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 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.
Tollefsen
S E
SE
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
Stoszek
R M
RM
Thompson
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017451:Receptors, Somatomedin
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D007334:Insulin-Like Growth Factor I; D000255:Adenosine Triphosphate