Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Abstract

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58–IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

How IGF-II Binds to the Human Type 1 Insulin-like Growth Factor Receptor

“…Details of the above steps and assays are provided in STAR Methods . We note that the IC 50 values reported here for IGFs binding to holoIGF-1R differ from those reported by, for example, Macháčková et al. (2019) yet broadly concur with those reported earlier by Surinya et al.…”

“…The bioavailability of IGF-I and IGF-II is controlled by six insulin-like growth factor-binding proteins ( Baxter, 2014 ), and IGF-II is sequestered by the membrane-anchored type 2 insulin-like growth factor receptor (IGF-2R) that can also influence signaling via G-protein interaction ( El-Shewy and Luttrell, 2009 ). The affinity of IGF-II for IGF-1R is reported to be up to an order of magnitude lower than that of IGF-I ( Pandini et al., 2002 , Surinya et al., 2008 , Henderson et al., 2015 , Macháčková et al., 2019 ), with the lower affinity appearing to arise at least in part from differences in the length and amino acid composition of the C domains of the respective growth factors ( Denley et al., 2004 , Henderson et al., 2015 , Hexnerová et al., 2016 ) ( Figure 1 B). IGF-1R itself is closely related in structure to its homolog, the human insulin receptor (IR; Figure 1 A).…”

A radioligand binding assay for the insulin-like growth factor 2 receptor

“…The results are shown in S6 Fig in S1 File. In general, the abilities of all ligands to induce phosphorylation of both IR-A and IGF1R followed the trends of their binding affinities for these receptors and were in agreement with our previous data [44,46,48]. Leu19-IGF2 mutant exhibited a reduced capability to activate IR-A, compared to that of native IGF2 (S6A Fig in S1 File), which coincides well with its reduced binding.…”

“…Leu19-IGF2 analog was produced, according to our previously published protocols [48,55]. Briefly, Leu19-IGF2 was cloned into a modified pRSFDuet-1 expression vector as the fusion with an N-terminally His6 tagged-GB1 protein and TEV protease cleavage site.…”

“…The determination of binding affinities of hormones to their relevant receptors plays a pivotal role in the design of new analogs with altered properties for structure-activity studies with receptors or for therapeutic applications. The highly sensitive binding assays employing 125 Ilabeled hormones for unequivocal determination of binding affinities of the hormones toward both isoforms of insulin receptor as well as toward IGF1 receptor have been established [40] and are routinely used in our studies [41][42][43][44][45][46][47][48].…”