Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide antibodies

https://doi.org/10.1016/0303-7207(94)90226-7Get rights and content

Abstract

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the α-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two HIR α-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) directed against a sequence in the carboxy-termi- nal region of the α-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA-12 linearises the Scatchard plot of binding data, and retards the dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.

References (34)

  • L. Benzi et al.

    J. Chromatogr.

    (1986)
  • P. De Meyts et al.

    J. Biol. Chem.

    (1976)
  • Y. Ebina et al.

    Cell

    (1985)
  • R. Gherzi et al.

    J. Biol. Chem.

    (1989)
  • J.L. Gu et al.

    Biochem Biophys. Res. Commun.

    (1988)
  • L.M. Keefer et al.

    Biochem. Biophys. Res. Commun.

    (1981)
  • A. Pessino et al.

    Biochem. Biophys. Res. Commun.

    (1989)
  • R. Schumacher et al.

    J. Biol. Chem.

    (1991)
  • S. Seino et al.

    Biochem. Biophys. Res. Commun.

    (1989)
  • B. Vogt et al.

    Biochem. Biophys. Res. Commun.

    (1991)
  • C.C. Yip et al.

    Biochem. Biophys. Res. Commun.

    (1988)
  • A.S. Andersen et al.

    Biochemistry

    (1990)
  • D. Accili et al.

    EMBO J.

    (1989)
  • P. De Meyts et al.

    Mol. Endocrinol.

    (1990)
  • J.R. Forsayeth et al.

    J. Biol. Chem.

    (1987)
  • T.A. Gustafson et al.

    J. Biol. Chem.

    (1990)
  • M. Kellerer et al.

    Biochemistry

    (1992)
  • View full text