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Eur J Biochem. 1995 Oct 1;233(1):299-309.

Mutations at positions 11 and 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.

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  • 1University Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, England.


Insulin-like growth factor 1 (IGF-1) and three analogues ([V11I]IGF-1, [V11T]IGF-1, and [Y60F]IGF-1), constructed by site-directed mutagenesis, were expressed as fusion proteins and secreted into the periplasmic space of Escherichia coli. Purified IGF were obtained following IgG Sepharose affinity and cation-exchange chromatographies of the products of hydroxylamine cleavage of fusion proteins. The properties of the mutants were assessed using (a) quantification of affinities for the human insulin receptor overexpressed on NIH 3T3 cells, (b) quantification of affinities for the type I IGF receptor via competition for binding to a monolayer of MDA-MB-231 cells, (c) promotion of the in vitro mitogenesis of growth-arrested MCF-7 cells in the presence of 17-beta-oestradiol, and (d) a competition assay for binding to IGF-binding proteins secreted by MCF-7 cells. The mutants exhibited decreases in affinity for the insulin receptor, relative to IGF-1, of 2.6-, 3.8- and, 8.8-fold for [Y60F]IGF-1, [V11I]IGF-1, and [V11T]IGF-1, respectively. IGF-1, [V11I]IGF-1, and [Y60F]IGF-1 were of equal potency in the growth assay and in affinity for the type I IGF receptor. [V11T]IGF-1 exhibited 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 [Y60F]IGF-1 at the type I IGF receptor, in contrast to the weakened receptor affinity of IGF-1 with a Leu substitution at this position, indicates a requirement for an aromatic ring, rather than a hydroxyl group, in the interaction of IGF-1 with the type I IGF receptor. The decrease in affinity for the insulin receptor of all the mutants indicates that, 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 mutated at equivalent sites: 3-fold and 5-10-fold reductions in biological activity for [VB12I]insulin and [YA19F]insulin, respectively. Thus, the results obtained using the mutants indicate important differences between the IGF-1/type I IGF receptor and insulin/insulin receptor interactions.

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