Tyrosine Kinase Activity of a Chimeric Insulin-Like-Growth-Factor-l Receptor Containing the Insulin Receptor C-Terminal Domain. Comparison with the Tyrosine Kinase Activities of the Insulin and Msulin-Like-Growth-Factor-1 Receptors Using a Cell-Free System

In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the β subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-F., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449–11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin re-ceptor-(1142–1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor β subunit plays a key role in regulation of the tyrosine kinase activity. The fine-tuning of the tyrosine kinase by the C-terminal tail could participate in the receptor specificity.