Interaction of the alpha beta dimers of the insulin-like growth factor I receptor is required for receptor autophosphorylation

Biochemistry. 1991 Jan 8;30(1):48-54. doi: 10.1021/bi00215a008.

Abstract

We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Autoradiography
  • Blotting, Western
  • Cell Membrane / metabolism
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Humans
  • Insulin-Like Growth Factor I / metabolism*
  • Macromolecular Substances
  • Molecular Weight
  • Phosphorus Radioisotopes
  • Phosphorylation
  • Placenta / metabolism
  • Pregnancy
  • Receptors, Cell Surface / isolation & purification
  • Receptors, Cell Surface / metabolism*
  • Receptors, Somatomedin

Substances

  • Macromolecular Substances
  • Phosphorus Radioisotopes
  • Receptors, Cell Surface
  • Receptors, Somatomedin
  • Insulin-Like Growth Factor I
  • Adenosine Triphosphate