title: N-terminal deletion mutants of insulin-like growth factor-II (IGF-II) show Thr7 and Leu8 important for binding to insulin and IGF-I receptors and Leu8 critical for all IGF-II functions.
abstract: To define the role of the N-terminal region of insulin-like 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 Gly8 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 > or = 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to approximately 10% and approximately 3%, respectively, of that of rIGF-II. When the octapeptide including Leu8 was removed prior to the Cys9-Cys47 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 Gly8 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 Thr7 and Leu8 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 [Gly8]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 Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions.
authors.affiliation: Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Hashimoto
authors.forename: H
authors.initials: H
authors.lastname: Fujiwara
authors.forename: N
authors.initials: N
authors.lastname: Higashihashi
authors.forename: T
authors.initials: T
authors.lastname: Enjoh-Kimura
authors.forename: H
authors.initials: H
authors.lastname: Terasawa
authors.forename: Y
authors.initials: Y
authors.lastname: Fujita-Yamaguchi
authors.forename: F
authors.initials: F
authors.lastname: Inagaki
authors.forename: J F
authors.initials: JF
authors.lastname: Perdue
authors.forename: K
authors.initials: K
authors.lastname: Sakano
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D017384:Sequence Deletion; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
chemical_list: D013912:Threonine; D007335:Insulin-Like Growth Factor II; D004247:DNA; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D007930:Leucine