Abstract
Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain.
Highlights
Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their cognate cell surface receptors, together with IGF-binding proteins (IGFBP-1−6), form a complex hormonal/signaling system that is essential for the development and functioning of humans
The extracellular α parts of the IR contain hormone-binding regions, and the membrane-anchored parts β contain intracellular tyrosine kinase domains.[7−9] The functional heterogeneity of IR arises from an alternative splicing of exon 11 located at the Cterminus of the α subunit, which results in two isoforms, IR-B and IR-A, with different C-terminal αCT peptides (IR-B and IR-A, ±12 amino acids)
Insulin’s A chain C-terminal AsnA21 has been substituted in analogues 1−3 with alanine that is present in both IGFs at that site; the A21 site could be considered here as part of the D domains
Summary
Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their cognate cell surface receptors, together with IGF-binding proteins (IGFBP-1−6), form a complex hormonal/signaling system that is essential for the development and functioning of humans. IGF-1 and IGF-2 are single-chain hormones (70 and 67 amino acids, respectively) involved mainly in cell growth and protection against apoptosis.[4,5] Both IGFs share a high degree of sequence homology, which is extended to insulin (see Figure 1). They consist of four domains, B, C, A, and D, with their B and A domains corresponding to the B and A chains of insulin. The recent crystallographic studies showed that site 1 on both IR and IGF1R receptors is similar, involving some L1 domain surface and the αCT peptide, which interact with the respective binding sites 1 on insulin and IGFs (in A and B chains/domains)[17,18] or IGF-1.19 the nature of IR/IGF-1R-binding site 2 is still understood only on the basis of mutagenesis studies.[20−23]
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