The preparation of specifically iodine-125 (125I)-labeled peptides of high purity and specific activity represents a key tool for the detailed characterization of their binding properties in interaction with their binding partners. Early synthetic methods for the incorporation of iodine faced challenges such as harsh reaction conditions, the use of strong oxidants and low reproducibility. Herein, we review well-established radiolabeling strategies available to incorporate radionuclide into a protein of interest, and our long-term experience with a mild, simple and generally applicable technique of 125I late-stage-labeling of biomolecules using the Pierce iodination reagent for the direct solid-phase oxidation of radioactive iodide. General recommendations, tips, and details of optimized chromatographic conditions to isolate pure, specifically 125I-mono-labeled biomolecules are illustrated on a diverse series of (poly)peptides, ranging up to 7.6 kDa and 67 amino acids (aa). These series include peptides that contain at least one tyrosine or histidine residue, along with those featuring disulfide crosslinking or lipophilic derivatization. This mild and straightforward late-stage-labeling technique is easily applicable to longer and more sensitive proteins, as demonstrated in the cases of the insulin-like growth factor binding protein-3 (IGF-BP-3) (29 kDa and 264 aa) and the acid-labile subunit (ALS) (93 kDa and 578 aa).

• Keywords
• 125I-labeling of peptides, High specific activity, Intramolecular effect of 125I decay, Late-stage peptide labeling, Radiochemical stability, Radiohalogenated prosthetic groups, Site-specific labeling,
• Publication type
• Journal Article MeSH
• Review MeSH

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-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