Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation

Abstract

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.

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.…”

“…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].…”

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

“…We prepared the IGF-1 lacking the D domain residues PLKPAKSA (des(63-70)-IGF-1) with the aim to reduce the number of primary amines available for reaction with the succinimide ester in Sulfo-SDA and thus simplify the MS analysis (Figure 2). Also, an additional N -terminal glycine residue Gly(-1) was incorporated into IGF-1 to facilitate production of des(63-70)-IGF-1 (13).…”

“…Human IGF-1 was purchased from Tercica Inc. Human IGF-1 lacking the C-terminal D domain amino acids PLKPAKSA (des(63-70)-IGF-1) (IGF-1 UniprotKB entry P05019, amino acids 49–110) was cloned similarly to our previous work (13). Briefly, modified pRSFDuet-1 harboring Gly-1-IGF-1 was amplified by PCR using ACYCDuetUP1 (5′-GGATCTCGACGCTCTCCCT−3′) and IGF-1del PLKPAKSA Rev (5′-GCAGGTGAATTCATTACGCGCAATACATTTCCAG−3′) primers and cloned back into NcoI/EcoRI sites of pRSFDuet-1.…”

A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding

“…Our laboratory is interested in the design of insulin and IGFs analogs, which should map the structure–activity relationship among ligands and their receptors and potentially serve medicinal purposes ( 22 , 23 , 29 , 32 – 35 ). In the course of routine testing of our analogs, we have detected the unexpectedly high binding and stimulation of IGF-1R by [ d -His B24 ]-insulin ( 29 ).…”