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

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

• Klíčová slova
• Förster resonance energy transfer, Nkrp1, cysteine, dimerization, disulfide bond arrangement,
• MeSH
• antigeny Ly analýza   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• Jurkat buňky MeSH
• lektinové receptory NK-buněk - podrodina B analýza   chemie   MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• receptory imunologické analýza   MeSH
• refolding proteinů MeSH
• rezonanční přenos fluorescenční energie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny Ly MeSH
• Klrb1a protein, mouse MeSH Prohlížeč
• Klrb1c protein, mouse MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• Nkrp1f protein, mouse MeSH Prohlížeč
• receptory imunologické 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

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

• MeSH
• alkyny chemie   MeSH
• azidy chemie   MeSH
• cykloadiční reakce MeSH
• inzulin chemie   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• protein - isoformy MeSH
• receptor IGF typ 1 chemie   metabolismus   MeSH
• receptor inzulinu chemie   metabolismus   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkyny MeSH
• azidy MeSH
• inzulin MeSH
• protein - isoformy MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH