signal transduction pathways

    Annu. Rev. Cell Biol.

    (1994)
  • D.J. Riese II et al.

    Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-β

    Oncogene

    (1996)
  • D.J. Riese II et al.

    Activation of ErbB4 by the bifunctional epidermal growth factor family hormone epiregulin is regulated by ErbB2

    J. Biol. Chem.

    (1998)
  • D.J. Riese II et al.

    The epidermal growth factor receptor couples transforming growth factor-α, heparin-binding epidermal growth factor-like factor, and amphiregulin to Neu, ErbB-3, and ErbB-4

    J. Biol. Chem.

    (1996)
  • Cited by (0)

    Abbreviations: BTC, betacellulin; BTCe, the EGF-like domain of BTC; CSI, chemical shift index; DQF-COSY, double quantum filtered correlation spectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HRG-α, heregulin-α; HRG-αe, EGF-like domain of heregulin-α; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RMSD, root mean square deviation; SA, simulated annealing; SD, standard deviation; TGF-α, transforming growth factor-α; TOCSY, total correlation spectroscopy. I lost my Debit Card last week, All the particulars like Debit Card No., T-pin No. etc., I have remember my ATM Pin number only. I don't remember my Debit Card No., so I find difficulty to contact by telephone, While I was trying by phone, to communicate

    1

    Current address: Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan.

    2

    Current address: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.

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