MUTATIONS AT POSITION-11 AND POSITION-60 OF INSULIN-LIKE-GROWTH-FACTOR-1 REVEAL DIFFERENCES BETWEEN ITS INTERACTIONS WITH THE TYPE-I INSULIN-LIKE-GROWTH-FACTOR RECEPTOR AND THE INSULIN-RECEPTOR

Insulin-like growth factor 1 (IGF-1) and three analogues ([V11I]IGF-I, [V11T]IGF-1, and [Y60F]IGF-1), constructed by site-directed mutagenes is, were expressed as fusion proteins and secreted into the periplasmi c space of Escherichia coli. Purified IGF were obtained following IgG Sepharose affinity and cation-exchange chromatographies of the product s of hydroxylamine cleavage of fusion proteins. The properties of the mutants were assessed using (a) quantification of affinities for the h uman insulin receptor overexpressed on NIH 3T3 cells, (b) quantificati on of affinities for the type I IGF receptor via competition for bindi ng to a monolayer of MDA-MB-231 cells, (c) promotion of the in vitro m itogenesis of growth-arrested MCF-7 cells in the presence of 17-beta-o estradiol, and (d) a competition assay for binding to IGF-binding prot eins secreted by MCF-7 cells. The mutants exhibited decreases in affin ity for the insulin receptor, relative to IGF-I, of 2.6-, 3.8- and, 8. 8-fold for [Y60F]IGF-1, [V11I]IGF-1, and [V11T]IG-1, respectively. IGF -1, [V11I]IGF-I, and [Y60F]IGF-1 were of equal potency in the growth a ssay and in affinity for the type I IGF receptor. [V11T]IGF-1 exhibite d a three fold loss of potency in the type I IGF receptor-binding and growth assays. The mutants did not differ significantly from IGF-1 in their affinities for the IGF-binding proteins. The full-activity of [Y 60F]IGF-1 at the type I IGF receptor, in contrast to the weakened rece ptor affinity of IGF-1 with a Leu substitution at this position, indic ates a requirement for an aromatic ring, rather than a hydroxyl group, in the interaction of IGF-I with the type I IGF receptor. The decreas e in affinity for the insulin receptor of all the mutants indicates th at, as in insulin, the residues Val11 and Tyr60 are important for the interaction of IGF-1 with the insulin receptor. The unchanged or minor changes in the affinities of the mutants for the type I IGF receptor contrast with the more deleterious effects of the mutations on insulin receptor binding and with the properties of analogues of insulin muta ted at equivalent sites: 3-fold and 5-10-fold reductions in biological activity for [VB12I]insulin and [YA19F]insulin, respectively Thus, th e results obtained using the mutants indicate important differences be tween the IGF-1/type I IGF receptor and insulin/insulin receptor inter actions.