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.

• Klíčová slova
• mutagenesis in vitro, peptide hormone, receptor modification, receptor tyrosine kinase, structure–function,
• MeSH
• elektronová kryomikroskopie MeSH
• insulinu podobný růstový faktor I genetika   chemie   metabolismus   MeSH
• inzulin metabolismus   MeSH
• ligandy MeSH
• receptor IGF typ 1 * genetika   chemie   metabolismus   MeSH
• receptor inzulinu * genetika   chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• insulinu podobný růstový faktor I MeSH
• inzulin MeSH
• ligandy MeSH
• receptor IGF typ 1 * MeSH
• receptor inzulinu * 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.

• Klíčová slova
• Adipose tissue, GLUT4, Glucose metabolism, IGF-1, Insulin, VILPs, Viral insulin, Viral mimicry,
• MeSH
• bílá tuková tkáň metabolismus   MeSH
• buněčné linie MeSH
• CD antigeny MeSH
• fosforylace MeSH
• glukosa metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• inzulin genetika   metabolismus   MeSH
• inzuliny metabolismus   MeSH
• Iridovirus genetika   MeSH
• iridoviry genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• receptor IGF typ 1 genetika   metabolismus   MeSH
• receptor inzulinu metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CD antigeny MeSH
• glukosa MeSH
• IGF1 protein, human MeSH Prohlížeč
• IGF1R protein, human MeSH Prohlížeč
• Igf1r protein, mouse MeSH Prohlížeč
• INSR protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• inzulin MeSH
• inzuliny MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu 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.

• Klíčová slova
• 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
• insulinu podobný růstový faktor I chemie   genetika   MeSH
• insulinu podobný růstový faktor II chemie   genetika   MeSH
• inzulin analogy a deriváty   chemická syntéza   chemie   genetika   MeSH
• lidé MeSH
• mnohočetné abnormality genetika   MeSH
• mutace genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• poruchy růstu genetika   MeSH
• proteinové domény genetika   MeSH
• receptor IGF typ 1 chemie   genetika   MeSH
• receptor inzulinu chemie   genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• vazba proteinů genetika   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IGF1R protein, human MeSH Prohlížeč
• IGF2 protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• insulinu podobný růstový faktor II MeSH
• inzulin MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH

Cone snails have evolved a variety of insulin-like molecules that may help with the development of better treatments for diabetes.

• Klíčová slova
• biochemistry, chemical biology, cone snail, diabetes, hypoglycemic shock, insulin, receptors, venom,
• MeSH
• diabetes mellitus * MeSH
• jedy měkkýšů MeSH
• ligandy MeSH
• obratlovci MeSH
• peptidy MeSH
• receptor inzulinu MeSH
• živočišné jedy * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• Názvy látek
• jedy měkkýšů MeSH
• ligandy MeSH
• peptidy MeSH
• receptor inzulinu MeSH
• živočišné jedy * 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.

• Klíčová slova
• Site 1, binding, insulin, insulin receptor, insulin-like growth factor (IGF), kinetics, protein design, structure-function,
• MeSH
• insulinu podobný růstový faktor I chemie   genetika   metabolismus   MeSH
• inzulin agonisté   metabolismus   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu chemie   genetika   metabolismus   MeSH
• receptory somatomedinů chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IGF1R protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• inzulin MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH
• receptory somatomedinů MeSH

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.

• Klíčová slova
• insulin, insulin receptor, insulin-like growth factor (IGF), nuclear magnetic resonance (NMR), structural biology, structure-function,
• MeSH
• CD antigeny chemie   genetika   metabolismus   MeSH
• insulinu podobný růstový faktor II chemie   genetika   metabolismus   MeSH
• lidé MeSH
• missense mutace MeSH
• protein - isoformy chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• receptor IGF typ 1 chemie   genetika   metabolismus   MeSH
• receptor inzulinu chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CD antigeny MeSH
• IGF2 protein, human MeSH Prohlížeč
• INSR protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor II MeSH
• protein - isoformy MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH
• rekombinantní proteiny MeSH