INTERACTION OF THE ALPHA-BETA DIMERS OF THE INSULIN-LIKE GROWTH FACTOR-I RECEPTOR IS REQUIRED FOR RECEPTOR AUTOPHOSPHORYLATION

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 I-125-labeled alpha-IR-3, a monoclonal antibody to the IGFI 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.