The insulin receptor (IR, with its isoforms IR-A and IR-B) and the insulin-like growth factor 1 receptor (IGF-1R) are related tyrosine kinase receptors. Recently, the portfolio of solved hormone-receptor structures has grown extensively thanks to advancements in cryo-electron microscopy. However, the dynamics of how these receptors transition between their inactive and active state are yet to be fully understood. The C-terminal part of the alpha subunit (αCT) of the receptors is indispensable for the formation of the hormone-binding site. We mutated the αCT residues Arg717 and His710 of IR-A and Arg704 and His697 of IGF-1R. We then measured the saturation binding curves of ligands on the mutated receptors and their ability to become activated. Mutations of Arg704 and His697 to Ala in IGF-1R decreased the binding of IGF-1. Moreover, the number of binding sites for IGF-1 on the His697 IGF-1R mutant was reduced to one-half, demonstrating the presence of two binding sites. Both mutations of Arg717 and His710 to Ala in IR-A inactivated the receptor. We have proved that Arg717 is important for the binding of insulin to its receptor, which suggests that Arg717 is a key residue for the transition to the active conformation.

• Keywords
• mutagenesis in vitro, peptide hormone, receptor modification, receptor tyrosine kinase, structure–function,
• MeSH
• Cryoelectron Microscopy MeSH
• Insulin-Like Growth Factor I genetics   chemistry   metabolism   MeSH
• Insulin metabolism   MeSH
• Ligands MeSH
• Receptor, IGF Type 1 * genetics   chemistry   metabolism   MeSH
• Receptor, Insulin * genetics   chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insulin-Like Growth Factor I MeSH
• Insulin MeSH
• Ligands MeSH
• Receptor, IGF Type 1 * MeSH
• Receptor, Insulin * MeSH

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.

• Keywords
• antagonism, dicarba, disulfide mimetics, insulin mimetic peptide, insulin receptor, staple, triazole,
• MeSH
• Disulfides chemistry   MeSH
• Insulin * metabolism   MeSH
• Peptides chemistry   MeSH
• Receptor, Insulin * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Disulfides MeSH
• Insulin * MeSH
• Peptides MeSH
• Receptor, Insulin * MeSH

UNLABELLED: Insulin-like growth factor 1 (IGF-1) and its IGF-1 receptor (IGF-1R) belong to an important biological system that is involved in the regulation of normal growth, but that has also been recognized as playing a role in cancer. IGF-1R antagonists could be interesting for the testing of their potential antiproliferative properties as an alternative to IGF-1R tyrosine-kinase inhibitors or anti-IGF-1R monoclonal antibodies. In this study, we were inspired by the successful development of insulin dimers capable of antagonizing insulin effects on the insulin receptor (IR) by simultaneous binding to two separated binding sites and by blocking structural rearrangement of the IR. We designed and produced in Escherichia coli three different IGF-1 dimers in which IGF-1 monomers are interlinked through their N- and C-termini, with linkers having 8, 15 or 25 amino acids. We found that the recombinant products were susceptible to the formation of misfolded or reduced variants, but that some of them were able to bind IGF-1R in low nanomolar affinities and all of them activate IGF-1R proportionally to their binding affinities. Overall, our work can be considered as a pilot study that, although it did not lead to the discovery of new IGF-1R antagonists, explored the possibility of recombinant production of IGF-1 dimers and led to the preparation of active compounds. This work could inspire further studies dealing, for example, with the preparation of IGF-1 conjugates with specific proteins for the study of the hormone and its receptor or for therapeutic applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-023-10499-1.

• Keywords
• Binding, Dimer, Hormone, IGF-1, Insulin, Phosphorylation, Receptor,
• Publication type
• Journal Article MeSH

OBJECTIVE: Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. METHODS: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. RESULTS: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. CONCLUSIONS: Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

• Keywords
• Adipose tissue, GLUT4, Glucose metabolism, IGF-1, Insulin, VILPs, Viral insulin, Viral mimicry,
• MeSH
• Adipose Tissue, White metabolism   MeSH
• Cell Line MeSH
• Antigens, CD MeSH
• Phosphorylation MeSH
• Glucose metabolism   MeSH
• Adipose Tissue, Brown metabolism   MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin genetics   metabolism   MeSH
• Insulins metabolism   MeSH
• Iridovirus genetics   MeSH
• Iridoviridae genetics   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Receptor, IGF Type 1 genetics   metabolism   MeSH
• Receptor, Insulin metabolism   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antigens, CD MeSH
• Glucose MeSH
• IGF1 protein, human MeSH Browser
• IGF1R protein, human MeSH Browser
• Igf1r protein, mouse MeSH Browser
• INSR protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin MeSH
• Insulins MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin MeSH

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

• MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin-Like Growth Factor II metabolism   MeSH
• Binding, Competitive MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Iodine Radioisotopes MeSH
• Radioligand Assay methods   MeSH
• Receptor, IGF Type 2 immunology   ultrastructure   MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1 protein, human MeSH Browser
• IGF2 protein, human MeSH Browser
• IGF2R protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin-Like Growth Factor II MeSH
• Iodine-125 MeSH Browser
• Iodine Radioisotopes MeSH
• Receptor, IGF Type 2 MeSH

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.

• Keywords
• NMR structure, complex, hormone analog, insulin, insulin-like growth factor (IGF), molecular dynamics, mutagenesis, peptide hormone, receptor autophosphorylation, receptor binding, receptor tyrosine kinase, structural biology, structure-function,
• MeSH
• Insulin-Like Growth Factor I chemistry   genetics   MeSH
• Insulin-Like Growth Factor II chemistry   genetics   MeSH
• Insulin analogs & derivatives   chemical synthesis   chemistry   genetics   MeSH
• Humans MeSH
• Abnormalities, Multiple genetics   MeSH
• Mutation genetics   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Growth Disorders genetics   MeSH
• Protein Domains genetics   MeSH
• Receptor, IGF Type 1 chemistry   genetics   MeSH
• Receptor, Insulin chemistry   genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• Protein Binding genetics   MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1R protein, human MeSH Browser
• IGF2 protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin-Like Growth Factor II MeSH
• Insulin MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin MeSH

Insulin and insulin-like growth factor 1 (IGF-1) are closely related hormones involved in the regulation of metabolism and growth. They elicit their functions through activation of tyrosine kinase-type receptors: insulin receptors (IR-A and IR-B) and IGF-1 receptor (IGF-1R). Despite similarity in primary and three-dimensional structures, insulin and IGF-1 bind the noncognate receptor with substantially reduced affinity. We prepared [d-HisB24, GlyB31, TyrB32]-insulin, which binds all three receptors with high affinity (251 or 338% binding affinity to IR-A respectively to IR-B relative to insulin and 12.4% binding affinity to IGF-1R relative to IGF-1). We prepared other modified insulins with the aim of explaining the versatility of [d-HisB24, GlyB31, TyrB32]-insulin. Through structural, activity, and kinetic studies of these insulin analogs, we concluded that the ability of [d-HisB24, GlyB31, TyrB32]-insulin to stimulate all three receptors is provided by structural changes caused by a reversed chirality at the B24 combined with the extension of the C terminus of the B chain by two extra residues. We assume that the structural changes allow the directing of the B chain C terminus to some extra interactions with the receptors. These unusual interactions lead to a decrease of dissociation rate from the IR and conversely enable easier association with IGF-1R. All of the structural changes were made at the hormones' Site 1, which is thought to interact with the Site 1 of the receptors. The results of the study suggest that merely modifications of Site 1 of the hormone are sufficient to change the receptor specificity of insulin.

• Keywords
• Site 1, binding, insulin, insulin receptor, insulin-like growth factor (IGF), kinetics, protein design, structure-function,
• MeSH
• Insulin-Like Growth Factor I chemistry   genetics   metabolism   MeSH
• Insulin agonists   metabolism   MeSH
• Kinetics MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin chemistry   genetics   metabolism   MeSH
• Receptors, Somatomedin chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1R protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin MeSH
• Receptor, IGF Type 1 MeSH
• Receptor, Insulin MeSH
• Receptors, Somatomedin MeSH