N-TERMINAL DELETION MUTANTS OF INSULIN-LIKE GROWTH FACTOR-II (IGF-II) SHOW THR(7) AND LEU(8) IMPORTANT FOR BINDING TO INSULIN AND IGF-I RECEPTORS AND LEU(8) CRITICAL FOR ALL IGF-II FUNCTIONS

To define the role of the N-terminal region of insulinlike growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly(8) for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors remained at greater than or equal to 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to similar to 10% and similar to 3%, respectively, of that of rIGF-II. When the octapeptide including Leu(8) was removed prior to the Cys(9)-Cys(47) intrachain bond, the relative affinity of this deletion mutant, des(1-8)-rIGF-II, for these receptors dramatically decreased to <1% of that of rIGF-II. Substituting Gly(8) for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These results suggest that the side chains of Thr(7) and Leu(8) may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the relative affinity of des-(1-8)- or [Gly(8)]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also <1% of that of rIGF-II, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II. These results clearly demonstrate that Thr(7) and Leu(8) are important for binding to insulin and IGF-I receptors and Leu(8) is critical for expression of all IGF-II functions.