Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Abstract

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.

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

“…By contrast, in holoIGF-1R.IGF-I, the C-terminal segment of IGF-I C domain appears to lack any stabilizing interactions with either the growth factor or the receptor; indeed, IGF-I residues 38–40 are unmodeled in holoIGF-1R.IGF-I ( Li et al., 2019 ). We note that lengthening of the IGF-II C domain—by insertion of elements of the IGF-I C domain—increases the affinity of IGF-II for IGF-1R ( Henderson et al., 2015 , Hexnerová et al., 2016 ).…”

“…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).…”

Cross-Linking/Mass Spectrometry Uncovers Details of Insulin-Like Growth Factor Interaction With Insect Insulin Binding Protein Imp-L2

“…Des(63-70)-IGF-1was created with an additional N -terminal Gly-1 to facilitate TEV cleavage. Des(63-70)-IGF-1 was produced in E. coli BL21(DE3), purified and characterized as previously published (14).…”

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

“…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 binding affinities of analogs were determined with receptors in the intact cells. Specifically, binding affinities for IGF-1R were determined with mouse fibroblasts transfected with human IGF-1R and with deleted mouse IGF-1R, according to Hexnerova et al [55]. The [ 125 I]-monoiodotyrosyl-IGF1 was used as a radiotracer.…”